AJP: Cell Physiology recent issues

"Systems biology"-What's in a name?

Brown, D. — 2008-06-09

Mitochondria, oxygen sensing, and the regulation of HIF-2{alpha}. Focus on "Induction of HIF-2{alpha} is dependent on mitochondrial O2 consumption in an O2-sensitive adrenomedullary chromaffin cell line"

Taylor, C. T. — 2008-06-09

Maintaining the integrity of trophoblast during growth of the placenta. Focus on "Insulin-like growth factor I and II regulate the life cycle of trophoblast in the developing human placenta"

Carter, A. M. — 2008-06-09

Induction of HIF-2{alpha} is dependent on mitochondrial O2 consumption in an O2-sensitive adrenomedullary chromaffin cell line

Brown, S. T., Nurse, C. A. — 2008-06-09

During low O₂ (hypoxia), hypoxia-inducible factor (HIF)- is stabilized and translocates to the nucleus, where it regulates genes critical for survival and/or adaptation in low O₂. While it appears that mitochondria play a critical role in HIF induction, controversy surrounds the underlying mechanism(s). To address this, we monitored HIF-2 expression and oxygen consumption in an O₂-sensitive immortalized rat adrenomedullary chromaffin (MAH) cell line. Hypoxia (2–8% O₂) caused a concentration- and time-dependent increase in HIF-2 induction, which was blocked in MAH cells with either RNA interference knockdown of the Rieske Fe-S protein, a component of complex III, or knockdown of cytochrome-c oxidase subunit of complex IV, or defective mitochondrial DNA (0 cells). Additionally, pharmacological inhibitors of mitochondrial complexes I, III, IV, i.e., rotenone (1 µM), myxothiazol (1 µM), antimycin A (1 µg/ml), and cyanide (1 mM), blocked HIF-2 induction in control MAH cells. Interestingly, the inhibitory effects of the mitochondrial inhibitors were dependent on O₂ concentration such that at moderate-to-severe hypoxia (6% O₂), HIF-2 induction was blocked by low inhibitor concentrations that were ineffective at more severe hypoxia (2% O₂). Manipulation of the levels of reactive oxygen species (ROS) had no effect on HIF-2 induction. These data suggest that in this O₂-sensitive cell line, mitochondrial O₂ consumption, rather than changes in ROS, regulates HIF-2 during hypoxia.

Insulin-like growth factor I and II regulate the life cycle of trophoblast in the developing human placenta

Forbes, K., Westwood, M., Baker, P. N., Aplin, J. D. — 2008-06-09

The main disorders of human pregnancy are rooted in defective placentation. Normal placental development depends on proliferation, differentiation, and fusion of cytotrophoblasts to form and maintain an overlying syncytiotrophoblast. There is indirect evidence that the insulin-like growth factors (IGFs), which are aberrant in pregnancy disorders, are involved in regulating trophoblast turnover, but the processes that control human placental growth are poorly understood. Using an explant model of human first-trimester placental villus in which the spatial and ontological relationships between cell populations are maintained, we demonstrate that cytotrophoblast proliferation is enhanced by IGF-I/IGF-II and that both factors can rescue cytotrophoblast from apoptosis. Baseline cytotrophoblast proliferation ceases in the absence of syncytiotrophoblast, although denuded cytotrophoblasts can proliferate when exposed to IGF and the rate of cytotrophoblast differentiation/fusion and, consequently, syncytial regeneration, increases. Use of signaling inhibitors suggests that IGFs mediate their effect on cytotrophoblast proliferation/syncytial formation through the MAPK pathway, whereas effects on survival are regulated by the phosphoinositide 3-kinase pathway. These results show that directional contact between cytotrophoblast and syncytium is important in regulating the relative amounts of the two cell populations. However, IGFs can exert an exogenous regulatory influence on placental growth/development, suggesting that manipulation of the placental IGF axis may offer a potential therapeutic route to the correction of inadequate placental growth.

Enhanced exocytotic-like insertion of Orai1 into the plasma membrane upon intracellular Ca2+ store depletion

Woodard, G. E., Salido, G. M., Rosado, J. A. — 2008-06-09

Ca⁺ release-activated Ca²⁺ (CRAC) channels are activated when free Ca²⁺ concentration in the intracellular stores is substantially reduced and mediate sustained Ca²⁺ entry. Recent studies have identified Orai1 as a CRAC channel subunit. Here we demonstrate that passive Ca²⁺ store depletion using the inhibitor of the sarcoendoplasmic reticulum Ca²⁺-ATPase, thapsigargin (TG), enhances the surface expression of Orai1, a process that depends on rises in cytosolic free Ca²⁺ concentration, as demonstrated in cells loaded with dimethyl BAPTA, an intracellular Ca²⁺ chelator that prevented TG-evoked cytosolic free Ca²⁺ concentration elevation. Similar results were observed with a low concentration of carbachol. Cleavage of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor, synaptosomal-assiciated protein-25 (SNAP-25), with botulinum neurotoxin A impaired TG-induced increase in the surface expression of Orai1. In addition, SNAP-25 cleaving by botulinum neurotoxin A reduces the maintenance but not the initial stages of store-operated Ca²⁺ entry. In aggregate, these findings demonstrate that store depletion enhances Orai1 plasma membrane expression in an exocytotic manner that involves SNAP-25, a process that contributes to store-dependent Ca²⁺ entry.

Specific signals involved in the long-term maintenance of radiation-induced fibrogenic differentiation: a role for CCN2 and low concentration of TGF-{beta}1

Haydont, V., Riser, B. L., Aigueperse, J., Vozenin-Brotons, M.-C. — 2008-06-09

The fibrogenic differentiation of resident mesenchymal cells is a key parameter in the pathogenesis of radiation fibrosis and is triggered by the profibrotic growth factors transforming growth factor (TGF)-β1 and CCN2. TGF-β1 is considered the primary inducer of fibrogenic differentiation and is thought to control its long-term maintenance, whereas CCN2 is considered secondary effector of TGF-β1. Yet, in long-term established fibrosis like that associated with delayed radiation enteropathy, in situ TGF-β1 deposition is low, whereas CCN2 expression is high. To explore this apparent paradox, cell response to increasing doses of TGF-β1 was investigated in cells modeling initiation and maintenance of fibrosis, i.e., normal and fibrosis-derived smooth muscle cells, respectively. Activation of cell-specific signaling pathways by low TGF-β1 doses was demonstrated with a main activation of the Rho/ROCK pathway in fibrosis-derived cells, whereas the Smad pathway was mainly activated in normal cells. This leads to subsequent and cell-specific regulation of the CCN2 gene. These results suggested a specific profibrotic role of CCN2 in fibrosis-initiated cells. Furthermore, the modulation of CCN2 expression by itself and the combination of TGF-β1 and CCN2 was investigated in fibrosis-derived cells. In fibrosis-initiated cells CCN2 triggered its autoinduction; furthermore, low concentration of TGF-β1-potentiated CCN2 autoinduction. Our findings showed a differential requirement and action of TGF-β1 in the fibrogenic response of normal vs. fibrosis-derived cells. This study defines a novel Rho/ROCK but Smad3-independent mode of TGF-β signaling that may operate during the chronic stages of fibrosis and provides evidence of both specific and combinatorial roles of low TGF-β1 dose and CCN2.

Zinc-induced G2/M blockage is p53 and p21 dependent in normal human bronchial epithelial cells

Wong, S. H. K., Shih, R. S. M., Schoene, N. W., Lei, K. Y. — 2008-06-09

The involvement of p53 and p21 signal pathway in the G2/M cell cycle progression of zinc-supplemented normal human bronchial epithelial (NHBE) cells was examined using the small interferring RNA (siRNA) approach. Cells were cultured for one passage in a different concentration of zinc: <0.4 µM (ZD) as zinc deficient; 4 µM as normal zinc level (ZN) in culture medium; 16 µM (ZA) as normal human plasma zinc level; and 32 µM (ZS) as the high end of plasma zinc attainable by oral supplementation. Nuclear p21 protein and mRNA levels as well as promoter activity in ZS cells, but not in ZD cells, were markedly elevated to almost twofold compared with ZN control cells. G2/M blockage in ZS cells was coupled with the observation of elevated p21 gene expression. In ZS cells, the abrogation of p21 protein induction by the transfection of p21 siRNA was shown to alleviate the G2/M blockage, demonstrating the positive linkage of p21 elevation and G2/M blockage. Abolishment of the increase in p53 protein in ZS cells with transfection of p53 siRNA normalized the elevated p21 protein to a similar level as in ZN control cells, which demonstrated that the p21 induction is p53 dependent. Furthermore, the normalization of p53 protein by siRNA treatment in ZS cells alleviated cell growth depression and G2/M blockage, which demonstrated that p53 was involved in the high zinc status-induced G2/M blockage and growth depression. Thus high zinc status in NHBE cells upregulates p53 expression which in turn elevates p21 that eventually induces G2/M blockage.

