AJP: Cell Physiology current issue

AJP-Cell Physiology: the whole is greater than the sum of its parts

Insel, P. A. — 2008-07-16

Hypoxia enhances lysosomal TNF-{alpha} degradation in mouse peritoneal macrophages

2008-07-16

Infection, simulated by lipopolysaccharide (LPS), is a potent stimulator of tumor necrosis factor- (TNF-) production, and hypoxia often synergizes with LPS to induce higher levels of the secreted cytokine. However, we show that in primary mouse peritoneal macrophages and in three mouse peritoneal macrophage cell lines (RAW 264.7, J774A.1, and PMJ-2R), hypoxia (O₂ < 0.3%) reduces the secretion of LPS-induced TNF- (P < 0.01). In RAW 264.7 cells this reduction was not regulated transcriptionally as TNF- mRNA levels remained unchanged. Rather, hypoxia and LPS reduced the intracellular levels of TNF- by twofold (P < 0.01) by enhancing its degradation in the lysosomes and inhibiting its secretion via secretory lysosomes, as shown by confocal microscopy and verified by the use of the lysosome inhibitor Bafilomycin A1. In addition, although hypoxia did not change the accumulation of the soluble receptor TNF-RII, it increased its binding to the secreted TNF- by twofold (P < 0.05). We suggest that these two posttranslational regulatory checkpoints coexist in hypoxia and may partially explain the reduced secretion and diminished biological activity of TNF- in hypoxic peritoneal macrophages.

The class V myosin motor, myosin 5c, localizes to mature secretory vesicles and facilitates exocytosis in lacrimal acini

2008-07-16

We investigated the role of the actin-based myosin motor, myosin 5c (Myo5c) in vesicle transport in exocrine secretion. Lacrimal gland acinar cells (LGAC) are the major source for the regulated secretion of proteins from the lacrimal gland into the tear film. Confocal fluorescence and immunogold electron microscopy revealed that Myo5c was associated with secretory vesicles in primary rabbit LGAC. Upon stimulation of secretion with the muscarinic agonist, carbachol, Myo5c was also detected in association with actin-coated fusion intermediates. Adenovirus-mediated expression of green fluorescent protein (GFP) fused to the tail domain of Myo5c (Ad-GFP-Myo5c-tail) showed that this protein was localized to secretory vesicles. Furthermore, its expression induced a significant (P ≤ 0.05) decrease in carbachol-stimulated release of two secretory vesicle content markers, secretory component and syncollin-GFP. Adenovirus-mediated expression of GFP appended to the full-length Myo5c (Ad-GFP-Myo5c-full) was used in parallel with adenovirus-mediated expression of GFP-Myo5c-tail in LGAC to compare various parameters of secretory vesicles labeled with either GFP-labeled protein in resting and stimulated LGAC. These studies revealed that the carbachol-stimulated increase in secretory vesicle diameter associated with compound fusion of secretory vesicles that was also exhibited by vesicles labeled with GFP-Myo5c-full was impaired in vesicles labeled with GFP-Myo5c-tail. A significant decrease in GFP labeling of actin-coated fusion intermediates was also seen in carbachol-stimulated LGAC transduced with GFP-Myo5c-tail relative to LGAC transduced with GFP-Myo5c-full. These results suggest that Myo5c participates in apical exocytosis of secretory vesicles.

O2-sensing signal cascade: clamping of O2 respiration, reduced ATP utilization, and inducible fumarate respiration

2008-07-16

These studies explore the consequences of activating the prolyl hydroxylase (PHD) O₂-sensing pathway in spontaneously twitching neonatal cardiomyocytes. Full activation of the PHD pathway was achieved using the broad-spectrum PHD inhibitor (PHI) dimethyloxaloylglycine (DMOG). PHI treatment of cardiomyocytes caused an 85% decrease in O₂ consumption and a 300% increase in lactic acid production under basal conditions. This indicates a ~75% decrease in ATP turnover rate, inasmuch as the increased ATP generation by glycolysis is inadequate to compensate for the lower respiration. To determine the extent to which decreased ATP turnover underlies the suppressed O₂ consumption, mitochondria were uncoupled with 2,4-dinitrophenol. We were surprised to find that 2,4-dinitrophenol failed to increase O₂ consumption by PHI-treated cells, indicating that electron transport chain activity, rather than ATP turnover rate, limits respiration in PHI-treated cardiomyocytes. Silencing of hypoxia-inducible factor-1 (HIF-1) expression restored the ability of uncoupled PHI-treated myocytes to increase O₂ consumption; however, basal O₂ uptake rates remained low because of the unabated suppression of cellular ATP consumption. Thus it appears that respiration is actively "clamped" through an HIF-dependent mechanism, whereas HIF-independent mechanisms are responsible for downregulation of ATP consumption. In addition, we find that PHD pathway activation enables mitochondria to utilize fumarate as a terminal electron acceptor when cytochrome c oxidase is inactive. The source of fumarate for this unusual respiration is derived from aspartate via the purine nucleotide cycle. In sum, these studies show that the O₂-sensing pathway is sufficient to actively "clamp" O₂ consumption and independently suppress cellular ATP consumption. The PHD pathway also enables the mitochondria to utilize fumarate for respiration.

Rho-ROCK signaling differentially regulates chondrocyte spreading on fibronectin and bone sialoprotein

Gill, K. S., Beier, F., Goldberg, H. A. — 2008-07-16

The mammalian growth plate is a dynamic structure rich in extracellular matrix (ECM). Interactions of growth plate chondrocytes with ECM proteins regulate cell behavior. In this study, we compared chondrocyte adhesion and spreading dynamics on fibronectin (FN) and bone sialoprotein (BSP). Chondrocyte adhesion and spreading were also compared with fibroblasts to analyze potential cell-type-specific effects. Chondrocyte adhesion to BSP is independent of posttranslational modifications but is dependent on the RGD sequence in BSP. Whereas chondrocytes and fibroblasts adhered at similar levels on FN and BSP, cells displayed more actin-dependent spread on FN despite a 16x molar excess of BSP adsorbed to plastic. To identify intracellular mediators responsible for this difference in spreading, we investigated focal adhesion kinase (FAK)-Src and Rho-Rho kinase (ROCK) signaling. Although activated FAK localized to the vertices of adhered chondrocytes, levels of FAK activation did not correlate with the extent of spreading. Furthermore, Src inhibition reduced chondrocyte spreading on both FN and BSP, suggesting that FAK-Src signaling is not responsible for less cell spreading on BSP. In contrast, inhibition of Rho and ROCK in chondrocytes increased cell spreading on BSP and membrane protrusiveness on FN but did not affect cell adhesion. In fibroblasts, Rho inhibition increased fibroblast spreading on BSP while ROCK inhibition changed membrane protrusiveness of FN and BSP. In summary, we identify a novel role for Rho-ROCK signaling in regulating chondrocyte spreading and demonstrate both cell- and matrix molecule-specific mechanisms controlling cell spreading.

