AJP: Cell Physiology current issue

Calcium and diabetic vascular dysfunction. Focus on "Elevated Ca2+ sparklet activity during acute hyperglycemia and diabetes in cerebral arterial smooth muscle cells"

Dunn, K. M., Nelson, M. T. — Fri, 22 Jan 2010 16:04:01 PST

Filamin and filamin-binding proteins in integrin-regulation and adhesion. Focus on: "FilaminA is required for vimentin-mediated cell adhesion and spreading"

MacPherson, M., Fagerholm, S. C. — Fri, 22 Jan 2010 16:04:01 PST

From blood typing to a transport metabolon at a crossroad. Focus on "Ammonium-dependent sodium uptake in mitochondrion-rich cells of medaka (Oryzias latipes) larvae"

Hirose, S., Nakada, T. — Fri, 22 Jan 2010 16:04:01 PST

Elevated Ca2+ sparklet activity during acute hyperglycemia and diabetes in cerebral arterial smooth muscle cells

Navedo, M. F., Takeda, Y., Nieves-Cintron, M., Molkentin, J. D., Santana, L. F. — Fri, 22 Jan 2010 16:04:01 PST

Ca⁺ sparklets are subcellular Ca²⁺ signals produced by the opening of L-type Ca²⁺ channels (LTCCs). In cerebral arterial myocytes, Ca²⁺ sparklet activity varies regionally, resulting in low and high activity, "persistent" Ca²⁺ sparklet sites. Although increased Ca²⁺ influx via LTCCs in arterial myocytes has been implicated in the chain of events contributing to vascular dysfunction during acute hyperglycemia and diabetes, the mechanisms underlying these pathological changes remain unclear. Here, we tested the hypothesis that increased Ca²⁺ sparklet activity contributes to higher Ca²⁺ influx in cerebral artery smooth muscle during acute hyperglycemia and in an animal model of non-insulin-dependent, type 2 diabetes: the dB/dB mouse. Consistent with this hypothesis, acute elevation of extracellular glucose from 10 to 20 mM increased the density of low activity and persistent Ca²⁺ sparklet sites as well as the amplitude of LTCC currents in wild-type cerebral arterial myocytes. Furthermore, Ca²⁺ sparklet activity and LTCC currents were higher in dB/dB than in control myocytes. We found that activation of PKA contributed to higher Ca²⁺ sparklet activity during hyperglycemia and diabetes. In addition, we found that the interaction between PKA and the scaffolding protein A-kinase anchoring protein was critical for the activation of persistent Ca²⁺ sparklets by PKA in cerebral arterial myocytes after hyperglycemia. Accordingly, PKA inhibition equalized Ca²⁺ sparklet activity between dB/dB and wild-type cells. These findings suggest that hyperglycemia increases Ca²⁺ influx by increasing Ca²⁺ sparklet activity via a PKA-dependent pathway in cerebral arterial myocytes and contributes to vascular dysfunction during diabetes.

Filamin A is required for vimentin-mediated cell adhesion and spreading

Kim, H., Nakamura, F., Lee, W., Shifrin, Y., Arora, P., McCulloch, C. A. — Fri, 22 Jan 2010 16:04:01 PST

Cell adhesion and spreading are regulated by complex interactions involving the cytoskeleton and extracellular matrix proteins. We examined the interaction of the intermediate filament protein vimentin with the actin cross-linking protein filamin A in regulation of spreading in HEK-293 and 3T3 cells. Filamin A and vimentin-expressing cells were well spread on collagen and exhibited numerous cell extensions enriched with filamin A and vimentin. By contrast, cells treated with small interfering RNA (siRNA) to knock down filamin A or vimentin were poorly spread; both of these cell populations exhibited >50% reductions of cell adhesion, cell surface β1 integrin expression, and β1 integrin activation. Knockdown of filamin A reduced vimentin phosphorylation and blocked recruitment of vimentin to cell extensions, whereas knockdown of filamin and/or vimentin inhibited the formation of cell extensions. Reduced vimentin phosphorylation, cell spreading, and β1 integrin surface expression, and activation were phenocopied in cells treated with the protein kinase C inhibitor bisindolylmaleimide; cell spreading was also reduced by siRNA knockdown of protein kinase C-. By immunoprecipitation of cell lysates and by pull-down assays using purified proteins, we found an association between filamin A and vimentin. Filamin A also associated with protein kinase C-, which was enriched in cell extensions. These data indicate that filamin A associates with vimentin and to protein kinase C-, thereby enabling vimentin phosphorylation, which is important for β1 integrin activation and cell spreading on collagen.