Repetitive deformation activates Src-independent FAK-dependent ERK motogenic signals in human Caco-2 intestinal epithelial cells

Chaturvedi, L. S., Gayer, C. P., Marsh, H. M., Basson, M. D. — 2008-06-09

Repetitive deformation due to villous motility or peristalsis may support the intestinal mucosa, stimulating intestinal epithelial proliferation under normal circumstances and restitution in injured and inflamed mucosa rich in tissue fibronectin. Cyclic strain enhances Caco-2 and IEC-6 intestinal epithelial cell migration across fibronectin via ERK. However, the upstream mediators of ERK activation are unknown. We investigated whether Src and FAK mediate strain-induced ERK phosphorylation and migration in human Caco-2 intestinal epithelial cells on fibronectin. Monolayers on tissue fibronectin-precoated membranes were subjected to an average 10% repetitive deformation at 10 cycles/min. Phosphorylation of Src-Tyr⁴¹⁸, FAK-Tyr³⁹⁷-Tyr⁵⁷⁶-Tyr⁹²⁵, and ERK were significantly increased by deformation. The stimulation of wound closure by strain was prevented by Src blockade with PP2 (10 µmol/l) or specific short interfering (si)RNA. Src inhibition also prevented strain-induced FAK phosphorylation at Tyr³⁹⁷ and Tyr⁵⁷⁶ but not FAK-Tyr⁹²⁵ or ERK phosphorylation. Reducing FAK by siRNA inhibited strain-induced ERK phosphorylation. Transfection of NH₂-terminal tyrosine phosphorylation-deficient FAK mutants Y397F, Y576F-Y577F, and Y397F-Y576F-Y577F did not prevent the activation of ERK2 by cyclic strain, but a FAK mutant at the COOH terminal (Y925F) prevented the strain-induced activation of ERK2. Although the Y397F-Y576F-Y577F FAK construct exhibited less basal FAK-Tyr⁹²⁵ phosphorylation under static conditions, it nevertheless exhibited increased FAK-Tyr⁹²⁵ phosphorylation in response to strain. These results suggest that repetitive deformation stimulates intestinal epithelial motility across fibronectin in a manner that requires both Src activation and a novel Src-independent FAK-Tyr⁹²⁵-dependent pathway that activates ERK. This pathway may be an important target for interventions to promote mucosal healing in settings of intestinal ileus or fasting.

Induction of Na+/K+/2Cl- cotransporter expression mediates chronic potentiation of intestinal epithelial Cl- secretion by EGF

O'Mahony, F., Toumi, F., Mroz, M. S., Ferguson, G., Keely, S. J. — 2008-06-09

Alterations in EGF receptor (EGFR) signaling occur in intestinal disorders associated with dysregulated epithelial transport. In the present study, we investigated a role for the EGFR in the chronic regulation of intestinal epithelial secretory function. Epithelial Cl^– secretion was measured as changes in short-circuit current (I_sc) across voltage-clamped monolayers of T₈₄ cells in Ussing chambers. Acute treatment of T₈₄ cells with EGF (100 ng/ml, 15 min) chronically enhanced I_sc responses to a broad range of secretagogues. This effect was apparent within 3 h, maximal by 6 h, and sustained for 24 h after treatment with EGF. The Na⁺/K⁺/2Cl^– cotransporter (NKCC1) inhibitor bumetanide (100 µM) abolished the effect of EGF, indicating increased responses are due to potentiated Cl^– secretion. Neither basal nor agonist-stimulated levels of intracellular Ca²⁺ or PKA activity were altered by EGF, implying that the effects of the growth factor are not due to chronic alterations in levels of second messengers. EGF increased the expression of NKCC1 with a time course similar to that of its effects on Cl^– secretion. This effect of EGF was maximal after 6 h, at which time NKCC1 expression in EGF-treated cells was 199.9 ± 21.9% of that in control cells (n = 21, P < 0.005). EGF-induced NKCC1 expression was abolished by actinomycin D, and RT-PCR analysis demonstrated EGF increased expression of NKCC1 mRNA. These data increase our understanding of mechanisms regulating intestinal fluid and electrolyte transport and reveal a novel role for the EGFR in the chronic regulation of epithelial secretory capacity through upregulation of NKCC1 expression.

Bestrophin Cl- channels are highly permeable to HCO3-

Qu, Z., Hartzell, H. C. — 2008-06-09

Bestrophin-1 (Best1) is a Cl^– channel that is linked to various retinopathies in both humans and dogs. Dysfunction of the Best1 Cl^– channel has been proposed to cause retinopathy because of altered Cl^– transport across the retinal pigment epithelium (RPE). In addition to Cl^–, many Cl^– channels also transport HCO₃^–. Because HCO₃^– is physiologically important in pH regulation and in fluid and ion transport across the RPE, we measured the permeability and conductance of bestrophins to HCO₃^– relative to Cl^–. Four human bestrophin homologs (hBest1, hBest2, hBest3, and hBest4) and mouse Best2 (mBest2) were expressed in HEK cells, and the relative HCO₃^– permeability (P_HCO₃/P_Cl) and conductance (G_HCO₃/G_Cl) were determined. P_HCO₃/P_Cl was calculated from the change in reversal potential (E_rev) produced by replacing extracellular Cl^– with HCO₃^–. hBest1 was highly permeable to HCO₃^– (P_HCO₃/P_Cl = ~0.44). hBest2, hBest4, and mBest2 had an even higher relative HCO₃^– permeability (P_HCO₃/P_Cl = 0.6–0.7). All four bestrophins had HCO₃^– conductances that were nearly the same as Cl^– (G_HCO₃/G_Cl = 0.9–1.1). Extracellular Na⁺ did not affect the permeation of hBest1 to HCO₃^–. At physiological HCO₃^– concentration, HCO₃^– was also highly conductive. The hBest1 disease-causing mutations Y85H, R92C, and W93C abolished both Cl^– and HCO₃^– currents equally. The V78C mutation changed P_HCO₃/P_Cl and G_HCO₃/G_Cl of mBest2 channels. These results raise the possibility that disease-causing mutations in hBest1 produce disease by altering HCO₃^– homeostasis as well as Cl^– transport in the retina.

NO-induced regulation of human trabecular meshwork cell volume and aqueous humor outflow facility involve the BKCa ion channel

Dismuke, W. M., Mbadugha, C. C., Ellis, D. Z. — 2008-06-09

Nitric oxide (NO) donors decrease intraocular pressure (IOP) by increasing aqueous outflow facility in the trabecular meshwork (TM) and/or Schlemm's canal. However, the cellular mechanisms are unknown. Cellular mechanisms known to regulate outflow facility include changes in cell volume and cellular contractility. In this study, we investigated the effects of NO donors on outflow facility and NO-induced effects on TM cell volume. We tested the involvement of soluble guanylate cyclase (sGC), cGMP, PKG, and the large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel using inhibitors and activators. Cell volume was measured using calcein AM fluorescent dye, detected by confocal microscopy, and quantified using NIH ImageJ software. An anterior segment organ perfusion system measured outflow facility. NO increased outflow facility in porcine eye anterior segments (0.4884–1.3956 µl·min^–1·mmHg^–1) over baseline (0.2373–0.5220 µl·min^–1·mmHg^–1) within 10 min of drug application. These NO-induced increases in outflow facility were inhibited by the the BK_Ca channel inhibitor IBTX. Exposure of TM cells to NO resulted in a 10% decrease in cell volume, and these decreases were abolished by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and IBTX, suggesting the involvement of sGC and K⁺ eflux, respectively. NO-induced decreases in cell volume were mimicked by 8-Br-cGMP and abolished by the PKG inhibitor (RP)-8-Br-PET-cGMP-S, suggesting the involvement cGMP and PKG. Additionally, the time course for NO-induced decreases in TM cell volume correlated with NO-induced increases in outflow facility, suggesting that the NO-induced alterations in cell volume may influence outflow facility.

Endogenous heparan sulfate and heparin modulate bone morphogenetic protein-4 signaling and activity

Khan, S. A., Nelson, M. S., Pan, C., Gaffney, P. M., Gupta, P. — 2008-06-09

Bone morphogenetic proteins (BMPs) and their endogenous antagonists are important for brain and bone development and tumor initiation and progression. Heparan sulfate (HS) proteoglycans (HSPG) modulate the activities of BMPs and their antagonists. How glycosaminoglycans (GAGs) influence BMP activity in various malignancies and in inherited abnormalities of GAG metabolism, and the structural features of GAGs essential for modulation of BMP signaling, remain incompletely defined. We examined whether chemically modified soluble heparins, the endogenous HS in malignant cells and the HS accumulated in Hurler syndrome cells influence BMP-4 signaling and activity. We show that both exogenous (soluble) and endogenous GAGs modulate BMP-4 signaling and activity, and that this effect is dependent on specific sulfate residues of GAGs. Our studies suggest that endogenous sulfated GAGs promote the proliferation and impair differentiation of malignant human cells, providing the rationale for investigating whether pharmacological agents that inhibit GAG synthesis or function might reverse this effect. Our demonstration of impairment of BMP-4 signaling by GAGs in multipotent stem cells in human Hurler syndrome identifies a mechanism that might contribute to the progressive neurological and skeletal abnormalities in Hurler syndrome and related mucopolysaccharidoses.