Regulation of H2O2-induced necrosis by PKC and AMP-activated kinase signaling in primary cultured hepatocytes

2008-07-16

Recent studies have suggested that, in certain cases, necrosis, like apoptosis, may be programmed, involving the activation and inhibition of many signaling pathways. In this study, we examined whether necrosis induced by H₂O₂ is regulated by signaling pathways in primary hepatocytes. A detailed time course revealed that H₂O₂ treated to hepatocytes is consumed within minutes, but hepatocytes undergo necrosis several hours later. Thus, H₂O₂ treatment induces a "lag phase" where signaling changes occur, including PKC activation, Akt (PKB) downregulation, activation of JNK, and downregulation of AMP-activated kinase (AMPK). Investigation of various inhibitors demonstrated that PKC inhibitors were effective in reducing necrosis caused by H₂O₂ (~80%). PKC inhibitor treatment decreased PKC activity but, surprisingly, also upregulated Akt and AMPK, suggesting that various PKC isoforms negatively regulate Akt and AMPK. Akt did not appear to play a significant role in H₂O₂-induced necrosis, since PKC inhibitor treatment protected hepatocytes from H₂O₂ even when Akt was inhibited. On the other hand, compound C, a selective AMPK inhibitor, abrogated the protective effect of PKC inhibitors against necrosis induced by H₂O₂. Furthermore, AMPK activators protected against H₂O₂-induced necrosis, suggesting that much of the protective effect of PKC inhibition was mediated through the upregulation of AMPK. Work with PKC inhibitors suggested that atypical PKC downregulates AMPK in response to H₂O₂. Knockdown of PKC- using antisense oligonucleotides also slightly protected (~22%) against H₂O₂. Taken together, our data demonstrate that the modulation of signaling pathways involving PKC and AMPK can alter H₂O₂-induced necrosis, suggesting that a signaling "program" is important in mediating H₂O₂-induced necrosis in primary hepatocytes.

Transmembrane IV of the high-affinity sodium-glucose cotransporter participates in sugar binding

Liu, T., Lo, B., Speight, P., Silverman, M. — 2008-07-16

Investigation of the structure/function relationships of the sodium-glucose transporter (SGLT1) is crucial to understanding the cotransporter mechanism. In the present study, we used cysteine-scanning mutagenesis and chemical modification by methanethiosulfonate (MTS) derivatives to test whether predicted transmembrane IV participates in sugar binding. Five charged and polar residues (K139, Q142, T156, K157, and D161) and two glucose/galactose malabsorption missense mutations (I147 and S159) were replaced with cysteine. Mutants I147C, T156C, and K157C exhibited sufficient expression to be studied in detail using the two-electrode voltage-clamp method in Xenopus laevis oocytes and COS-7 cells. I147C was similar in function to wild-type and was not studied further. Mutation of lysine-157 to cysteine (K157C) causes loss of phloridzin and -methyl-d-glucopyranoside (MG) binding. These functions are restored by chemical modification with positively charged (2-aminoethyl) methanethiosulfonate hydrobromide (MTSEA). Mutation of threonine-156 to cysteine (T156C) reduces the affinity of MG and phloridzin for T156C by ~5-fold and ~20-fold, respectively. In addition, phloridzin protects cysteine-156 in T156C from alkylation by MTSEA. Therefore, the presence of a positive charge or a polar residue at 157 and 156, respectively, affects sugar binding and sugar-induced Na⁺ currents.

Regulation of the voltage-gated K+ channels KCNQ2/3 and KCNQ3/5 by serum- and glucocorticoid-regulated kinase-1

Schuetz, F., Kumar, S., Poronnik, P., Adams, D. J. — 2008-07-16

The voltage-gated KCNQ2/3 and KCNQ3/5 K⁺ channels regulate neuronal excitability. We recently showed that KCNQ2/3 and KCNQ3/5 channels are regulated by the ubiquitin ligase Nedd4-2. Serum- and glucocorticoid-regulated kinase-1 (SGK-1) plays an important role in regulation of epithelial ion transport. SGK-1 phosphorylation of Nedd4-2 decreases the ability of Nedd4-2 to ubiquitinate the epithelial Na⁺ channel, which increases the abundance of channel protein in the cell membrane. In this study, we investigated the mechanism(s) of SGK-1 regulation of M-type KCNQ channels expressed in Xenopus oocytes. SGK-1 significantly upregulated the K⁺ current amplitudes of KCNQ2/3 and KCNQ3/5 channels ~1.4- and ~1.7-fold, respectively, whereas the kinase-inactive SGK-1 mutant had no effect. The cell surface levels of KCNQ2-hemagglutinin/3 were also increased by SGK-1. Deletion of the KCNQ3 channel COOH terminus in the presence of SGK-1 did not affect the K⁺ current amplitude of KCNQ2/3/5-mediated currents. Coexpression of Nedd4-2 and SGK-1 with KCNQ2/3 or KCNQ3/5 channels did not significantly alter K⁺ current amplitudes. Only the Nedd4-2 mutant ^S448ANedd4-2 exhibited a significant downregulation of the KCNQ2/3/5 K⁺ current amplitudes. Taken together, these results demonstrate a potential mechanism for regulation of KCNQ2/3 and KCNQ3/5 channels by SGK-1 regulation of the activity of the ubiquitin ligase Nedd4-2.