Ammonium-dependent sodium uptake in mitochondrion-rich cells of medaka (Oryzias latipes) larvae

Fri, 22 Jan 2010 16:04:02 PST

In this study, a scanning ion-selective electrode technique (SIET) was applied to measure H⁺, Na⁺, and NH₄⁺ gradients and apparent fluxes at specific cells on the skin of medaka larvae. Na⁺ uptake and NH₃/NH₄⁺ excretion were detected at most mitochondrion-rich cells (MRCs). H⁺ probing at MRCs revealed two group of MRCs, i.e., acid-secreting and base-secreting MRCs. Treatment with EIPA (100 µM) blocked 35% of the NH₃/NH₄⁺ secretion and 54% of the Na⁺ uptake, suggesting that the Na⁺/H⁺ exchanger (NHE) is involved in Na⁺ and NH₃/NH₄⁺ transport. Low-Na⁺ water (<0.001 mM) or high-NH₄⁺ (5 mM) acclimation simultaneously increased Na⁺ uptake and NH₃/NH₄⁺ excretion but decreased or even reversed the H⁺ gradient at the skin and MRCs. The correlation between NH₄⁺ production and H⁺ consumption at the skin surface suggests that MRCs excrete nonionic NH₃ (base) by an acid-trapping mechanism. Raising the external NH₄⁺ significantly blocked NH₃/NH₄⁺ excretion and Na⁺ uptake. In contrast, raising the acidity of the water (pH 7 to pH 6) enhanced NH₃/NH₄⁺ excretion and Na⁺ uptake by MRCs. In situ hybridization and real-time PCR showed that the mRNAs of the Na⁺/H⁺ exchanger (slc9a3) and Rhesus glycoproteins (Rhcg1 and Rhbg) were colocalized in MRCs of medaka, and their expressions were induced by low-Na⁺ acclimation. This study suggests a novel Na⁺/NH₄⁺ exchange pathway in apical membranes of MRCs, in which a coupled NHE and Rh glycoprotein is involved and the Rh glycoprotein may drive the NHE by generating H⁺ gradients across apical membranes of MRCs.

Protein-protein interaction between cPLA2 and splice variants of {alpha}-subunit of BK channels

Li, J., Al-Khalili, O., Ramosevac, S., Eaton, D. C., Denson, D. D. — Fri, 22 Jan 2010 16:04:02 PST

Altering the splice variant composition of large-conductance Ca²⁺-activated potassium (BK) channels can alter their activity and apparent sensitivity to Ca²⁺ and other regulators of activity. We hypothesized that differences in the responsiveness to arachidonic acid of GH3 and GH4 cells was due to a difference in two splice variants, one present in GH3 cells and the other in GH4 cells. The sequences of the two splice variants differ from one another in several ways, but the largest difference is the presence or absence of 27 amino acids in the COOH terminus of the BK -subunit. Open probability of the variant containing the 27 amino acids is significantly increased by arachidonic acid, while the variant lacking the 27 amino acids is insensitive to arachidonic acid. In addition, sensitivity of BK channels to arachidonic acid depends on cytosolic phospholipase A₂ (cPLA₂). Here we used the Mammalian Matchmaker two-hybrid assay and two BK -subunit constructs with [rSlo(27)] and without [rSlo(0)] the 27-amino acid motif to determine whether cPLA₂ associates with one construct [rSlo(27)] and not the other. We hypothesized that differential association of cPLA₂ might explain the differing responsiveness of the two constructs and GH3 and GH4 cells to arachidonic acid. We found that cPLA₂ is strongly associated with the COOH terminus of rSlo(27) and only very weakly associated with rSlo(0). We also found that arachidonic acid has a lower affinity for rSlo(0) than for rSlo(27). We conclude that the lack of response of BK channels in GH4 cells to arachidonic acid can be explained, in part, by the poor binding of cPLA₂ to the COOH terminus of the rSlo(0) -subunit, which is very similar to the splice variant found in the arachidonic acid-insensitive GH4 cells.