IK1 channel activity contributes to cisplatin sensitivity of human epidermoid cancer cells

Lee, E. L., Hasegawa, Y., Shimizu, T., Okada, Y. — 2008-06-09

Cisplatin, a platinum-based drug, is an important weapon against many types of cancer. It induces apoptosis by forming adducts with DNA, although many aspects of its mechanism of action remain to be clarified. Previously, we found a role for the volume-sensitive, outwardly rectifying Cl^– channel in cisplatin-induced apoptosis. To investigate the possibility that cation channels also have a role in the cellular response to cisplatin, we examined the activity of cation channels in cisplatin-sensitive KB-3-1 (KB) epidermoid cancer cells by the whole cell patch-clamp method. A cation channel in KB cells, activated by hypotonic stress, was identified as the Ca²⁺-activated, intermediate-conductance K⁺ (IK1) channel on the basis of its requirement for intracellular Ca²⁺, its blockage by the blockers clotrimazole and triarylmethane-34, and its suppression by a dominant-negative construct. Activity of this channel was not observed in KCP-4 cells, a cisplatin-resistant cell line derived from KB cells, and its molecular expression, observed by semiquantitative RT-PCR and immunostaining, appeared much reduced. Cell volume measurements confirmed a physiological role for the IK1 channel as a component of the volume-regulatory machinery in KB cells. A possible role of the IK1 channel in cisplatin-induced apoptosis was investigated. It was found that clotrimazole and triarylmethane-34 inhibited a cisplatin-induced decrease in cell viability and increase in caspase-3/7 activity, whereas 1-ethyl-2-benzimidazolinone, an activator of the channel, had the opposite effect. Thus IK1 channel activity appears to mediate, at least in part, the response of KB cells to cisplatin treatment.

Asymmetric dimethylarginine inhibits HSP90 activity in pulmonary arterial endothelial cells: role of mitochondrial dysfunction

Sud, N., Wells, S. M., Sharma, S., Wiseman, D. A., Wilham, J., Black, S. M. — 2008-06-09

Increased asymmetric dimethylarginine (ADMA) levels have been implicated in the pathogenesis of a number of conditions affecting the cardiovascular system. However, the mechanism(s) by which ADMA exerts its effect has not been adequately elucidated. Thus the purpose of this study was to determine the effect of increased ADMA on nitric oxide (NO) signaling and to begin to elucidate the mechanism by which ADMA acts. Our initial data demonstrated that ADMA increased NO synthase (NOS) uncoupling in both recombinant human endothelial NO synthase (eNOS) and pulmonary arterial endothelial cells (PAEC). Furthermore, we found that this endothelial NOS (eNOS) uncoupling increased 3-nitrotyrosine levels preferentially in the mitochondria of PAEC due to a redistribution of eNOS from the plasma membrane to the mitochondria. This increase in nitration in the mitochondria was found to induce mitochondrial dysfunction as determined by increased mitochondrial-derived reactive oxygen species and decreased generation of ATP. Finally, we found that the decrease in ATP resulted in a reduction in the chaperone activity of HSP90 resulting in a decrease in its interaction with eNOS. In conclusion increased levels of ADMA causes mitochondrial dysfunction and a loss of heat shock protein-90 chaperone activity secondary to an uncoupling of eNOS. Mitochondrial dysfunction may be an understudied component of the endothelial dysfunction associated with various cardiovascular disease states.

Aberrant cell-to-cell coupling in Ca2+-overloaded guinea pig ventricular muscles

2008-06-09

To investigate how intercellular coupling can be changed during Ca²⁺ overloading of ventricular muscle, we studied Ca²⁺ signals in individual cells and the histochemistry of the major gap junction channel, connexin43 (Cx43), using multicellular preparations. Papillary muscles were obtained from guinea pig ventricles and loaded with rhod-2. Sequential Ca²⁺ images of surface cells were obtained with a confocal microscope. In intact muscles, all cells showed simultaneous Ca²⁺ transients in response to field stimulation over a field of view of 0.3 x 0.3 mm². In severely Ca²⁺-overloaded muscles, obtained by high-frequency stimulation in nonflowing Krebs solution, cells became less responsive to stimulation. Furthermore, nonsimultaneous but serial onsets of Ca²⁺ transients were often detected, suggesting a propagation delay of action potentials. The time lag of the onset between two aligned cells was sometimes as long as 100 ms. Similar lags were also observed in muscles with gap junction channels inhibited by heptanol. To investigate whether the phosphorylation state of Cx43 is affected in Ca²⁺-overloaded muscles, the distributions of phosphorylated and nonphosphorylated Cx43 were determined using specific antibodies. Most of the Cx43 was phosphorylated in the nonoverloaded muscles, whereas nonphosphorylated Cx43 was significantly elevated in severely Ca²⁺-overloaded muscles. Our results suggest that the propagation delay of action potential within a small area, a few square millimeters, can be a cause of abnormal conduction and a microreentry in Ca²⁺-overloaded heart. Inactivation of Na⁺ channels and inhibition of gap junctional communication may underlie the cell-to-cell propagation delay.

Surface change of the mammalian lens during accommodation

Zamudio, A. C., Candia, O. A., Kong, C. W., Wu, B., Gerometta, R. — 2008-06-09

Classical theories suggest that the surface area of the crystalline lens changes during accommodation while the lens volume remains constant. Our recent work challenged this view by showing that the lens volume decreases as the lens flattens during unaccommodation. In this paper we investigate 1) the magnitude of changes in the surface of the in vitro isolated cow lens during simulated accommodation, as well as that of human lens models, determined from lateral photographs and the application of the first theorem of Pappus; and 2) the velocity of the equatorial diameter recovery of prestretched cow and rabbit lenses by using a custom-built software-controlled stretching apparatus synchronized to a digital camera. Our results showed that the in vitro cow lens surface changed in an unexpected manner during accommodation depending on how much tension was applied to flatten the lens. In this case, the anterior surface initially collapsed with a reduction in surface followed by an increase in surface, when the stretching was applied. In the human lens model, the surface increased when the lens unaccommodated. The lens volume always decreases as the lens flattens. An explanation for the unexpected surface change is presented and discussed. Furthermore, we determined that the changes in lens volume, as reflected by the speed of the equatorial diameter recovery in in vitro cow and rabbit lenses during simulated accommodation, occurred within a physiologically relevant time frame (200 ms), implying a rapid movement of fluid to and from the lens during accommodation.

Neuroinflammation facilitates LIF entry into brain: role of TNF

Pan, W., Yu, C., Hsuchou, H., Zhang, Y., Kastin, A. J. — 2008-06-09

Leukemia inhibitory factor (LIF) is a proinflammatory cytokine mediating a variety of central nervous system (CNS) responses to inflammatory stimuli. During lipopolysaccharide (LPS)-induced inflammation, blood concentrations of LIF increase, correlating with lethality of sepsis. Circulating LIF crosses the blood-brain barrier (BBB) by a saturable transport system. Here we determine how this transport system is regulated in neuroinflammation. Using transport assays that quantify the influx rate and volume of distribution of LIF in mice, we show that LPS facilitated the permeation of LIF from the blood to the brain without compromising the paracellular permeability of the BBB as determined by coadministration of fluorescein. Concurrently, gp130 (shared by the interleukin-6 family of cytokines), but not gp190 (the specific receptor for LIF) or cilliary neutrophic factor (CNTF-R, a unique receptor for cilliary neurotrophic factor that also uses gp130 and gp190), showed increased levels of mRNA and protein expression in cerebral microvessels from the LPS-treated mice. The upregulation of gp130 by LPS was at least partially mediated by vascular tumor necrosis factor receptor (TNFR)1 and TNFR2. This was shown by elevated TNFR1 and TNFR2 mRNA and protein in cerebral microvessels after LPS and by the absence of the LPS effect on gp130 in knockout mice lacking these receptors. The results show that neuroinflammation by LPS induces endothelial signaling and enhances cytokine transport across the BBB.

Apical adenosine regulates basolateral Ca2+-activated potassium channels in human airway Calu-3 epithelial cells

Wang, D., Sun, Y., Zhang, W., Huang, P. — 2008-06-09

In airway epithelial cells, apical adenosine regulates transepithelial anion secretion by activation of apical cystic fibrosis transmembrane conductance regulator (CFTR) via adenosine receptors and cAMP/PKA signaling. However, the potent stimulation of anion secretion by adenosine is not correlated with its modest intracellular cAMP elevation, and these uncorrelated efficacies have led to the speculation that additional signaling pathways may be involved. Here, we showed that mucosal adenosine-induced anion secretion, measured by short-circuit current (I_sc), was inhibited by the PLC-specific inhibitor U-73122 in the human airway submucosal cell line Calu-3. In addition, the I_sc was suppressed by BAPTA-AM (a Ca²⁺ chelator) and 2-aminoethoxydiphenyl borate (2-APB; an inositol 1,4,5-trisphosphate receptor blocker), but not by PKC inhibitors, suggesting the involvement of PKC-independent PLC/Ca²⁺ signaling. Ussing chamber and patch-clamp studies indicated that the adenosine-induced PLC/Ca²⁺ signaling stimulated basolateral Ca²⁺-activated potassium (K_Ca) channels predominantly via A_2B adenosine receptors and contributed substantially to the anion secretion. Thus, our data suggest that apical adenosine activates contralateral K⁺ channels via PLC/Ca²⁺ and thereby increases the driving force for transepithelial anion secretion, synergizing with its modulation of ipsilateral CFTR via cAMP/PKA. Furthermore, the dual activation of CFTR and K_Ca channels by apical adenosine resulted in a mixed secretion of chloride and bicarbonate, which may alter the anion composition in the secretion induced by secretagogues that elicit extracellular ATP/adenosine release. Our findings provide novel mechanistic insights into the regulation of anion section by adenosine, a key player in the airway surface liquid homeostasis and mucociliary clearance.