Phosphatidylinositol 4,5-bisphosphate hydrolysis mediates histamine-induced KCNQ/M current inhibition

Liu, B., Liang, H., Liu, L., Zhang, H. — 2008-07-16

The M-type potassium channel, of which its molecular basis is constituted by KCNQ2-5 homo- or heteromultimers, plays a key role in regulating neuronal excitability and is modulated by many G protein-coupled receptors. In this study, we demonstrate that histamine inhibits KCNQ2/Q3 currents in human embryonic kidney (HEK)293 cells via phosphatidylinositol 4,5-bisphosphate (PIP₂) hydrolysis mediated by stimulation of H₁ receptor and phospholipase C (PLC). Histamine inhibited KCNQ2/Q3 currents in HEK293 cells coexpressing H₁ receptor, and this effect was totally abolished by H₁ receptor antagonist mepyramine but not altered by H₂ receptor antagonist cimetidine. The inhibition of KCNQ currents was significantly attenuated by a PLC inhibitor U-73122 but not affected by depletion of internal Ca²⁺ stores or intracellular Ca²⁺ concentration ([Ca²⁺]_i) buffering via pipette dialyzing BAPTA. Moreover, histamine also concentration dependently inhibited M current in rat superior cervical ganglion (SCG) neurons by a similar mechanism. The inhibitory effect of histamine on KCNQ2/Q3 currents was entirely reversible but became irreversible when the resynthesis of PIP₂ was impaired with phosphatidylinsitol-4-kinase inhibitors. Histamine was capable of producing a reversible translocation of the PIP₂ fluorescence probe PLC₁-PH-GFP from membrane to cytosol in HEK293 cells by activation of H₁ receptor and PLC. We concluded that the inhibition of KCNQ/M currents by histamine in HEK293 cells and SCG neurons is due to the consumption of membrane PIP₂ by PLC.

Inhibition of transient receptor potential A1 channel by phosphatidylinositol-4,5-bisphosphate

Kim, D., Cavanaugh, E. J., Simkin, D. — 2008-07-16

Membrane phosphatidylinositol-4,5-bisphosphate (PIP2) is critical for the function of many transient receptor potential (TRP) ion channels. The role of PIP2 in TRPA1 function is not well known. The effect of PIP2 on TRPA1 was investigated by direct application of PIP2 and by using polylysine and PIP2 antibody that sequester PIP2. In inside-out patches from HeLa cells expressing mouse TRPA1, polytriphosphate (PPPi) was added to the bath solution to keep TRPA1 sensitive to allyl isothiocyanate (AITC; mustard oil). Direct application of PIP2 (10 µM) to inside-out patches did not activate TRPA1, but AITC and ⁹-tetrahydrocannabinol (THC) produced strong activation. In inside-out patches in which TRPA1 was first activated with AITC (in the presence of PPPi), further addition of PIP2 produced a concentration-dependent inhibition of TRPA1 [agonist concentration producing half-maximal activity (K_1/2), 2.8 µM]. Consistent with the inhibition of TRPA1 by PIP2, AITC activated a large whole cell current when polylysine or PIP2 antibody was added to the pipette but a markedly diminished current when PIP2 was added to the pipette. In inside-out patches with PPPi in the bath solution, application of PIP2 antibody or polylysine caused activation of TRPA1, and this was blocked by PIP2. However, TRPA1 was not activated by polylysine and PIP2 antibody under whole cell conditions, suggesting a more complex regulation of TRPA1 by PIP2 in intact cells. These results show that PIP2 inhibits TRPA1 and reduces the sensitivity of TRPA1 to AITC.

Ouabain-induced stimulation of sodium-hydrogen exchange in rat optic nerve astrocytes

Mandal, A., Delamere, N. A., Shahidullah, M. — 2008-07-16

Sodium-dependent transporters are inhibited indirectly by the Na-K-ATPase inhibitor ouabain. Here we report stimulation of sodium-hydrogen exchange (NHE) in ouabain-treated cells. BCECF was used to measure cytoplasmic pH in cultured rat optic nerve astrocytes. Ammonium chloride was applied to acid load the cells. On removal of ammonium chloride, cytoplasmic pH fell abruptly, then gradually recovered toward baseline. Ouabain (1 µM) did not change cell sodium content, but the rate of pH recovery increased by 68%. Ouabain speeded pH recovery both in the presence and absence of bicarbonate. In bicarbonate-free medium, dimethylamiloride, an NHE inhibitor, eliminated the effect of 1 µM ouabain on pH recovery. Western blot analysis showed an NHE1 immunoreactive band but not NHE2, NHE3, or NHE4. Immunoprecipitation studies showed phosphorylation of NHE1 in cells treated with 1 µM ouabain. Ouabain evoked an increase of cAMP, and the effect of 1 µM ouabain on pH recovery was abolished by H-89, a protein kinase A inhibitor. 8-Bromoadenosine-cAMP increased the pH recovery rate, and this recovery was not further increased by ouabain. Although 1 µM ouabain did not alter cytoplasmic calcium concentration, it stimulated calcium entry after store depletion, a response abolished by 2-APB. Ouabain-induced stimulation of pH recovery was suppressed by inhibitors of capacitative calcium entry, SKF-96365, and 2-APB, as well as the cytoplasmic calcium chelator BAPTA. The cAMP increase in ouabain-treated cells was abolished by BAPTA and 2-APB. Taken together, the results are consistent with increased capacitative calcium entry and subsequent cAMP-PKA-dependent stimulation of NHE1 in ouabain-treated cells.

Rat aquaporin-5 4.3-kb 5'-flanking region differentially regulates expression in salivary gland and lung in vivo

2008-07-16

We previously cloned a 4.3-kb genomic fragment encompassing 5'-flanking regulatory elements of rat aquaporin-5 (Aqp5) that demonstrated preferential transcriptional activity in lung and salivary cells in vitro. To investigate the ability of Aqp5 regulatory elements to direct transgene expression in vivo, transgenic (TG) mice and rats were generated in which the 4.3-kb Aqp5 fragment directed the expression of enhanced green fluorescent protein (EGFP). RT-PCR revealed relative promoter specificity for the lung and salivary glands in TG mice. Immunofluorescence microscopy showed strong EGFP expression in salivary acinar cells but not in lung type I (AT1) cells, both known sites of endogenous AQP5 expression. Similar results were obtained in TG rats generated by lentiviral transgenesis. EGFP mRNA was detected in both salivary glands and lung. Robust EGFP fluorescence was observed in frozen sections of the rat salivary gland but not in the lung or other tested tissues. The percentage of EGFP-positive acinar cells was increased in parotid and submandibular glands of TG rats receiving a chronic injection of the β-adrenergic receptor agonist isoproterenol. EGFP-positive cells in the lung that were also reactive with the AT1-cell specific monoclonal antibody VIIIB2 were identified by flow cytometry. These findings demonstrate that the 4.3-kb Aqp5 promoter/enhancer directs strong cell-specific transgene expression in salivary gland and low-level AT1 cell-specific expression in the lung. While these Aqp5 regulatory elements should be useful for functional studies in salivary glands, additional upstream or intronic cis-active elements are likely required for robust expression in the lung.