Mechanisms of disease pathogenesis in long QT syndrome type 5

Harmer, S. C., Wilson, A. J., Aldridge, R., Tinker, A. — Fri, 22 Jan 2010 16:04:02 PST

KCNE1 associates with the pore-forming -subunit KCNQ1 to generate the slow (I_Ks) current in cardiac myocytes. Mutations in either KCNQ1 or KCNE1 can alter the biophysical properties of I_Ks and mutations in KCNE1 underlie cases of long QT syndrome type 5 (LQT5). We previously investigated a mutation in KCNE1, T58P/L59P, which causes severe attenuation of I_Ks. However, how T58P/L59P acts to disrupt I_Ks has not been determined. In this study, we investigate and compare the effects of T58P/L59P with three other LQT5 mutations (G52R, S74L, and R98W) on the biophysical properties of the current, trafficking of KCNQ1, and assembly of the I_Ks channel. G52R and T58P/L59P produce currents that lack the kinetic behavior of I_Ks. In contrast, S74L and R98W both produce I_Ks-like currents but with rightward shifted voltage dependence of activation. All of the LQT5 mutants express protein robustly, and T58P/L59P and R98W cause modest, but significant, defects in the trafficking of KCNQ1. Despite defects in trafficking, in the presence of KCNQ1, T58P/L59P and the other LQT5 mutants are present at the plasma membrane. Interestingly, in comparison to KCNE1 and the other LQT5 mutants, T58P/L59P associates only weakly with KCNQ1. In conclusion, we identify the disease mechanisms for each mutation and reveal that T58P/L59P causes disease through a novel mechanism that involves defective I_Ks complex assembly.

Phosphatidylinositol 4,5-bisphosphate and loss of PLC{gamma} activity inhibit TRPM channels required for oscillatory Ca2+ signaling

Xing, J., Strange, K. — Fri, 22 Jan 2010 16:04:02 PST

The Caenorhabditis elegans intestinal epithelium generates rhythmic inositol 1,4,5-trisphosphate (IP₃)-dependent Ca²⁺ oscillations that control muscle contractions required for defecation. Two highly Ca²⁺-selective transient receptor potential (TRP) melastatin (TRPM) channels, GON-2 and GTL-1, function with PLC in a common signaling pathway that regulates IP₃-dependent intracellular Ca²⁺ release. A second PLC, PLCβ, is also required for IP₃-dependent Ca²⁺ oscillations, but functions in an independent signaling mechanism. PLC generates IP₃ that regulates IP₃ receptor activity. We demonstrate here that PLC via hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) also regulates GON-2/GTL-1 function. Knockdown of PLC but not PLCβ activity by RNA interference (RNAi) inhibits channel activity ~80%. Inhibition is fully reversed by agents that deplete PIP₂ levels. PIP₂ added to the patch pipette has no effect on channel activity in PLC RNAi cells. However, in control cells, 10 µM PIP₂ inhibits whole cell current ~80%. Channel inhibition by phospholipids is selective for PIP₂ with an IC₅₀ value of 2.6 µM. Elevated PIP₂ levels have no effect on channel voltage and Ca²⁺ sensitivity and likely inhibit by reducing channel open probability, single-channel conductance, and/or trafficking. We conclude that hydrolysis of PIP₂ by PLC functions in the activation of both the IP₃ receptor and GON-2/GTL-1 channels. GON-2/GTL-1 functions as the major intestinal cell Ca²⁺ influx pathway. Calcium influx through the channel feedback regulates its activity and likely functions to modulate IP₃ receptor function. PIP₂-dependent regulation of GON-2/GTL-1 may provide a mechanism to coordinate plasma membrane Ca²⁺ influx with PLC and IP₃ receptor activity as well as intracellular Ca²⁺ store depletion.