Distinct pathways of ERK activation by the muscarinic agonists pilocarpine and carbachol in a human salivary cell line

Lin, A. L., Zhu, B., Zhang, W., Dang, H., Zhang, B.-X., Katz, M. S., Yeh, C.-K. — 2008-06-09

Cholinergic-muscarinic receptor agonists are used to alleviate mouth dryness, although the cellular signals mediating the actions of these agents on salivary glands have not been identified. We examined the activation of ERK1/2 by two muscarinic agonists, pilocarpine and carbachol, in a human salivary cell line (HSY). Immunoblot analysis revealed that both agonists induced transient activation of ERK1/2. Whereas pilocarpine induced phosphorylation of the epidermal growth factor (EGF) receptor, carbachol did not. Moreover, ERK activation by pilocarpine, but not carbachol, was abolished by the EGF receptor inhibitor AG-1478. Downregulation of PKC by prolonged treatment of cells with the phorbol ester PMA diminished carbachol-induced ERK phosphorylation but had no effect on pilocarpine responsiveness. Depletion of intracellular Ca²⁺ ([Ca²⁺]_i) by EGTA did not affect ERK activation by either agent. In contrast to carbachol, pilocarpine did not elicit [Ca²⁺]_i mobilization in HSY cells. Treatment of cells with the muscarinic receptor subtype 3 (M₃) antagonist N-(3-chloropropyl)-4-piperidnyl diphenylacetate decreased ERK responsiveness to both agents, whereas the subtype 1 (M₁) antagonist pirenzepine reduced only the carbachol response. Stimulation of ERKs by pilocarpine was also decreased by M₃, but not M₁, receptor small interfering RNA. The Src inhibitor PP2 blocked pilocarpine-induced ERK activation and EGF receptor phosphorylation, without affecting ERK activation by carbachol. Our results demonstrate that the actions of pilocarpine and carbachol in salivary cells are mediated through two distinct signaling mechanisms—pilocarpine acting via M₃ receptors and Src-dependent transactivation of EGF receptors, and carbachol via M₁/M₃ receptors and PKC—converging on the ERK pathway.

CaM kinase II{delta}2-dependent regulation of vascular smooth muscle cell polarization and migration

Mercure, M. Z., Ginnan, R., Singer, H. A. — 2008-06-09

Previous studies indicate involvement of the multifunctional Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) in vascular smooth muscle (VSM) cell migration. In the present study, molecular loss-of-function studies were used specifically to assess the role of the predominant CaMKII₂ isoform on VSM cell migration using a scratch wound healing assay. Targeted CaMKII₂ knockdown using siRNA or inhibition of activity by overexpressing a kinase-negative mutant resulted in attenuation of VSM cell migration. Temporal and spatial assessments of kinase autophosphorylation indicated rapid and transient activation in response to wounding, in addition to a sustained activation in the leading edge of migrating and spreading cells. Furthermore, siRNA-mediated suppression of CaMKII₂ resulted in the inhibition of wound-induced Rac activation and Golgi reorganization, and disruption of leading edge morphology, indicating an important function for CaMKII₂ in regulating VSM cell polarization. Numerous previous reports link activation of CaMKII to ERK1/2 signaling in VSM. Wound-induced ERK1/2 activation was also found to be dependent on CaMKII; however, ERK activity did not account for effects of CaMKII in regulating Golgi polarization, indicating alternative mechanisms by which CaMKII affects the complex events involved in cell migration. Wounding a VSM cell monolayer results in CaMKII₂ activation, which positively regulates VSM cell polarization and downstream signaling, including Rac and ERK1/2 activation, leading to cell migration.

A Kv channel with an altered activation gate sequence displays both "fast" and "slow" activation kinetics

Labro, A. J., Grottesi, A., Sansom, M. S. P., Raes, A. L., Snyders, D. J. — 2008-06-09

The Kv1–4 families of K⁺ channels contain a tandem proline motif (PXP) in the S6 helix that is crucial for channel gating. In human Kv1.5, replacing the first proline by an alanine resulted in a nonfunctional channel. This mutant was rescued by introducing another proline at a nearby position, changing the sequence into AVPP. This resulted in a channel that activated quickly (ms range) upon the first depolarization. However, thereafter, the channel became trapped in another gating mode that was characterized by slow activation kinetics (s range) with a shallow voltage dependence. The switch in gating mode was observed even with very short depolarization steps, but recovery to the initial "fast" mode was extremely slow. Computational modeling suggested that switching occurred during channel deactivation. To test the effect of the altered PXP sequence on the mobility of the S6 helix, we used molecular dynamics simulations of the isolated S6 domain of wild type (WT) and mutants starting from either a closed or open conformation. The WT S6 helix displayed movements around the PXP region with simulations starting from either state. However, the S6 with a AVPP sequence displayed flexibility only when started from the closed conformation and was rigid when started from the open state. These results indicate that the region around the PXP motif may serve as a "hinge" and that changing the sequence to AVPP results in channels that deactivate to a state with an alternate configuration that renders them "reluctant" to open subsequently.

Depletion of {beta}-COP reveals a role for COP-I in compartmentalization of secretory compartments and in biosynthetic transport of caveolin-1

Styers, M. L., O'Connor, A. K., Grabski, R., Cormet-Boyaka, E., Sztul, E. — 2008-06-09

We have utilized small interfering RNA (siRNA)-mediated depletion of the β-COP subunit of COP-I to explore COP-I function in organellar compartmentalization and protein traffic. Reduction in β-COP levels causes the colocalization of markers for the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC), Golgi, trans-Golgi network (TGN), and recycling endosomes in large, globular compartments. The lack of spatial differentiation of these compartments is not due to a general collapse of all cellular organelles since markers for the early endosomes and lysosomes do not redistribute to the common structures. Anterograde trafficking of the transmembrane cargo vesicular stomatitis virus membrane glycoprotein and of a subset of soluble cargoes is arrested within the common globular compartments. Similarly, recycling traffic of transferrin through the common compartment is perturbed. Furthermore, the trafficking of caveolin-1 (Cav1), a structural protein of caveolae, is arrested within the globular structures. Importantly, Cav1 coprecipitates with the -subunit of COP-I, suggesting that Cav1 is a COP-I cargo. Our findings suggest that COP-I is required for the compartmentalization of the ERGIC, Golgi, TGN, and recycling endosomes and that COP-I plays a novel role in the biosynthetic transport of Cav1.

Complex regulation of store-operated Ca2+ entry pathway by PKC-{varepsilon} in vascular SMCs

Smani, T., Patel, T., Bolotina, V. M. — 2008-06-09

The role of PKC in the regulation of store-operated Ca²⁺ entry (SOCE) is rather controversial. Here, we used Ca²⁺-imaging, biochemical, pharmacological, and molecular techniques to test if Ca²⁺-independent PLA₂β (iPLA₂β), one of the transducers of the signal from depleted stores to plasma membrane channels, may be a target for the complex regulation of SOCE by PKC and diacylglycerol (DAG) in rabbit aortic smooth muscle cells (SMCs). We found that the inhibition of PKC with chelerythrine resulted in significant inhibition of thapsigargin (TG)-induced SOCE in proliferating SMCs. Activation of PKC by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) caused a significant depletion of intracellular Ca²⁺ stores and triggered Ca²⁺ influx that was similar to TG-induced SOCE. OAG and TG both produced a PKC-dependent activation of iPLA₂β and Ca²⁺ entry that were absent in SMCs in which iPLA₂β was inhibited by a specific chiral enantiomer of bromoenol lactone (S-BEL). Moreover, we found that PKC regulates TG- and OAG-induced Ca²⁺ entry only in proliferating SMCs, which correlates with the expression of the specific PKC- isoform. Molecular downregulation of PKC- impaired TG- and OAG-induced Ca²⁺ influx in proliferating SMCs but had no effect in confluent SMCs. Our results demonstrate that DAG (or OAG) can affect SOCE via multiple mechanisms, which may involve the depletion of Ca²⁺ stores as well as direct PKC--dependent activation of iPLA₂β, resulting in a complex regulation of SOCE in proliferating and confluent SMCs.