Ubiquitination regulates the plasma membrane expression of renal UT-A urea transporters

Stewart, G. S., O'Brien, J. H., Smith, C. P. — 2008-07-16

The renal UT-A urea transporters UT-A1, UT-A2, and UT-A3 are known to play an important role in the urinary concentrating mechanism. The control of the cellular localization of UT-A transporters is therefore vital to overall renal function. In the present study, we have investigated the effect of ubiquitination on UT-A plasma membrane expression in Madin-Darby canine kidney (MDCK) cell lines expressing each of the three renal UT-A transporters. Inhibition of the ubiquitin-proteasome pathway caused an increase in basal transepithelial urea flux across MDCK-rat (r)UT-A1 and MDCK-mouse (m)UT-A2 monolayers (P < 0.01, n = 3, ANOVA) and also increased dimethyl urea-sensitive, arginine vasopressin-stimulated urea flux (P < 0.05, n = 3, ANOVA). Inhibition of the ubiquitin-proteasome pathway also increased basolateral urea flux in MDCK-mUT-A3 monolayers (P < 0.01, n = 4, ANOVA) in a concentration-dependent manner. These increases in urea flux corresponded to a significant increase in UT-A transporter expression in the plasma membrane (P < 0.05, n = 3, ANOVA). Further analysis of the MDCK-mUT-A3 cell line confirmed that vasopressin specifically increased UT-A3 expression in the plasma membrane (P < 0.05, n = 3, ANOVA). However, preliminary data suggested that vasopressin produces this effect through an alternative route to that of the ubiquitin-proteasome pathway. In conclusion, our study suggests that ubiquitination regulates the plasma membrane expression of all three major UT-A urea transporters, but that this is not the mechanism primarily used by vasopressin to produce its physiological effects.

Collagen phagocytosis is regulated by the guanine nucleotide exchange factor Vav2

Arora, P. D., Marignani, P. A., McCulloch, C. A. — 2008-07-16

Collagen phagocytosis is a crucial 2β1-integrin-dependent process that mediates extracellular matrix remodeling by fibroblasts. We showed previously that after initial contact with collagen, activated Rac1 accelerates collagen phagocytosis but the Rac guanine nucleotide exchange factors (GEFs) that regulate Rac are not defined. We examined here the GEFs that regulate collagen phagocytosis in mouse fibroblasts. Collagen binding enhanced Rac1 activity (5–20 min) but not Cdc42 or RhoA activity. Analysis of collagen bead-associated proteins showed enrichment with Vav2, which correlated temporally with increased Rac1 activity. Knockdown of Vav2 prevented Rac activation, recruitment of Rac1 to collagen bead binding sites, and collagen bead binding, but knockdown of Sos-1 or β-Pix had no effect on Rac activation or collagen binding. Vav2 was associated with the nucleotide-free Rac1 mutant (G15ARac1) after collagen binding. Collagen bead binding promoted phosphorylation of Vav2, which temporally correlated with Rac1 activation and which required Src kinase activity. Blockage of Src activity prevented collagen bead-induced Rac activation and collagen bead binding. Collectively these data indicate that Vav2 regulates the Rac1 activity associated with the binding step of collagen phagocytosis.

The effect of angiotensin II on intracellular pH is mediated by AT1 receptor translocation

Thieme, K., Eguti, D. M. N., Mello-Aires, M., Oliveira-Souza, M. — 2008-07-16

The effect of ANG II on intracellular pH (pH_i) recovery rate and AT₁ receptor translocation was investigated in transfected MDCK cells. The pH_i recovery rate was evaluated by fluorescence microscopy using the fluorescent probe BCECF-AM. The human angiotensin II receptor isoform 1 (hAT₁) translocation was analyzed by immunofluorescence and confocal microscope. Our data show that transfected cells in control situation have a pH_i recovery rate of 0.219 ± 0.017 pH U/min (n = 11). This value was similar to nontransfected cells [0.211 ± 0.009 pH U/min (n = 12)]. Both values were significantly increased with ANG II (10^–9 M) but not with ANG II (10^–6 M). Losartan (10^–7 M) and dimethyl-BAPTA-AM (10^–7 M) decreased significantly the stimulatory effect of ANG II (10^–9 M) and induced an increase in Na⁺/H⁺ exchanger 1 (NHE-1) activity with ANG II (10^–6 M). Immunofluorescence studies indicated that in control situation, the hAT₁ receptor was predominantly expressed in cytosol. However, it was translocated to plasma membrane with ANG II (10^–9 M) and internalized with ANG II (10^–6 M). Losartan (10^–7 M) induced hAT₁ translocation to plasma membrane in all studied groups. Dimethyl-BAPTA-AM (10^–7 M) did not change the effect of ANG II (10^–9 M) on the hAT₁ receptor distribution but induced its accumulation at plasma membrane in cells treated with ANG II (10^–6 M). With ionomycin (10^–6 M), the receptor was accumulated in cytosol. The results indicate that, in MDCK cells, the effect of ANG II on NHE-1 activity is associated with ligand binding to AT₁ receptor and intracellular signaling events related to AT₁ translocation.