The GPA-dependent, spherostomatocytosis mutant AE1 E758K induces GPA-independent, endogenous cation transport in amphibian oocytes

Fri, 22 Jan 2010 16:04:02 PST

The previously undescribed heterozygous missense mutation E758K was discovered in the human AE1/SLC4A1/band 3 gene in two unrelated patients with well-compensated hereditary spherostomatocytic anemia (HSt). Oocyte surface expression of AE1 E758K, in contrast to that of wild-type AE1, required coexpressed glycophorin A (GPA). The mutant polypeptide exhibited, in parallel, strong GPA dependence of DIDS-sensitive ³⁶Cl^– influx, trans-anion-dependent ³⁶Cl^– efflux, and Cl^–/HCO₃^– exchange activities at near wild-type levels. AE1 E758K expression was also associated with GPA-dependent increases of DIDS-sensitive pH-independent SO₄^2– uptake and oxalate uptake with altered pH dependence. In marked contrast, the bumetanide- and ouabain-insensitive ⁸⁶Rb⁺ influx associated with AE1 E758K expression was largely GPA-independent in Xenopus oocytes and completely GPA-independent in Ambystoma oocytes. AE1 E758K-associated currents in Xenopus oocytes also exhibited little or no GPA dependence. ⁸⁶Rb⁺ influx was higher but inward cation current was lower in oocytes expressing AE1 E758K than previously reported in oocytes expressing the AE1 HSt mutants S731P and H734R. The pharmacological inhibition profile of AE1 E758K-associated ³⁶Cl^– influx differed from that of AE1 E758K-associated ⁸⁶Rb⁺ influx, as well as from that of wild-type AE1-mediated Cl^– transport. Thus AE1 E758K-expressing oocytes displayed GPA-dependent surface polypeptide expression and anion transport, accompanied by substantially GPA-independent, pharmacologically distinct Rb⁺ flux and by small, GPA-independent currents. The data strongly suggest that most of the increased cation transport associated with the novel HSt mutant AE1 E758K reflects activation of endogenous oocyte cation permeability pathways, rather than cation translocation through the mutant polypeptide.

Extensive mononuclear infiltration and myogenesis characterize recovery of dysferlin-null skeletal muscle from contraction-induced injuries

Roche, J. A., Lovering, R. M., Roche, R., Ru, L. W., Reed, P. W., Bloch, R. J. — Fri, 22 Jan 2010 16:04:02 PST

We studied the response of dysferlin-null and control skeletal muscle to large- and small-strain injuries to the ankle dorsiflexors in mice. We measured contractile torque and counted fibers retaining 10-kDa fluorescein dextran, necrotic fibers, macrophages, and fibers with central nuclei and expressing developmental myosin heavy chain to assess contractile function, membrane resealing, necrosis, inflammation, and myogenesis. We also studied recovery after blunting myogenesis with X-irradiation. We report that dysferlin-null myofibers retain 10-kDa dextran for 3 days after large-strain injury but are lost thereafter, following necrosis and inflammation. Recovery of dysferlin-null muscle requires myogenesis, which delays the return of contractile function compared with controls, which recover from large-strain injury by repairing damaged myofibers without significant inflammation, necrosis, or myogenesis. Recovery of control and dysferlin-null muscles from small-strain injury involved inflammation and necrosis followed by myogenesis, all of which were more pronounced in the dysferlin-null muscles, which recovered more slowly. Both control and dysferlin-null muscles also retained 10-kDa dextran for 3 days after small-strain injury. We conclude that dysferlin-null myofibers can survive contraction-induced injury for at least 3 days but are subsequently eliminated by necrosis and inflammation. Myogenesis to replace lost fibers does not appear to be significantly compromised in dysferlin-null mice.