Glucosamine protects neonatal cardiomyocytes from ischemia-reperfusion injury via increased protein O-GlcNAc and increased mitochondrial Bcl-2

Champattanachai, V., Marchase, R. B., Chatham, J. C. — 2008-06-09

We have previously reported that glucosamine protected neonatal rat ventricular myocytes against ischemia-reperfusion (I/R) injury, and this was associated with an increase in protein O-linked-N-acetylglucosamine (O-GlcNAc) levels. However, the protective effect of glucosamine could be mediated via pathways other that O-GlcNAc formation; thus the initial goal of the present study was to determine whether increasing O-GlcNAc transferase (OGT) expression, which catalyzes the formation of O-GlcNAc, had a protective effect similar to that of glucosamine. To better understand the potential mechanism underlying O-GlcNAc-mediated cytoprotection, we examined whether increased O-GlcNAc levels altered the expression and translocation of members of the Bcl-2 protein family. Both glucosamine (5 mM) and OGT overexpression increased basal and I/R-induced O-GlcNAc levels, significantly decreased cellular injury, and attenuated loss of cytochrome c. Both interventions also attenuated the loss of mitochondrial membrane potential induced by H₂O₂ and were also associated with an increase in mitochondrial Bcl-2 levels but had no effect on Bad or Bax levels. Compared with glucosamine and OGT overexpression, NButGT (100 µM), an inhibitor of O-GlcNAcase, was less protective against I/R and H₂O₂ and did not affect Bcl-2 expression, despite a 5- to 10-fold greater increase in overall O-GlcNAc levels. Decreased OGT expression resulted in lower basal O-GlcNAc levels, prevented the I/R-induced increase in O-GlcNAc and mitochondrial Bcl-2, and increased cellular injury. These results demonstrate that the protective effects of glucosamine are mediated via increased formation of O-GlcNAc and suggest that this is due, in part, to enhanced mitochondrial Bcl-2 translocation.

Attenuation of retinal endothelial cell migration and capillary morphogenesis in the absence of bcl-2

Kondo, S., Tang, Y., Scheef, E. A., Sheibani, N., Sorenson, C. M. — 2008-06-09

Apoptosis plays a critical role during development and in the maintenance of the vascular system. B-cell leukemia lymphoma 2 (bcl-2) protects endothelial cells (EC) from apoptosis in response to a variety of stimuli. Previous work from this laboratory demonstrated attenuation of postnatal retinal vascular development and retinal neovascularization during oxygen-induced ischemic retinopathy in bcl-2-deficient (bcl-2^–/–) mice. To gain further insight into the function of bcl-2 in the endothelium, we isolated retinal EC from bcl-2^+/+ and bcl-2^–/– mice. Retinal EC lacking bcl-2 demonstrated reduced cell migration, tenascin-C expression, and adhesion to vitronectin and fibronectin. The bcl-2^–/– retinal EC also failed to undergo capillary morphogenesis in Matrigel. In addition, using an ex vivo angiogenesis assay, we observed reduced sprouting from aortic rings grown in culture from bcl-2^–/– mice compared with bcl-2^+/+ mice. Furthermore, reexpression of bcl-2 was sufficient to restore migration and capillary morphogenesis defects observed in bcl-2^–/– retinal EC. Mechanistically, bcl-2^–/– cells expressed significantly less endothelial nitric oxide synthase, an important downstream effecter of proangiogenic signaling. This may be attributed to increased oxidative stress in the absence of bcl-2. In fact, incubation of retinal EC or aortic rings from bcl-2^–/– mice with the antioxidant N-acetylcysteine rescued their capillary morphogenesis and sprouting defects. Thus, bcl-2-mediated cellular functions play important roles not only in survival but also in proangiogenic phenotype of EC with a significant impact on vascular development and angiogenesis.

Estrogen and the Ca2+-mobilizing agonist ATP evoke acute NO synthesis via distinct pathways in an individual human vascular endothelium-derived cell

Sheng, J.-Z., Arshad, F., Braun, J. E., Braun, A. P. — 2008-06-09

In this study, we have systematically evaluated the signaling mechanisms underlying stimulated nitric oxide (NO) synthesis by estrogen (E₂) and other vasoactive agents at the level of a single endothelium-derived cell. To do so, we have characterized and contrasted rapid E₂-evoked NO synthesis with that of ATP using single-cell microfluorimetry and patch-clamp recordings to monitor stimulated changes in cellular NO synthesis (via 4-amino-5-methylamino-2',7'-difluorofluorescein), Ca²⁺ transients (via Fluo-3), and membrane hyperpolarization in cultured human EA.hy926 cells. E₂-evoked NO synthesis in single cells (EC₅₀ ~0.3 nM) was blocked by the E₂ receptor antagonist ICI 182,780 and the NO synthase inhibitor N-nitro-l-arginine methyl ester. Although both E₂ and ATP stimulated comparable Ca²⁺ transients, E₂-induced NO synthesis was insensitive to intracellular BAPTA-AM or removal of external Ca²⁺. In contrast, ATP-evoked NO production was abolished by either one of these treatments. ATP-evoked hyperpolarizations (~20 mV) and NO production were both inhibited by the respective small-conductance and intermediate-conductance calcium- activated K⁺ channel blockers apamin and charybdotoxin. E₂ minimally affected membrane potential, and stimulated NO synthesis was insensitive to calcium-activated K⁺ channel blockers. Exposure to either the phosphatidylinositol 3-kinase inhibitor LY-294001 or the MAP kinase inhibitor PD-98059 abolished the NO response to E₂, but not that to ATP. Finally, the NO response evoked by a combined stimulus of E₂ plus ATP was similar to that of ATP alone. In conclusion, our data directly demonstrate that an individual human EA.hy926 cell contains at least two distinct mechanisms for stimulated NO synthesis that depend on either calcium or protein kinase signaling events.

Octylphenol stimulates resistin gene expression in 3T3-L1 adipocytes via the estrogen receptor and extracellular signal-regulated kinase pathways

2008-06-09

Resistin is known as an adipocyte-specific secretory hormone that can cause insulin resistance and decrease adipocyte differentiation. It can be regulated by sexual hormones. Whether environmental estrogens regulate the production of resistin is still not clear. Using 3T3-L1 adipocytes, we found that octylphenol upregulated resistin mRNA expression in dose- and time-dependent manners. The concentration of octylphenol that increased resistin mRNA levels by 50% was ~100 nM within 6 h of treatment. The basal half-life of resistin mRNA induced by actinomycin D was lengthened by octylphenol treatment, suggesting that octylphenol decreases the rate of resistin mRNA degradation. In addition, octylphenol stimulated resistin protein expression and release. The basal half-life of resistin protein induced by cycloheximide was lengthened by octylphenol treatment, suggesting that octylphenol decreases the rate of resistin protein degradation. While octylphenol was shown to increase activities of the estrogen receptor (ER) and MEK1, signaling was demonstrated to be blocked by pretreatment with either ICI-182780 (an ER antagonist) or U-0126 (a MEK1 inhibitor), in which both inhibitors prevented octylphenol-stimulated phosphorylation of ERK. These results imply that ER and ERK are necessary for the octylphenol stimulation of resistin mRNA expression. Moreover, U-0126 antagonized the octylphenol-increased resistin protein expression and release. These data suggest that the way octylphenol signaling increases resistin protein levels is similar to that by which it increases resistin mRNA levels; it is likely mediated through an ERK-dependent pathway. In vivo, octylphenol increased adipose resistin mRNA expression and serum resistin and glucose levels, supporting its in vitro effect.

Volume-sensitive NADPH oxidase activity and taurine efflux in NIH3T3 mouse fibroblasts

Friis, M. B., Vorum, K. G., Lambert, I. H. — 2008-06-09

Reactive oxygen species (ROS) are produced in NIH3T3 fibroblasts during hypotonic stress, and H₂O₂ potentiates the concomitant release of the organic osmolyte taurine (Lambert IH. J Membr Biol 192: 19–32, 2003). The increase in ROS production [5-(and-6)-carboxy-2', 7'-dichlorodihydrofluorescein diacetate fluorescence] is detectable after a reduction in the extracellular osmolarity from 335 mosM (isotonic) to 300 mosM and reaches a maximal value after a reduction to 260 mosM. The swelling-induced ROS production is reduced by the flavoprotein inhibitor diphenylene iodonium chloride (25 µM) but is unaffected by the nitric oxide synthase inhibitor N-nitro-l-arginine methyl ester, indicating that the volume-sensitive ROS production is NADPH oxidase dependent. NIH3T3 cells express the NADPH oxidase components: p22^phox, a NOX4 isotype; p47^phox; and p67^phox (real-time PCR). Exposure to the Ca²⁺-mobilizing agonist ATP (10 µM) potentiates the release of taurine but has no effect on ROS production under hypotonic conditions. On the other hand, addition of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 100 nM) or the lipid messenger lysophosphatidic acid (LPA, 10 nM) potentiates the swelling-induced taurine release as well as the ROS production. Overexpression of Rac1 or p47^phox or p47^phox knockdown [small interfering (si)RNA] had no effect on the swelling-induced ROS production or taurine release. NOX4 knockdown (siRNA) impairs the increase in the ROS production and the concomitant taurine release following osmotic exposure. It is suggested that a NOX4 isotype plus p22^phox account for the swelling-induced increase in the ROS production in NIH3T3 cells and that the oxidase activity is potentiated by PKC and LPA but not by Ca²⁺.