Poloxamer 188 reduces the contraction-induced force decline in lumbrical muscles from mdx mice

Ng, R., Metzger, J. M., Claflin, D. R., Faulkner, J. A. — 2008-07-16

Duchenne Muscular Dystrophy is a genetic disease caused by the lack of the protein dystrophin. Dystrophic muscles are highly susceptible to contraction-induced injury, and following contractile activity, have disrupted plasma membranes that allow leakage of calcium ions into muscle fibers. Because of the direct relationship between increased intracellular calcium concentration and muscle dysfunction, therapeutic outcomes may be achieved through the identification and restriction of calcium influx pathways. Our purpose was to determine the contribution of sarcolemmal lesions to the force deficits caused by contraction-induced injury in dystrophic skeletal muscles. Using isolated lumbrical muscles from dystrophic (mdx) mice, we demonstrate for the first time that poloxamer 188 (P188), a membrane-sealing poloxamer, is effective in reducing the force deficit in a whole mdx skeletal muscle. A reduction in force deficit was also observed in mdx muscles that were exposed to a calcium-free environment. These results, coupled with previous observations of calcium entry into mdx muscle fibers during a similar contraction protocol, support the interpretation that extracellular calcium enters through sarcolemmal lesions and contributes to the force deficit observed in mdx muscles. The results provide a basis for potential therapeutic strategies directed at membrane stabilization of dystrophin-deficient skeletal muscle fibers.

{alpha}4{beta}1-Integrin regulates directionally persistent cell migration in response to shear flow stimulation

2008-07-16

₄β₁-Integrin plays a pivotal role in cell migration in vivo. This integrin has been shown to regulate the front-back polarity of migrating cells via localized inhibition of ₄-integrin/paxillin binding by phosphorylation at the ₄-integrin cytoplasmic tail. Here, we demonstrate that ₄β₁-integrin regulates directionally persistent cell migration via a more complex mechanism in which ₄-integrin phosphorylation and paxillin binding act via both cooperative and independent pathways. We show that, in response to shear flow, ₄β₁-integrin binding to the CS-1 region of fibronectin was necessary and sufficient to promote directionally persistent cell migration when this integrin was ectopically expressed in CHO cells. Under shear flow, the ₄β₁-integrin-expressing cells formed a fan shape with broad lamellipodia at the front and retracted trailing edges at the back. This "fanning" activity was enhanced by disrupting paxillin binding alone and inhibited by disrupting phosphorylation alone or together with disrupting paxillin binding. Notably, the phosphorylation-disrupting mutation and the double mutation resulted in the formation of long trailing tails, suggesting that ₄-integrin phosphorylation is required for trailing edge retraction/detachment independent of paxillin binding. Furthermore, the stable polarity and directional persistence of shear flow-stimulated cells were perturbed by the double mutation but not the single mutations alone, indicating that paxillin binding and ₄-integrin phosphorylation can facilitate directionally persistent cell migration in an independent and compensatory manner. These findings provide a new insight into the mechanism by which integrins regulate directionally persistent cell migration.

Role for stress fiber contraction in surface tension development and stretch-activated channel regulation in C2C12 myoblasts

2008-07-16

Membrane-cytoskeleton interaction regulates transmembrane currents through stretch-activated channels (SACs); however, the mechanisms involved have not been tested in living cells. We combined atomic force microscopy, confocal immunofluorescence, and patch-clamp analysis to show that stress fibers (SFs) in C2C12 myoblasts behave as cables that, tensed by myosin II motor, activate SACs by modifying the topography and the viscoelastic (Young's modulus and hysteresis) and electrical passive (membrane capacitance, C_m) properties of the cell surface. Stimulation with sphingosine 1-phosphate to elicit SF formation, the inhibition of Rho-dependent SF formation by Y-27632 and of myosin II-driven SF contraction by blebbistatin, showed that not SF polymerization alone but the generation of tensional forces by SF contraction were involved in the stiffness response of the cell surface. Notably, this event was associated with a significant reduction in the amplitude of the cytoskeleton-mediated corrugations in the cell surface topography, suggesting a contribution of SF contraction to plasma membrane stretching. Moreover, C_m, used as an index of cell surface area, showed a linear inverse relationship with cell stiffness, indicating participation of the actin cytoskeleton in plasma membrane remodeling and the ability of SF formation to cause internalization of plasma membrane patches to reduce C_m and increase membrane tension. SF contraction also increased hysteresis. Together, these data provide the first experimental evidence for a crucial role of SF contraction in SAC activation. The related changes in cell viscosity may prevent SAC from abnormal activation.

Effect of low pH on single skeletal muscle myosin mechanics and kinetics

Debold, E. P., Beck, S. E., Warshaw, D. M. — 2008-07-16

Acidosis (low pH) is the oldest putative agent of muscular fatigue, but the molecular mechanism underlying its depressive effect on muscular performance remains unresolved. Therefore, the effect of low pH on the molecular mechanics and kinetics of chicken skeletal muscle myosin was studied using in vitro motility (IVM) and single molecule laser trap assays. Decreasing pH from 7.4 to 6.4 at saturating ATP slowed actin filament velocity (V_actin) in the IVM by 36%. Single molecule experiments, at 1 µM ATP, decreased the average unitary step size of myosin (d) from 10 ± 2 nm (pH 7.4) to 2 ± 1 nm (pH 6.4). Individual binding events at low pH were consistent with the presence of a population of both productive (average d = 10 nm) and nonproductive (average d = 0 nm) actomyosin interactions. Raising the ATP concentration from 1 µM to 1 mM at pH 6.4 restored d (9 ± 3 nm), suggesting that the lifetime of the nonproductive interactions is solely dependent on the [ATP]. V_actin, however, was not restored by raising the [ATP] (1–10 mM) in the IVM assay, suggesting that low pH also prolongs actin strong binding (t_on). Measurement of t_on as a function of the [ATP] in the single molecule assay suggested that acidosis prolongs t_on by slowing the rate of ADP release. Thus, in a detachment limited model of motility (i.e., V_actin ~ d/t_on), a slowed rate of ADP release and the presence of nonproductive actomyosin interactions could account for the acidosis-induced decrease in V_actin, suggesting a molecular explanation for this component of muscular fatigue.