Angiotensin II exerts glucose-dependent effects on Kv currents in mouse pancreatic {beta}-cells via angiotensin II type 2 receptors

Fri, 22 Jan 2010 16:04:02 PST

Hyperglycemia-associated glucotoxicity induces β-cell apoptosis but the underlying mechanisms are unknown. Interestingly, prolonged exposure to high glucose upregulates the expression and function of the renin-angiotensin system (RAS). We hypothesize that the voltage-gated outward potassium (K_v) current, which governs β-cell membrane potential and insulin secretion, has a role in glucotoxicity. In this study, we investigated the effects of prolonged exposure to high glucose on mouse pancreatic β-cells and concurrent effects on the RAS by examining changes in expression of angiotensin II (ANG II) receptors and changes in the expression and activity of K_v channels. β-Cells were incubated in high glucose medium for 1–7 days and then were examined with electrophysiological and molecular biology techniques. Prolonged exposure to high glucose produced a marked increase in β-cell primary K_v channel subunit, K_v2.1, expression and K_v current amplitude. Enhanced expression of ANG II type 1 receptor (AT₁R) was also observed under high glucose conditions, whereas blockade of AT₁R by losartan did not alter K_v channel expression. External application of ANG II reduced K_v current amplitude under normal, but not high, glucose conditions. The effect of ANG II on K_v channel gating was abolished by ANG II type 2 receptor (AT₂R) antagonism. These data suggest that hyperglycemia alters β-cell function through modification of the K_v channel which may be associated with the RAS.

Gq-mediated Ca2+ signals inhibit adenylyl cyclases 5/6 in vascular smooth muscle cells

Fri, 22 Jan 2010 16:04:02 PST

cAMP and Ca²⁺ are antagonistic intracellular messengers for the regulation of vascular smooth muscle tone; rising levels of Ca²⁺ lead to vasoconstriction, whereas an increase of cAMP induces vasodilatation. Here we investigated whether Ca²⁺ interferes with cAMP signaling by regulation of phophodiesterases (PDEs) or adenylyl cyclases (ACs). We studied regulation of cAMP concentrations by Ca²⁺ signals evoked by endogenous purinergic receptors in vascular smooth muscle cells (VSMCs). The fluorescence resonance energy transfer (FRET)-based cAMP sensor Epac1-camps allowed the measurement of cAMP levels in single-living VSMCs with subsecond temporal resolution. Moreover, in vitro calibration of Epac1-camps enabled us to estimate the absolute cytosolic cAMP concentrations. Stimulation of purinergic receptors decreased cAMP levels in the presence of the β-adrenergic agonist isoproterenol. Simultaneous imaging of cAMP with Epac1-camps and of Ca²⁺ with Fura 2 revealed a rise of intracellular Ca²⁺ in response to purinergic stimulation followed by a decline of cAMP. Chelation of intracellular Ca²⁺ and overexpression of Ca²⁺-independent AC4 antagonized this decline of cAMP, whereas pharmacological inhibition of Ca²⁺-activated PDE1 had no effect. AC assays with VSMC membranes revealed a significant attenuation of isoproterenol-stimulated cAMP production by the presence of 2 µM Ca²⁺. Furthermore, small interfering RNA (siRNA) knockdown of AC5 and AC6 (the two ACs known to be inhibited by Ca²⁺), significantly reduced the decrease of cAMP upon purinergic stimulation of isoproterenol-prestimulated VSMCs. Taken together, these results implicate a Ca²⁺-mediated inhibition of AC5 and 6 as an important mechanism of purinergic receptor-induced decline of cAMP and show a direct cross talk of these signaling pathways in VSMCs.

Cytoskeletal structure regulates endothelial cell immunogenicity independent of fluid shear stress

Vartanian, K. B., Berny, M. A., McCarty, O. J. T., Hanson, S. R., Hinds, M. T. — Fri, 22 Jan 2010 16:04:02 PST