Neuronal nitric oxide synthase signaling within cardiac myocytes targets phospholamban

2008-06-09

Studies have shown that neuronal nitric oxide synthase (nNOS, NOS1) knockout mice (NOS1^–/–) have increased or decreased contractility, but consistently have found a slowed rate of intracellular Ca²⁺ ([Ca²⁺]_i) decline and relengthening. Contraction and [Ca²⁺]_i decline are determined by many factors, one of which is phospholamban (PLB). The purpose of this study is to determine the involvement of PLB in the NOS1-mediated effects. Force-frequency experiments were performed in trabeculae isolated from NOS1^–/– and wild-type (WT) mice. We also simultaneously measured Ca²⁺ transients (Fluo-4) and cell shortening (edge detection) in myocytes isolated from WT, NOS1^–/–, and PLB^–/– mice. NOS1^–/– trabeculae had a blunted force-frequency response and prolonged relaxation. We observed similar effects in myocytes with NOS1 knockout or specific NOS1 inhibition with S-methyl-l-thiocitrulline (SMLT) in WT myocytes (i.e., decreased Ca²⁺ transient and cell shortening amplitudes and prolonged decline of [Ca²⁺]_i). Alternatively, NOS1 inhibition with SMLT in PLB^–/– myocytes had no effect. Acute inhibition of NOS1 with SMLT in WT myocytes also decreased basal PLB serine16 phosphorylation. Furthermore, there was a decreased SR Ca²⁺ load with NOS1 knockout or inhibition, which is consistent with the negative contractile effects. Perfusion with FeTPPS (peroxynitrite decomposition catalyst) mimicked the effects of NOS1 knockout or inhibition. β-Adrenergic stimulation restored the slowed [Ca²⁺]_i decline in NOS1^–/– myocytes, but a blunted contraction remained, suggesting additional protein target(s). In summary, NOS1 inhibition or knockout leads to decreased contraction and slowed [Ca²⁺]_i decline, and this effect is absent in PLB^–/– myocytes. Thus NOS1 signaling modulates PLB serine16 phosphorylation, in part, via peroxynitrite.

Shear stress influences spatial variations in vascular Mn-SOD expression: implication for LDL nitration

2008-06-09

Fluid shear stress modulates vascular production of endothelial superoxide anion (O₂^·–) and nitric oxide (^·NO). Whether the characteristics of shear stress influence the spatial variations in mitochondrial manganese superoxide dismutase (Mn-SOD) expression in vasculatures is not well defined. We constructed a three-dimensional computational fluid dynamics model simulating spatial variations in shear stress at the arterial bifurcation. In parallel, explants of arterial bifurcations were sectioned from the human left main coronary bifurcation and right coronary arteries for immunohistolocalization of Mn-SOD expression. We demonstrated that Mn-SOD staining was prominent in the pulsatile shear stress (PSS)-exposed and atheroprotective regions, but it was nearly absent in the oscillatory shear stress (OSS)-exposed regions and lateral wall of arterial bifurcation. In cultured bovine aortic endothelial cells, PSS at mean shear stress (_ave) of 23 dyn/cm² upregulated Mn-SOD mRNA expression at a higher level than did OSS at _ave = 0.02 dyn/cm² ± 3.0 dyn·cm^–2·s^–1 and at 1 Hz (PSS by 11.3 ± 0.4-fold vs. OSS by 5.0 ± 0.5-fold vs. static condition; P < 0.05, n = 4). By liquid chromatography and tandem mass spectrometry, it was found that PSS decreased the extent of low-density lipoprotein (LDL) nitration, whereas OSS increased nitration (P < 0.05, n = 4). In the presence of LDL, treatment with Mn-SOD small interfering RNA increased intracellular nitrotyrosine level (P < 0.5, n = 4), a fingerprint for nitrotyrosine formation. Our findings indicate that shear stress in the atheroprone versus atheroprotective regions regulates spatial variations in mitochondrial Mn-SOD expression with an implication for modulating LDL nitration.

Substance P enhances NF-{kappa}B transactivation and chemokine response in murine macrophages via ERK1/2 and p38 MAPK signaling pathways

Sun, J., Ramnath, R. D., Zhi, L., Tamizhselvi, R., Bhatia, M. — 2008-06-09

The neuropeptide substance P (SP), as a major mediator of neuroimmunomodulatory activity, modulates diverse functions of immune cells, including macrophages. In the current study, we focused on the yet uncertain role of SP in enhancing the inducible/inflammatory chemokine response of macrophages and the signaling mechanism involved. We studied the effect on the murine monocyte/macrophage cell line RAW 264.7 as well as isolated primary macrophages. Our data show that SP, at nanomolar concentrations, elicited selective chemokine production from murine macrophages. Among the chemokines examined, macrophage inflammatory protein-2 and monocyte chemoattractant protein-1 are two major chemokines that were synthesized by macrophages in response to SP. Furthermore, SP treatment strongly induced the classic pathway of IB-dependent NF-B activation and enhanced DNA binding as well as transactivation activity of the transcription factor. SP-evoked transcriptional induction of chemokines was specific, since it was blocked by treatment with selective neurokinin-1 receptor antagonists. Moreover, SP stimulation of macrophages activated the ERK1/2 and p38 MAPK but not JNKs. Blockade of these two MAPK pathways with specific inhibitors abolished SP-elicited nuclear translocation of phosphorylated NF-B p65 and NF-B-driven chemokine production, suggesting that the two MAPKs lie in the signaling pathways leading to the chemokine response. Collectively, our data demonstrate that SP enhances selective inflammatory chemokine production by murine macrophages via ERK/p38 MAPK-mediated NF-B activation.

Activation of cytosolic phospholipase A2 and fatty acid transacylase is essential but not sufficient for thrombin-induced smooth muscle cell proliferation

Gluck, N., Schwob, O., Krimsky, M., Yedgar, S. — 2008-06-09

Thrombin is a potent stimulant of smooth muscle cell (SMC) proliferation in inflammatory conditions, leading to pathological thickening of vascular walls in atherosclerosis and airway remodeling in asthma. Cell proliferation requires the formation and remodeling of cell membrane phospholipids (PLs), involving the activation of PL-metabolizing enzymes. Yet, the role of specific PL-metabolizing enzymes in SMC proliferation has hardly been studied. To bridge this gap, in the present study, we investigated the role of key enzymes involved in PL metabolism, the PL-hydrolyzing enzyme phospholipase A₂ (PLA₂) and the PL-synthesizing enzyme lysophosphatidic acid-fatty acid transacylase (LPAAT), in thrombin-induced proliferation of bovine aortic SMCs (BASMCs). Concomitantly with the induction of BASMC proliferation, thrombin activated cytosolic PLA₂ (cPLA₂-), expressed by selective release of arachidonic acid and mRNA expression, as well as LPAAT, expressed by nonselective incorporation of fatty acid and mRNA expression. Specific inhibitors of these enzymes, arachidonyl-trifluoromethyl-ketone for cPLA₂ and thimerosal for LPAAT, suppressed their activities, concomitantly with suppression of BASMC proliferation, suggesting a mandatory requirement for cPLA₂ and LPAAT activation in thrombin-induced SMC proliferation. Thrombin acts through the protease-activated receptor (PAR-1), and, accordingly, we found that thrombin-induced BASMC proliferation was suppressed by the PAR-1 inhibitor SCH-79797. However, the PAR-1 inhibitor did not prevent thrombin-induced mRNA expression of cPLA₂ and LPAAT, implying that the activation of cPLA₂ and LPAAT is essential but not sufficient for thrombin-induced proliferation of BASMCs.

Corrigendum

2008-06-09

Image acquisition for colocalization using optical microscopy

Scriven, D. R. L., Lynch, R. M., Moore, E. D. W. — 2008-05-06

Colocalization, in which images of two or more fluorescent markers are overlaid, and coincidence between the probes is measured or displayed, is a common analytical tool in cell biology. Interpreting the images and the meaning of this identified coincidence is difficult in the absence of basic information about the acquisition parameters. In this commentary, we highlight important factors in the acquisition of images used to demonstrate colocalization, and we discuss the minimum information that authors should include in a manuscript so that a reader can interpret both the fluorescent images and any observed colocalization.

Mitochondrial fluidity matters. Focus on "Inherited complex I deficiency is associated with faster protein diffusion in the matrix of moving mitochondria"

Benard, G., Rossignol, R. — 2008-05-06

Inherited complex I deficiency is associated with faster protein diffusion in the matrix of moving mitochondria

2008-05-06

Mitochondria continuously change shape, position, and matrix configuration for optimal metabolite exchange. It is well established that changes in mitochondrial metabolism influence mitochondrial shape and matrix configuration. We demonstrated previously that inhibition of mitochondrial complex I (CI or NADH:ubiquinone oxidoreductase) by rotenone accelerated matrix protein diffusion and decreased the fraction and velocity of moving mitochondria. In the present study, we investigated the relationship between inherited CI deficiency, mitochondrial shape, mobility, and matrix protein diffusion. To this end, we analyzed fibroblasts of two children that represented opposite extremes in a cohort of 16 patients, with respect to their residual CI activity and mitochondrial shape. Fluorescence correlation spectroscopy (FCS) revealed no relationship between residual CI activity, mitochondrial shape, the fraction of moving mitochondria, their velocity, and the rate of matrix-targeted enhanced yellow fluorescent protein (mitoEYFP) diffusion. However, mitochondrial velocity and matrix protein diffusion in moving mitochondria were two to three times higher in patient cells than in control cells. Nocodazole inhibited mitochondrial movement without altering matrix EYFP diffusion, suggesting that both activities are mutually independent. Unexpectedly, electron microscopy analysis revealed no differences in mitochondrial ultrastructure between control and patient cells. It is discussed that the matrix of a moving mitochondrion in the CI-deficient state becomes less dense, allowing faster metabolite diffusion, and that fibroblasts of CI-deficient patients become more glycolytic, allowing a higher mitochondrial velocity.