Endothelial cell respiration is affected by the oxygen tension during shear exposure: role of mitochondrial peroxynitrite

2008-07-16

Cultured vascular endothelial cell (EC) exposure to steady laminar shear stress results in peroxynitrite (ONOO^–) formation intramitochondrially and inactivation of the electron transport chain. We examined whether the "hyperoxic state" of 21% O₂, compared with more physiological O₂ tensions (Po₂), increases the shear-induced nitric oxide (NO) synthesis and mitochondrial superoxide (O₂^·–) generation leading to ONOO^– formation and suppression of respiration. Electron paramagnetic resonance oximetry was used to measure O₂ consumption rates of bovine aortic ECs sheared (10 dyn/cm², 30 min) at 5%, 10%, or 21% O₂ or left static at 5% or 21% O₂. Respiration was inhibited to a greater extent when ECs were sheared at 21% O₂ than at lower Po₂ or left static at different Po₂. Flow in the presence of an endothelial NO synthase (eNOS) inhibitor or a ONOO^– scavenger abolished the inhibitory effect. EC transfection with an adenovirus that expresses manganese superoxide dismutase in mitochondria, and not a control virus, blocked the inhibitory effect. Intracellular and mitochondrial O₂^·– production was higher in ECs sheared at 21% than at 5% O₂, as determined by dihydroethidium and MitoSOX red fluorescence, respectively, and the latter was, at least in part, NO-dependent. Accumulation of NO metabolites in media of ECs sheared at 21% O₂ was modestly increased compared with ECs sheared at lower Po₂, suggesting that eNOS activity may be higher at 21% O₂. Hence, the hyperoxia of in vitro EC flow studies, via increased NO and mitochondrial O₂^·– production, leads to enhanced ONOO^– formation intramitochondrially and suppression of respiration.

Comparative study of ezrin phosphorylation among different tissues: more is good; too much is bad

Zhu, L., Hatakeyama, J., Chen, C., Shastri, A., Poon, K., Forte, J. G. — 2008-07-16

In a comparison of three different tissues, the membrane cytoskeleton linker protein ezrin was found to assume high levels of phosphorylation on threonine-567 (T567) in the brush border membranes of renal proximal tubule cells and small intestine enterocytes, in contrast to the apical canalicular membrane of gastric parietal cells. Together with an earlier observation that increased T567 phosphorylation is associated with more elaborate microvilli in parietal cells, this comparative study suggested a higher phosphorylation level requirement for the denser and more uniform distribution of microvilli at brush border surfaces. Using a kinase inhibitor, staurosporin, and metabolic inhibitor, sodium azide, relatively high turnover of ezrin T567 phosphorylation was observed in all three epithelia. Aiming to understand the role of phosphorylation turnover in these tissues, detergent extraction analysis of gastric glands and proximal tubules revealed that an increased phosphorylation on ezrin T567 greatly enhanced its association with F-actin, while ezrin-membrane interaction persisted regardless of the changes of phosphorylation level on ezrin T567. Finally, expression of Thr567Asp mutant ezrin, which mimics the phospho-ezrin state but does not allow turnover, caused aberrant growth of membrane projections in cultured proximal tubule cells, consistent with what had previously been observed in several cell lines and gastric parietal cells. These results fit into a model of surface plasticity, which posits that the turnover of phosphorylation on T567 empowers ezrin to relax and reposition membrane to the underlying cytoskeleton under varying conditions of filament growth or rapid membrane expansion (or depletion).

Adiponectin is expressed by skeletal muscle fibers and influences muscle phenotype and function

2008-07-16

Adiponectin (Ad) is linked to various disease states and mediates antidiabetic and anti-inflammatory effects. While it was originally thought that Ad expression was limited to adipocytes, we demonstrate here that Ad is expressed in mouse skeletal muscles and within differentiated L6 myotubes, as assessed by RT-PCR, Western blot, and immunohistochemical analyses. Serial muscle sections stained for fiber type, lipid content, and Ad revealed that muscle fibers with elevated intramyocellular Ad expression were consistently type IIA and IID fibers with detectably higher intramyocellular lipid (IMCL) content. To determine the effect of Ad on muscle phenotype and function, we used an Ad-null [knockout (KO)] mouse model. Body mass increased significantly in 24-wk-old KO mice [+5.5 ± 3% relative to wild-type mice (WT)], with no change in muscle mass observed. IMCL content was significantly increased (+75.1 ± 25%), whereas epididymal fat mass, although elevated, was not different in the KO mice compared with WT (+35.1 ± 23%; P = 0.16). Fiber-type composition was unaltered, although type IIB fiber area was increased in KO mice (+25.5 ± 6%). In situ muscle stimulation revealed lower peak tetanic forces in KO mice relative to WT (–47.5 ± 6%), with no change in low-frequency fatigue rates. These data demonstrate that the absence of Ad expression causes contractile dysfunction and phenotypical changes in skeletal muscle. Furthermore, we demonstrate that Ad is expressed in skeletal muscle and that its intramyocellular localization is associated with elevated IMCL, particularly in type IIA/D fibers.

Transcription factor PU.1 is expressed in white adipose and inhibits adipocyte differentiation

Wang, F., Tong, Q. — 2008-07-16

PU.1 transcription factor is a critical regulator of hematopoiesis and leukemogenesis. Because PU.1 interacts with transcription factors GATA-2 and C/EBP, and both are involved in the regulation of adipogenesis, we investigated whether PU.1 plays a role in the regulation of adipocyte differentiation. Our data indicate that PU.1 is expressed in white adipose tissue. PU.1 protein can also be detected in cultured 3T3-L1 adipocytes. Forced expression of PU.1 in 3T3-L1 cells inhibits adipocyte differentiation, whereas deletion of the transactivation domain of PU.1 abolishes this effect. The inhibition of adipocyte differentiation by PU.1 is achieved, at least in part, through repression of the transcriptional activity of C/EBP and C/EBPβ. Furthermore, GATA-2 and PU.1 have an additive inhibitory effect on C/EBP transactivation and adipogenesis. Finally, the expression of PU.1 is increased in white adipose of obese mice.