The cardiovascular disease atherosclerosis is directly linked to the functions of endothelial cells (ECs), which are affected by fluid shear stress (FSS). High, unidirectional FSS causes EC elongation with aligned cytoskeletal components and nonimmunogenic EC functions that protect against atherosclerosis. In contrast, low, oscillatory FSS is associated with cobblestone-shaped ECs with randomly oriented cytoskeletons and proinflammatory EC functions that promote atherosclerosis. Whether EC shape plays a role in EC immunogenic functions, independent of FSS, has not been previously determined. The goal of this study was to determine the effect of EC elongation and cytoskeletal alignment on the expression of inflammatory genes and functions. With the use of micropatterned lanes, EC elongation and cytoskeletal alignment were achieved in the absence of FSS. EC gene expression of key inflammation markers determined that the elongation and cytoskeletal alignment of micropattern-elongated ECs (MPECs) alone significantly downregulated VCAM-1 while having no effect on E-selectin and ICAM-1. The positive control of FSS-elongated ECs promoted E-selectin and VCAM-1 downregulation and upregulation of ICAM-1. Functionally, monocytic U937 cells formed weaker interactions on the surface of MPECs compared with cobblestone ECs. Interestingly, MPEC expression of the known FSS-dependent transcription factor krüppel-like factor 2 (KLF2), which promotes a nonimmunogenic EC phenotype, was significantly upregulated in MPECs compared with cobblestone ECs. Cytoskeletal regulation of KLF2 expression was shown to be dependent on microtubules. Therefore, the cellular elongation and cytoskeletal alignment of MPECs regulated immunogenic gene expression and functions and may act synergistically with FSS to create an EC surface with reduced inflammatory capability.

Protein expression profiling of lens epithelial cells from Prdx6-depleted mice and their vulnerability to UV radiation exposure

Fri, 22 Jan 2010 16:04:02 PST

Oxidative stress is one of the causative factors in progression and etiology of age-related cataract. Peroxiredoxin 6 (Prdx6), a savior for cells from internal or external environmental stresses, plays a role in cellular signaling by detoxifying reactive oxygen species (ROS) and thereby controlling gene regulation. Using targeted inactivation of the Prdx6 gene, we show that Prdx6-deficient lens epithelial cells (LECs) are more vulnerable to UV-triggered cell death, a major cause of skin disorders including cataractogenesis, and these cells display abnormal protein profiles. PRDX6-depleted LECs showed phenotypic changes and formed lentoid body, a characteristic of terminal cell differentiation and epithelial-mesenchymal transition. Prdx6^–/– LECs exposed to UV-B showed higher ROS expression and were prone to apoptosis compared with wild-type LECs, underscoring a protective role for Prdx6. Comparative proteomic analysis using fluorescence-based difference gel electrophoresis along with mass spectrometry and database searching revealed a total of 13 proteins that were differentially expressed in Prdx6^–/– cells. Six proteins were upregulated, whereas expression of seven proteins was decreased compared with Prdx6^+/+ LECs. Among the cytoskeleton-associated proteins that were highly expressed in Prdx6-deficient LECs was tropomyosin (Tm)2β. Protein blot and real-time PCR validated dramatic increase of Tm2β and Tm1 expression in these cells. Importantly, Prdx6^+/+ LECs showed a similar pattern of Tm2β protein expression after transforming growth factor (TGF)-β or H₂O₂ treatment. An extrinsic supply of PRDX6 could restore Tm2β expression, demonstrating that PRDX6 may attenuate adverse signaling in cells and thereby maintain cellular homeostasis. Exploring redox-proteomics (Prdx6^–/–) and characterization and identification of abnormally expressed proteins and their attenuation by PRDX6 delivery should provide a basis for development of novel therapeutic interventions to postpone ROS-mediated abnormal signaling deleterious to cells or tissues.

Differential effects of hypoxia on osteochondrogenic potential of human adipose-derived stem cells

Fri, 22 Jan 2010 16:04:02 PST

Human adipose tissue-derived stem cells (hATSC) have been contemplated as reparative cells for cartilage engineering. Chondrogenic differentiation of hATSC can be induced by an enriched culture medium and a three-dimensional environment. Given that bone is vascularized and cartilage is not, oxygen tension has been suggested as a regulatory factor for osteochondrogenic differentiation. Our work aimed at determining whether hypoxia affects the osteochondrogenic potential of hATSC. hATSC were cultured in chondrogenic or osteogenic medium for 28 days, in pellets or monolayers, and under 5% or 20% oxygen tension. Cell differentiation was monitored by real-time PCR (COL2A1, aggrecan, Runx2, and osteocalcin). The chondrogenic differentiation was further evaluated by Alcian blue and immunohistological staining for glycosaminoglycans (GAGs) and type II collagen, respectively. Osteogenic differentiation was also assessed by the staining of mineralized matrix (Alizarin Red) and measurement of alkaline phosphatase (ALP) activity. The expression of chondrogenic markers was upregulated when hATSC were exposed to hypoxia in chondrogenic medium. Conversely, osteocalcin expression, mineralization, and ALP activity were severely reduced under hypoxic conditions even in the presence of osteogenic medium. Our data strongly suggest that hypoxia favors the chondrogenic differentiation of hATSC as evidenced by the expression of the chondrogenic markers, whereas it could alter their osteogenic potential. Our results highlight the differential regulatory role of hypoxia on the chondrogenic and osteogenic differentiation processes of hATSC. These data could help us exploit the potential of tissue engineering and stem cells to replace or restore the function of osteoarticular tissues.