Differentiation-dependent changes in the membrane properties of fiber cells isolated from the rat lens

Webb, K. F., Donaldson, P. J. — 2008-05-06

Impedance measurements in whole lenses showed that lens fiber cells possess different permeability properties to the epithelial cells from which they differentiate. To confirm these observations at the cellular level, we analyzed the membrane properties of fiber cells isolated in the presence of the nonselective cation channel inhibitor Gd³⁺. Isolated fiber cells were viable in physiological [Ca²⁺] and exhibited a range of lengths that reflected their stage of differentiation. Analysis of a large population of fiber cells revealed a subgroup of cells whose conductivity matched values measured in the whole lens (1). In this group of cells, membrane resistance, conductivity, and reversal potential all varied with cell length, suggesting that the process of differentiation is associated with a change in the membrane properties of fiber cells. Using pharmacology and ion substitution experiments, we showed that newly differentiated fiber cells (<150 µm) contained variable combinations of Ba²⁺-and tetraethylammonium-sensitive K⁺ currents. Longer fiber cells (150–650 µm) were dominated by a lyotropic anion conductance, which also appears to plays a role in the intact lens. Longer cells also exhibited a low-level, nonselective conductance that was eliminated by the replacement of extracellular Na⁺ with N-methyl-d-glucamine, indicating that the lens contains both Gd³⁺-sensitive and -insensitive nonselective cation conductances. Fiber cell differentiation is therefore associated with a shift in membrane permeability from a dominant K⁺ conductance(s) toward larger contributions from anion and nonselective cation conductances as fiber cells elongate.

Elucidation of the signaling network of COX-2 induction in sheared chondrocytes: COX-2 is induced via a Rac/MEKK1/MKK7/JNK2/c-Jun-C/EBP{beta}-dependent pathway

Healy, Z. R., Zhu, F., Stull, J. D., Konstantopoulos, K. — 2008-05-06

Shear stress is a pathophysiologically relevant mechanical signal in cartilage biology and tissue engineering. Cyclooxygenase-2 (COX-2) is a pivotal proinflammatory enzyme, which is induced by mechanical loading-derived shear stress in chondrocytes. In the present study, we investigated the transcriptional machinery and signaling pathway regulating shear-induced COX-2 expression in human chondrocytic cells. Deletion and mutation analyses of the human cox-2 promoter reveal that the CCAAT/enhancer-binding protein (C/EBP) and activator protein-1 (AP-1) predominantly contribute to the shear-induced cox-2 promoter activity. Supershift assays disclose that C/EBPβ, but not C/EBP or C/EBP, binds to the C/EBP site, whereas c-Jun binds to AP-1. Individual gene knockdown experiments demonstrate the direct regulation of C/EBPβ expression by c-Jun, and the critical roles of both c-Jun and C/EBPβ in shear-induced COX-2 synthesis. Our studies also indicate that Rac and, to a lesser extent, Cdc42 transactivate MEKK1, which is, in turn, responsible for activation of mitogen-activated protein kinase kinase 7 (MKK7). MKK7 regulates c-Jun NH₂-terminal kinase 2 activation, which, in turn, triggers the phosphorylation of c-Jun that controls shear-mediated COX-2 upregulation in chondrocytes. Reconstructing the signaling network regulating shear-induced COX-2 expression and inflammation may provide insights to optimize conditions for culturing artificial cartilage in bioreactors and for developing therapeutic interventions for arthritic disorders.

Prolactin stimulates transepithelial calcium transport and modulates paracellular permselectivity in Caco-2 monolayer: mediation by PKC and ROCK pathways

2008-05-06

Prolactin (PRL) was previously demonstrated to rapidly enhance calcium absorption in rat duodenum and the intestine-like Caco-2 monolayer. However, its mechanism was not completely understood. Here, we investigated nongenomic effects of PRL on the transepithelial calcium transport and paracellular permselectivity in the Caco-2 monolayer by Ussing chamber technique. PRL increased the transcellular and paracellular calcium fluxes and paracellular calcium permeability within 60 min after exposure but decreased the transepithelial resistance of the monolayer. The effects of PRL could not be inhibited by RNA polymerase II inhibitor (5,6-dichloro-1-β-d-ribobenzimidazole), confirming that PRL actions were nongenomic. Exposure to protein kinase C (PKC) or RhoA-associated coiled-coil forming kinase (ROCK) inhibitors (GF-109203X and Y-27632, respectively) abolished the stimulatory effect of PRL on transcellular calcium transport, whereas ROCK inhibitor, but not PKC inhibitor, diminished the PRL effect on paracellular calcium transport. Knockdown of the long isoform of PRL receptor (PRLR-L) also prevented the enhancement of calcium transport by PRL. In addition, PRL markedly increased paracellular sodium permeability and the permeability ratio of sodium to chloride, which are indicators of the paracellular charge-selective property and are known to be associated with the enhanced paracellular calcium transport. The permeability of other cations in the alkali metal series was also increased by PRL, and such increases were abolished by ROCK inhibitor. It could be concluded that PRL stimulated transepithelial calcium transport through PRLR-L and increased paracellular permeability to cations in the Caco-2 monolayer. These nongenomic actions of PRL were mediated by the PKC and ROCK signaling pathways.

Phosphorylation of {beta}-catenin by PKA promotes ATP-induced proliferation of vascular smooth muscle cells

Taurin, S., Sandbo, N., Yau, D. M., Sethakorn, N., Dulin, N. O. — 2008-05-06

Extracellular ATP stimulates proliferation of vascular smooth muscle cells (VSMC) through activation of G protein-coupled P2Y purinergic receptors. We have previously shown that ATP stimulates a transient activation of protein kinase A (PKA), which, together with the established mitogenic signaling of purinergic receptors, promotes proliferation of VSMC (Hogarth DK, Sandbo N, Taurin S, Kolenko V, Miano JM, Dulin NO. Am J Physiol Cell Physiol 287: C449–C456, 2004). We also have shown that PKA can phosphorylate β-catenin at two novel sites (Ser552 and Ser675) in vitro and in overexpression cell models (Taurin S, Sandbo N, Qin Y, Browning D, Dulin NO. J Biol Chem 281: 9971–9976, 2006). β-Catenin promotes cell proliferation by activation of a family of T-cell factor (TCF) transcription factors, which drive the transcription of genes implicated in cell cycle progression including cyclin D1. In the present study, using the phosphospecific antibodies against phospho-Ser552 or phospho-Ser675 sites of β-catenin, we show that ATP can stimulate PKA-dependent phosphorylation of endogenous β-catenin at both of these sites without affecting its expression levels in VSMC. This translates to a PKA-dependent stimulation of TCF transcriptional activity through an increased association of phosphorylated (by PKA) β-catenin with TCF-4. Using the PKA inhibitor PKI or dominant negative TCF-4 mutant, we show that ATP-induced cyclin D1 promoter activation, cyclin D1 protein expression, and proliferation of VSMC are all dependent on PKA and TCF activities. In conclusion, we show a novel mode of regulation of endogenous β-catenin through its phosphorylation by PKA, and we demonstrate the importance of this mechanism for ATP-induced proliferation of VSMC.

Syncoilin is required for generating maximum isometric stress in skeletal muscle but dispensable for muscle cytoarchitecture

2008-05-06

Syncoilin is a striated muscle-specific intermediate filament-like protein, which is part of the dystrophin-associated protein complex (DPC) at the sarcolemma and provides a link between the extracellular matrix and the cytoskeleton through its interaction with -dystrobrevin and desmin. Its upregulation in various neuromuscular diseases suggests that syncoilin may play a role in human myopathies. To study the functional role of syncoilin in cardiac and skeletal muscle in vivo, we generated syncoilin-deficient (syncoilin^–/–) mice. Our detailed analysis of these mice up to 2 yr of age revealed that syncoilin is entirely dispensable for cardiac and skeletal muscle development and maintenance of cellular structure but is required for efficient lateral force transmission during skeletal muscle contraction. Notably, syncoilin^–/– skeletal muscle generates less maximal isometric stress than wild-type (WT) muscle but is as equally susceptible to eccentric contraction-induced injury as WT muscle. This suggests that syncoilin may play a supportive role for desmin in the efficient coupling of mechanical stress between the myofibril and fiber exterior. It is possible that the reduction in isometric stress production may predispose the syncoilin skeletal muscle to a dystrophic condition.