Downregulation of connexin40 is associated with coronary endothelial cell dysfunction in streptozotocin-induced diabetic mice

Makino, A., Platoshyn, O., Suarez, J., Yuan, J. X.-J., Dillmann, W. H. — 2008-07-16

Vascular endothelial cells (ECs) play a major role in regulating vascular tone and in revascularization. There is increasing evidence showing endothelial dysfunction in diabetes, although little is known about the contribution of connexins (Cxs) to vascular complications in the diabetic heart. This study was designed to investigate the role of Cxs in coronary endothelial dysfunction in diabetic mice. Coronary ECs isolated from diabetic mice exhibit lowered protein levels of Cx37 and Cx40 (but not Cx43) and a loss of gap junction intercellular communication (GJIC). Vasodilatation induced by the assumed contribution of EC-dependent hyperpolarization was significantly reduced in the diabetic coronary artery (CA). Cx40-specific inhibitory peptide ⁴⁰GAP27 strongly attenuated endothelium-dependent relaxation in diabetic CA at the concentration that does not affect the relaxation in control CA, suggesting that the total amount of Cx40 is lower in diabetic CA than in control CA. In diabetic mice, coronary capillary density was significantly decreased in vivo. In vitro, GJIC inhibitor attenuated the ability of EC capillary network formation. High-glucose treatment caused a decrease in Cx40 protein expression in ECs and impaired endothelial capillary network formation, which was restored by Cx40 overexpression. Furthermore, we found that the hyperglycemia-induced decrease in Cx40 was associated with inhibited protein expression of Sp1, a transcriptional factor that regulates Cx40 expression. These data suggest that downregulation of Cx40 protein expression and resultant inhibition of GJIC contribute to coronary vascular dysfunction in diabetes.

Rho-family GTPases modulate Ca2+-dependent ATP release from astrocytes

Blum, A. E., Joseph, S. M., Przybylski, R. J., Dubyak, G. R. — 2008-07-16

Previously, we reported that activation of G protein-coupled receptors (GPCR) in 1321N1 human astrocytoma cells elicits a rapid release of ATP that is partially dependent on a G_q/phophospholipase C (PLC)/Ca²⁺ mobilization signaling cascade. In this study we assessed the role of Rho-family GTPase signaling as an additional pathway for the regulation of ATP release in response to activation of protease-activated receptor-1 (PAR1), lysophosphatidic acid receptor (LPAR), and M3-muscarinic (M3R) GPCRs. Thrombin (or other PAR1 peptide agonists), LPA, and carbachol triggered quantitatively similar Ca²⁺ mobilization responses, but only thrombin and LPA caused rapid accumulation of active GTP-bound Rho. The ability to elicit Rho activation correlated with the markedly higher efficacy of thrombin and LPA, relative to carbachol, as ATP secretagogues. Clostridium difficile toxin B and Clostridium botulinum C3 exoenzyme, which inhibit Rho-GTPases, attenuated the thrombin- and LPA-stimulated ATP release but did not decrease carbachol-stimulated release. Thus the ability of certain G_q-coupled receptors to additionally stimulate Rho-GTPases acts to strongly potentiate a Ca²⁺-activated ATP release pathway. However, pharmacological inhibition of Rho kinase I/II or myosin light chain kinase did not attenuate ATP release. PAR1-induced ATP release was also reduced twofold by brefeldin treatment suggesting the possible mobilization of Golgi-derived, ATP-containing secretory vesicles. ATP release was also markedly repressed by the gap junction channel inhibitor carbenoxolone in the absence of any obvious thrombin-induced change in membrane permeability indicative of hemichannel gating.

Role of caveolin-1 in the regulation of lipoprotein metabolism

Frank, P. G., Pavlides, S., Cheung, M. W.-C., Daumer, K., Lisanti, M. P. — 2008-07-16

Lipoprotein metabolism plays an important role in the development of several human diseases, including coronary artery disease and the metabolic syndrome. A good comprehension of the factors that regulate the metabolism of the various lipoproteins is therefore key to better understanding the variables associated with the development of these diseases. Among the players identified are regulators such as caveolins and caveolae. Caveolae are small plasma membrane invaginations that are observed in terminally differentiated cells. Their most important protein marker, caveolin-1, has been shown to play a key role in the regulation of several cellular signaling pathways and in the regulation of plasma lipoprotein metabolism. In the present paper, we have examined the role of caveolin-1 in lipoprotein metabolism using caveolin-1-deficient (Cav-1–/–) mice. Our data show that, while Cav-1–/– mice show increased plasma triglyceride levels, they also display reduced hepatic very low-density lipoprotein (VLDL) secretion. Additionally, we also found that a caveolin-1 deficiency is associated with an increase in high-density lipoprotein (HDL), and these HDL particles are enriched in cholesteryl ester in Cav-1–/– mice when compared with HDL obtained from wild-type mice. Finally, our data suggest that a caveolin-1 deficiency prevents the transcytosis of LDL across endothelial cells, and therefore, that caveolin-1 may be implicated in the regulation of plasma LDL levels. Taken together, our studies suggest that caveolin-1 plays an important role in the regulation of lipoprotein metabolism by controlling their plasma levels as well as their lipid composition. Thus caveolin-1 may also play an important role in the development of atherosclerosis.

Force deficits and breakage rates after single lengthening contractions of single fast fibers from unconditioned and conditioned muscles of young and old rats

Lynch, G. S., Faulkner, J. A., Brooks, S. V. — 2008-07-16

The deficit in force generation is a measure of the magnitude of damage to sarcomeres caused by lengthening contractions of either single fibers or whole muscles. In addition, permeabilized single fibers may suffer breakages. Our goal was to understand the interaction between breakages and force deficits in "young" and "old" permeabilized single fibers from control muscles of young and old rats and "conditioned" fibers from muscles that completed a 6-wk program of in vivo lengthening contractions. Following single lengthening contractions of old-control fibers compared with young-control fibers, the twofold greater force deficits at a 10% strain support the concept of an age-related increase in the susceptibility of fibers to mechanical damage. In addition, the much higher breakage rates for old fibers at all strains tested indicate an increase with aging in the number of fibers at risk of being severely injured during any given stretch. Following the 6-wk program of lengthening contractions, young-conditioned fibers and old-conditioned fibers were not different with respect to force deficit or the frequency of breakages. A potential mechanism for the increased resistance to stretch-induced damage of old-conditioned fibers is that, through intracellular damage and subsequent degeneration and regeneration, weaker sarcomeres were replaced by stronger sarcomeres. These data indicate that, despite the association of high fiber breakage rates and large force deficits with aging, the detrimental characteristics of old fibers were improved by a conditioning program that altered both sarcomeric characteristics as well as the overall structural integrity of the fibers.