Junctophilin damage contributes to early strength deficits and EC coupling failure after eccentric contractions

Fri, 22 Jan 2010 16:04:02 PST

Junctophilins (JP1 and JP2) are expressed in skeletal muscle and are the primary proteins involved in transverse (T)-tubule and sarcoplasmic reticulum (SR) membrane apposition. During the performance of eccentric contractions, the apposition of T-tubule and SR membranes may be disrupted, resulting in excitation-contraction (EC) coupling failure and thus reduced force-producing capacity. In this study, we made three primary observations: 1) through the first 3 days after the performance of 50 eccentric contractions in vivo by the left hindlimb anterior crural muscles of female mice, both JP1 and JP2 were significantly reduced by ~50% and 35%, respectively, while no reductions were observed after the performance of nonfatiguing concentric contractions; 2) following the performance of a repeated bout of 50 eccentric contractions in vivo, only JP1 was immediately reduced (~30%) but recovered by 3-day postinjury in tandem with the recovery of strength and EC coupling; and 3) following the performance of 10 eccentric contractions at either 15° or 35°C by isolated mouse extensor digitorum longus (EDL) muscle, isometric force, EC coupling, and JP1 and JP2 were only reduced after the eccentric contractions performed at 35°C. Regression analysis of JP1 and JP2 content in tibialis anterior and EDL muscles from each set of experiments indicated that JP damage is significantly associated with early (0–3 days) strength deficits after performance of eccentric contractions (R = 0.49; P < 0.001). As a whole, the results of this study indicate that JP damage plays a role in early force deficits due to EC coupling failure following the performance of eccentric contractions.

Identification of a novel phosphorylation site on TBC1D4 regulated by AMP-activated protein kinase in skeletal muscle

Fri, 22 Jan 2010 16:04:02 PST

TBC1D4 (also known as AS160) regulates glucose transporter 4 (GLUT4) translocation and glucose uptake in adipocytes and skeletal muscle. Its mode of action involves phosphorylation of serine (S)/threonine (T) residues by upstream kinases resulting in inactivation of Rab-GTPase-activating protein (Rab-GAP) activity leading to GLUT4 mobilization. The majority of known phosphorylation sites on TBC1D4 lie within the Akt consensus motif and are phosphorylated by insulin stimulation. However, the 5'-AMP-activated protein kinase (AMPK) and other kinases may also phosphorylate TBC1D4, and therefore we hypothesized the presence of additional phosphorylation sites. Mouse skeletal muscles were contracted or stimulated with 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), and muscle lysates were subjected to mass spectrometry analyses resulting in identification of novel putative phosphorylation sites on TBC1D4. The surrounding amino acid sequence predicted that S711 would be recognized by AMPK. Using a phosphospecific antibody against S711, we found that AICAR and contraction increased S711 phosphorylation in mouse skeletal muscle, and this increase was abolished in muscle-specific AMPK2 kinase-dead transgenic mice. Exercise in human vastus lateralis muscle also increased TBC1D4 S711 phosphorylation. Recombinant AMPK, but not Akt1, Akt2, or PKC, phosphorylated purified muscle TBC1D4 on S711 in vitro. Interestingly, S711 was also phosphorylated in response to insulin in an Akt2- and rapamycin-independent, but a wortmannin-sensitive, manner, suggesting this site is regulated by one or more additional upstream kinases. Despite increased S711 phosphorylation with AICAR, contraction, and insulin, mutation of S711 to alanine did not alter glucose uptake in response to these stimuli. S711 is a novel TBC1D4 phosphorylation site regulated by AMPK in skeletal muscle.