Endogenous interferon-{gamma} is required for efficient skeletal muscle regeneration

Cheng, M., Nguyen, M.-H., Fantuzzi, G., Koh, T. J. — 2008-05-06

The inflammatory response is thought to play important roles in tissue healing. The hypothesis of this study was that the inflammatory cytokine interferon (IFN)- is produced endogenously following skeletal muscle injury and promotes efficient healing. We show that IFN- is expressed at both mRNA and protein levels in skeletal muscle following injury, and that the time course of IFN- expression correlated with the accumulation of macrophages, T-cells, and natural killer cells, as well as myoblasts, in damaged muscle. Cells of each type were isolated from injured muscle, and IFN- expression was detected in each cell type. We also demonstrate that administration of an IFN- receptor blocking antibody to wild-type mice impaired induction of interferon response factor-1, reduced cell proliferation, and decreased formation of regenerating fibers. IFN- null mice showed similarly impaired muscle healing associated with impaired macrophage function and development of fibrosis. In vitro studies demonstrated that IFN- and its receptor are expressed in the C2C12 muscle cell line, and that the IFN- receptor blocking antibody reduced proliferation and fusion of these muscle cells. In summary, our results indicate that IFN- promotes muscle healing, in part, by stimulating formation of new muscle fibers.

Pyridoxine uptake by colonocytes: a specific and regulated carrier-mediated process

Said, Z. M., Subramanian, V. S., Vaziri, N. D., Said, H. M. — 2008-05-06

The water-soluble vitamin B₆ (pyridoxine) is important for normal cellular functions, growth, and development. The vitamin is obtained from two exogenous sources: a dietary source, which is absorbed in the small intestine, and a bacterial source, where the vitamin is synthesized in significant quantities by the normal microflora of the large intestine. Evidence exists to suggest the bioavailability of the latter source of the vitamin, but nothing is known about the mechanism involved and its regulation. In this study, we addressed these issues using young adult mouse colonic epithelial (YAMC) cells and human colonic apical membrane vesicles (AMV) as models and using [³H]pyridoxine as the uptake substrate. The results showed the initial rate of [³H]pyridoxine uptake by YAMC cells to be 1) energy- and temperature- (but not Na-) dependent and to occur without metabolic alteration in the transported substrate; 2) saturable as a function of concentration with an apparent K_m and V_max of 2.1 ± 0.5 µM and 53.4 ± 4.3 pmol·mg protein^–1·3 min^–1, respectively; 3) cis-inhibited by unlabeled pyridoxine and its structural analogs, but not by the unrelated compounds theophylline, penicillamine, and isoniazid; 4) trans-stimulated by unlabeled pyridoxine; 5) amiloride sensitive; and 6) regulated by extracellular and intracellular factors. Uptake of pyridoxine by native human colonic AMV was also found to involve a carrier-mediated process. These studies demonstrate, for the first time, the functional existence of a specific and regulatable carrier-mediated process for pyridoxine uptake by mammalian colonocytes.

IL-4 and IL-13 upregulate ornithine decarboxylase expression by PI3K and MAP kinase pathways in vascular smooth muscle cells

Wei, L. H., Yang, Y., Wu, G., Ignarro, L. J. — 2008-05-06

Ornithine decarboxylase (ODC) is the first and rate-controlling enzyme in the synthesis of polyamines, which are essential for normal cell growth. We have previously demonstrated that IL-4 and IL-13 can stimulate rat aortic smooth muscle cell (RASMC) proliferation. The objective of this study was to determine whether IL-4 and IL-13 induce cell proliferation by upregulating ODC expression in RASMC. The results revealed that incubation of RASMC with IL-4 and IL-13 for 24 h caused four- to fivefold induction of ODC catalytic activity. The increased ODC catalytic activity was attributed to the increased expression of ODC mRNA. Moreover, these observations were paralleled by increased production of polyamines. We further investigated the signal transduction pathways responsible for ODC induction by IL-4 and IL-13. The data illustrated that PD-98059, a MEK (MAPK kinase) inhibitor, LY-294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, and H-89, a protein kinase A (PKA) inhibitor, substantially decreased the induction of ODC catalytic activity and ODC mRNA expression induced by IL-4 and IL-13, suggesting positive regulation of the ODC gene by ERK, PI3K, and PKA pathways. Interestingly, dexamethasone, a known inhibitor of cell proliferation, completely abrogated the response of RASMC to IL-4 and IL-13. Furthermore, the inhibition of ODC by these inhibitors led to the reduced production of polyamines and decreased DNA synthesis as monitored by [³H]thymidine incorporation. Our data indicate that upregulation of ODC by IL-4 and IL-13 might play an important role in the pathophysiology of vascular disorders characterized by excessive smooth muscle growth.

The vitamin D receptor agonist elocalcitol upregulates L-type calcium channel activity in human and rat bladder

2008-05-06

Human bladder contraction mainly depends on Ca²⁺ influx via L-type voltage-gated Ca²⁺ channels and on RhoA/Rho kinase contractile signaling, which is upregulated in overactive bladder (OAB). Elocalcitol is a vitamin D receptor agonist inhibiting RhoA/Rho kinase signaling in rat and human bladder. Since in the normal bladder from Sprague-Dawley rats elocalcitol treatment delayed the carbachol-induced contraction without changing maximal responsiveness and increased sensitivity to the L-type Ca²⁺ channel antagonist isradipine, we investigated whether elocalcitol upregulated L-type Ca²⁺ channels in human bladder smooth muscle cells (hBCs). In hBCs, elocalcitol induced a rapid increase in intracellular [Ca²⁺], which was abrogated by the L-type Ca²⁺ channel antagonist verapamil. Moreover, hBCs exhibited L-type voltage-activated Ca²⁺ currents (I_Ca), which were selectively blocked by isradipine and verapamil and enhanced by the selective L-type agonist BAY K 8644. Addition of elocalcitol (10^–7 M) increased L-type I_Ca size and specific conductance by inducing faster activation and inactivation kinetics than control and BAY K 8644, while determining a significant negative shift of the activation and inactivation curves, comparable to BAY K 8644. These effects were strengthened in long-term treated hBCs with elocalcitol (10^–8 M, 48 h), which also showed increased mRNA and protein expression of pore-forming L-type _1C-subunit. In the bladder from Sprague-Dawley rats, BAY K 8644 induced a dose-dependent increase in tension, which was significantly enhanced by elocalcitol treatment (30 µg·kg^–1·day^–1, 2 wk). In conclusion, elocalcitol upregulated Ca²⁺ entry through L-type Ca²⁺ channels in hBCs, thus balancing its inhibitory effect on RhoA/Rho kinase signaling and suggesting its possible efficacy for the modulation of bladder contractile mechanisms.

Thrombin mediates mitogenesis and survival of human endothelial cells through distinct mechanisms

2008-05-06

Thrombin has been reported to play a pivotal role in the initiation of angiogenesis by indirectly regulating and organizing a network of angiogenic molecules. In addition, it has been proposed that thrombin can directly activate endothelial cell proliferation. However, in this report it was shown that thrombin is a poor growth factor for human endothelial cells, and its modest mitogenic activity is mediated indirectly by the release of heparin-binding epidermal growth factor, subsequent to proteinase-activated receptor 1 (PAR1) activation. On the other hand, it was demonstrated that thrombin is a potent anti-apoptotic factor for endothelial cells, pointing to a novel role of thrombin in vascular protection. Analysis by annexin V-propidium iodide double staining revealed that thrombin, specifically, promoted survival of serum-starved endothelial cells in a concentration-dependent manner. In contrast to its mitogenic effect, the anti-apoptotic effect of thrombin was largely independent of its catalytic activity and was mediated through interaction with β3 and 5β1 integrins, whereas the involvement of PAR1 was limited. These results provide new insights in understanding the role of thrombin in endothelial cell signaling and vascular biology.

H9c2 cardiomyoblasts produce thyroid hormone

2008-05-06

Thyroid hormone acts on a wide range of tissues. In the cardiovascular system, thyroid hormone is an important regulator of cardiac function and cardiovascular hemodynamics. Although some early reports in the literature suggested an unknown extrathyroidal source of thyroid hormone, it is currently thought to be produced exclusively in the thyroid gland, a highly specialized organ with the sole function of generating, storing, and secreting thyroid hormone. Whereas most of the proteins necessary for thyroid hormone synthesis are thought to be expressed exclusively in the thyroid gland, we now have found evidence that all of these proteins, i.e., thyroglobulin, DUOX1, DUOX2, the sodium-iodide symporter, pendrin, thyroid peroxidase, and thyroid-stimulating hormone receptor, are also expressed in cardiomyocytes. Furthermore, we found thyroglobulin to be transiently upregulated in an in vitro model of ischemia. When performing these experiments in the presence of ¹²⁵I, we found that ¹²⁵I was integrated into thyroglobulin and that under ischemia-like conditions the radioactive signal in thyroglobulin was reduced. Concomitantly we observed an increase of intracellularly produced, ¹²⁵I-labeled thyroid hormone. In conclusion, our findings demonstrate for the first time that cardiomyocytes produce thyroid hormone in a manner adapted to the cell's environment.