The inhibitory effect of HKa in endothelial cell tube formation is mediated by disrupting the uPA-uPAR complex and inhibiting its signaling and internalization

Liu, Y., Cao, D. J., Sainz, I. M., Guo, Y.-L., Colman, R. W. — 2008-07-16

In two-dimensional (2-D) culture systems, we have previously shown that cleaved two-chain high-molecular-weight kininogen (HKa) or its domain 5 induced apoptosis by disrupting urokinase plasminogen activator (uPA) receptor (uPAR)-integrin signal complex formation. In the present study, we used a three-dimensional (3-D) collagen-fibrinogen culture system to monitor the effects of HKa on tube formation. In a 3-D system, HKa significantly inhibited tube and vacuole formation as low as 10 nM, which represents 1.5% of the physiological concentration of high-molecular-weigh kininogen (660 nM), without apparent apoptosis. However, HKa (300 nM) completely inhibited tube formation and increased apoptotic cells about 2-fold by 20–24 h of incubation. uPA-dependent ERK activation and uPAR internalization regulate cell survival and migration. In a 2-D system, we found that exogenous uPA-induced ERK phosphorylation and uPAR internalization were blocked by HKa. In a 3-D system, we found that not only uPA-uPAR association but also the activation of ERK were inhibited by HKa. HKa disrupts the uPA-uPAR complex, inhibiting the signaling pathways, and also inhibits uPAR internalization and regeneration to the cell surface, thereby interfering with uPAR-mediated cell migration, proliferation, and survival. Thus, our data suggest that the suppression of ERK activation and uPAR internalization by HKa contributes to the inhibition of tube formation. We conclude that in this 3-D collagen-fibrinogen gel, HKa modulates the multiple functions of uPAR in endothelial cell tube formation, a process that is closely related to in vivo angiogenesis.

Extracellular matrix-specific focal adhesions in vascular smooth muscle produce mechanically active adhesion sites

Sun, Z., Martinez-Lemus, L. A., Hill, M. A., Meininger, G. A. — 2008-07-16

Integrin-mediated mechanotransduction in vascular smooth muscle cells (VSMCs) plays an important role in the physiological control of tissue blood flow and vascular resistance. To test whether force applied to specific extracellular matrix (ECM)-integrin interactions could induce myogenic-like mechanical activity at focal adhesion sites, we used atomic force microscopy (AFM) to apply controlled forces to specific ECM adhesion sites on arteriolar VSMCs. The tip of AFM probes were fused with a borosilicate bead (2~5 µm) coated with fibronectin (FN), collagen type I (CNI), laminin (LN), or vitronectin (VN). ECM-coated beads induced clustering of ₅- and β₃-integrins and actin filaments at sites of bead-cell contact indicative of focal adhesion formation. Step increases of an upward (z-axis) pulling force (800~1,600 pN) applied to the bead-cell contact site for FN-specific focal adhesions induced a myogenic-like, force-generating response from the VSMC, resulting in a counteracting downward pull by the cell. This micromechanical event was blocked by cytochalasin D but was enhanced by jasplakinolide. Function-blocking antibodies to ₅β₁- and _vβ₃-integrins also blocked the micromechanical cell event in a concentration-dependent manner. Similar pulling experiments with CNI, VN, or LN failed to induce myogenic-like micromechanical events. Collectively, these results demonstrate that mechanical force applied to integrin-FN adhesion sites induces an actin-dependent, myogenic-like, micromechanical event. Focal adhesions formed by different ECM proteins exhibit different mechanical characteristics, and FN appears of particular relevance in its ability to strongly attach to VSMCs and to induce myogenic-like, force-generating reactions from sites of focal adhesion in response to externally applied forces.

mCLCA4 ER processing and secretion requires luminal sorting motifs

2008-07-16

Ca⁺-activated Cl^– channel (CLCA) proteins are encoded by a family of highly related and clustered genes in mammals that are markedly upregulated in inflammation and have been shown to affect chloride transport. Here we describe the cellular processing and regulatory sequences underlying murine (m) CLCA4 proteins. The 125-kDa mCLCA4 gene product is cleaved to 90- and 40-kDa fragments, and the NH₂- and COOH-terminal fragments are secreted, where they are found in cell media and associated with the plasma membrane. The 125-kDa full-length protein is only found in the endoplasmic reticulum (ER), and specific luminal diarginine retention and dileucine forward trafficking signals contained within the CLCA4 sequence regulate export from the ER and proteolytic processing. Mutation of the dileucine luminal sequences resulted in ER trapping of the immaturely glycosylated 125-kDa peptide, indicating that proteolytic cleavage occurs following recognition of the trafficking motifs. Moreover, the mutated dileucine and diarginine signal sequences directed processing of a secreted form of enhanced green fluorescent protein in a manner consistent with the effects on mCLCA4.

Functional interaction of regulatory factors with the Pgc-1{alpha} promoter in response to exercise by in vivo imaging

Akimoto, T., Li, P., Yan, Z. — 2008-07-16

Real-time optical bioluminescence imaging is a powerful tool for studies of gene regulation in living animals. To elucidate exercise-induced signaling/transcriptional control of the peroxisome proliferator-activated receptor- coactivator-1 (Pgc-1) gene in skeletal muscle, we combined this technology with electric pulse-mediated gene transfer to cotransfect the Pgc-1 reporter gene with plasmid DNA encoding mutant/deletion forms of putative regulatory factors and, thereby, assess the responsiveness of the promoter to skeletal muscle contraction. We show that each of the myocyte enhancer factor 2 sites on the Pgc-1 promoter is required for contractile activity-induced Pgc-1 transcription. The responsiveness of the Pgc-1 promoter to contractile activity could be completely blocked by overexpression of the dominant-negative form of activating transcription factor 2 (ATF2), the signaling-resistant form of histone deacetylase (HDAC) 5 (HDAC5), or protein kinase D (PKD), but not by HDAC4. These findings provide in vivo evidence for functional interactions between PKD/HDAC5 and ATF2 regulatory factors and the Pgc-1 gene in adult skeletal muscle.