Extracellular osmolarity modulates G protein-coupled receptor-dependent ATP release from 1321N1 astrocytoma cells

Blum, A. E., Corbett Walsh, B., Dubyak, G. R. — Fri, 22 Jan 2010 16:04:02 PST

We previously reported that ATP release from 1321N1 human astrocytoma cells could be stimulated either by activation of G protein-coupled receptors (GPCR) or by hypotonic stress. Cheema et al. (Cheema TA, Ward CE, Fisher SK. J Pharmacol Exp Ther 315: 755–763, 2005) have demonstrated that thrombin activation of protease-activated receptor 1 (PAR1) in 1321N1 cells and primary astrocytes acts synergistically with hypotonic stress to gate the opening of volume-sensitive organic osmolyte and anion channels (VSOAC) and that hypertonic stress strongly inhibits PAR1 gating of VSOAC. We tested the hypothesis that a VSOAC-type permeability might comprise a GPCR-regulated pathway for ATP export by determining whether PAR1-sensitive ATP release from 1321N1 cells is similarly potentiated by hypotonicity but suppressed by hypertonic conditions. Strong hypotonic stress by itself elicited ATP release and positively modulated the response to thrombin. Thrombin-dependent ATP release was also potentiated by mild hypotonic stress that by itself did not stimulate ATP export. Notably, PAR1-sensitive ATP export was greatly inhibited in hypertonic medium. Neither the potency nor efficacy of thrombin as an activator of proximal PAR1 signaling was affected by hypotonicity or hypertonicity. 1,9-Dideoxyforskolin and carbenoxolone similarly attenuated PAR1-dependent ATP release and suppressed the PAR1-independent ATP elicited by strong hypotonic stress. Probenecid attenuated PAR1-stimulated ATP release under isotonic but not mild hypotonic conditions and had no effect on PAR1-independent release stimulated by strong hypotonicity. PAR1-dependent ATP export under all osmotic conditions required concurrent signaling by Ca²⁺ mobilization and Rho-GTPase activation. In contrast, PAR1-independent ATP release triggered by strong hypotonicity required neither of these intracellular signals. Thus, we provide the new finding that GPCR-regulated ATP release from 1321N1 astrocytoma cells is remarkably sensitive to both positive and negative modulation by extracellular osmolarity. This supports a model wherein GPCR stimulation and osmotic stress converge on an ATP release pathway in astrocytes that exhibits several features of VSOAC-type channels.

Electrophysiological and morphological features underlying neurotransmission efficacy at the splanchnic nerve-chromaffin cell synapse of bovine adrenal medulla

de Diego, A. M. G. — Fri, 22 Jan 2010 16:04:02 PST

The ability of adrenal chromaffin cells to fast-release catecholamines relies on their capacity to fire action potentials (APs). However, little attention has been paid to the requirements needed to evoke the controlled firing of APs. Few data are available in rodents and none on the bovine chromaffin cell, a model extensively used by researchers. The aim of this work was to clarify this issue. Short puffs of acetylcholine (ACh) were fast perifused to current-clamped chromaffin cells and produced the firing of single APs. Based on the currents generated by such ACh applications and previous literature, current waveforms that efficiently elicited APs at frequencies up to 20 Hz were generated. Complex waveforms were also generated by adding simple waveforms with different delays; these waveforms aimed at modeling the stimulation patterns that a chromaffin cell would conceivably undergo upon strong synaptic stimulation. Cholinergic innervation was assessed using the acetylcholinesterase staining technique on the supposition that the innervation pattern is a determinant of the kind of stimuli chromaffin cells can receive. It is concluded that 1) a reliable method to produce frequency-controlled APs by applying defined current injection waveforms is achieved; 2) the APs thus generated have essentially the same features as those spontaneously emitted by the cell and those elicited by fast-ACh perifusion; 3) the higher frequencies attainable peak at around 30 Hz; and 4) the bovine adrenal medulla shows abundant cholinergic innervation, and chromaffin cells show strong acetylcholinesterase staining, consistent with a tight cholinergic presynaptic control of firing frequency.