Gap junctions are clusters of transmembrane channels allowing a passive diffusion of ions and small molecules between adjacent cells. Connexin43, the main channel-forming protein expressed in ventricular myocytes, can associate with zonula occludens-1, a scaffolding protein linked to the actin cytoskeleton and to signal transduction molecules. The possible influence of Rho GTPases, major regulators of cellular junctions and of the actin cytoskeleton, in the modulation of gap junctional intercellular communication (GJIC) was examined. The activation of RhoA by cytoxic necrotizing factor 1 markedly enhanced GJIC, whereas its specific inhibition by the Clostridium botulinum C3 exoenzyme significantly reduced it. RhoA activity affects GJIC without major cellular redistribution of junctional plaques or changes in the Cx43 phosphorylation pattern. As these GTPases frequently act via the cortical cytoskeleton, the importance of F-actin in the modulation of GJIC was investigated by means of agents interfering with actin polymerization. Cytoskeleton stabilization by phalloidin slowed down the kinetics of channel rundown in the absence of ATP, whereas its disruption by cytochalasin D rapidly and markedly reduced GJIC despite ATP presence. Cytoskeleton stabilization by phalloidin markedly reduced the consequences of RhoA activation or inactivation. This mechanism appears to be the first described capable to both up-or down-regulate GJIC through RhoA activation or, conversely, inhibition. The inhibition of Rho downstream kinase effectors had no effect on GJIC. The present results provide further insight into the gating and regulation of junctional channels and identify a new downstream target for the small G-protein RhoA.

Background-Thrombin plays a role in mediating ischemic injury and cardiac arrhythmias, but the mechanisms involved are poorly understood. Because voltage-gated sodium channels (VGSCs) have not previously been considered, putative effects of thrombin on VGSC function were investigated in human isolated cardiomyocytes. Methods and Results-Sodium current (I Na ) was recorded by the whole-cell patch-clamp method. Thrombin increased peak I Na amplitude in an activity-dependent manner, from 1 to 100 U/mL, with an apparent EC 50 of 91Ϯ16 U/mL. When tested at 32 U/mL, thrombin-increased I Na was abolished by tetrodotoxin (50 mol/L). Thrombin effects on I Na were reversible and repeatable, and 100 U/mL doubled peak I Na amplitude. Thrombin (32 U/mL) shifted I Na activation to hyperpolarized potentials without affecting steady-state inactivation, producing unusually large increases in window current. Hirudin (320 U/mL) or haloenol lactone suicide substrate (10 mol/L) failed to significantly affect these effects of thrombin. In current-clamped cardiomyocytes, thrombin (32 U/mL) depolarized resting membrane potential by 10 mV. Conclusions-Facilitation of VGSC activation causing large increases in window current is a major mechanism by which thrombin may promote ischemic sodium loading and injury.

Recently, we reported indirect evidence that plasma membrane Ca2+-ATPase (PMCA) can mediate B-type Ca2+ channels of cardiac myocytes. In the present study, in order to bring more direct evidence, purified PMCA from human red blood cells (RBC) was reconstituted into giant azolectin liposomes amenable to the patch-clamp technique. Purified RBC PMCA was used because it is available pure in larger quantity than cardiac PMCA. The presence of B-type Ca2+ channels was first investigated in native membranes of human RBC. They were detected and share the characteristics of cardiac myocytes. They spontaneously appeared in scarce short bursts of activity, they were activated by chlorpromazine (CPZ) with an EC50 of 149 mmole/l or 1 mmole/l vanadate, and then switched off by 10 mmole/l eosin or dose-dependently blocked by 1-5 mmole/l ATP. Independent of membrane potential, the channel gating exhibited complex patterns of many conductance levels, with three most often observed conductance levels of 22, 47 and 80 pS. The activation by vanadate suggests that these channels could play a role in the influx of extracellular Ca2+ involved in the vanadate-induced Gardos effect. In PMCA-reconstituted proteoliposomes, nearly half of the ATPase activity was retained and clear "channel-like" openings of Ba2+- or Ca2+-conducting channels were detected. Channel activity could be spontaneously present, lasting the patch lifetime or, when previously quiescent, activity could be induced by application of 50 mmole/l CPZ only in presence of 25 U/ml calmodulin (CaM), or by application of 1 mmole/l vanadate alone. Eosin (10 mmole/l) and ATP (5 mmole/l) significantly reduced spontaneous activity. Channel gating characteristics were similar to those of RBC, with main conductance levels of 21, 40 and 72 pS. The lack of direct activation by CPZ alone might be attributed to a purification-induced modification or absence of unidentified regulatory component(s) of PMCA. Despite a few differences in results between RBC and reincorporated PMCA, most probably attributable to the decrease in ATPase activity following the procedure of reincorporation, the present experimental conditions appear to reveal a channel-mode of the PMCA that shares many similarities with the B-type Ca2+ channel.

We examined whether mutation of the ␦-sarcoglycan gene, which causes dilated cardiomyopathy, also alters the vascular smooth muscle cell (VSMC) phenotype and arterial function in the Syrian hamster CHF 147. Thoracic aorta media thickness showed marked variability in diseased hamsters with zones of atrophy and hypertrophied segments. CHF-147 VSMCs displayed a proliferating/"synthetic" phenotype characterized by the absence of the smooth muscle myosin heavy chain SM2, dystrophin, and Ca 2؉ -handling proteins, and the presence of cyclin D1. In freshly isolated VSMCs from CHF 147 hamsters, voltage-independent basal Ca 2؉ channels showed enhanced activity similar to that in proliferating wild-type (WT) cells. The transcription factor NFAT (nuclear factor of activated T cells) was spontaneously active in freshly isolated CHF 147 VSMCs, as in proliferating VSMCs from WT hamsters. Mibefradil inhibited B-type channels, NFAT activity, and VSMC proliferation. CHF 147 hamsters had abundant apoptotic cells distributed in patches along the aorta, and clusters of inactive mitochondria were observed in 25% of isolated CHF 147 cells, whereas no such clusters were seen in WT cells. In conclusion, mutation of the ␦-sarcoglycan gene increases plasma membrane permeability to Ca 2؉ , activates the Ca Disruption of the plasma membrane-associated sarcoglycan-sarcospan complex as a result of genetic defects causes muscular dystrophy and/or cardiomyopathy in humans (limb-girdle muscular dystrophy).1 There are six sarcoglycan family members: ␣-, ␤-, ␥-, ␦-, -, and -sarcoglycan.2 In hamster and mouse models, ␦-sarcoglycan gene deletion results in myopathy of cardiac and skeletal muscles, with focal areas of necrosis 3-5 and autophagic cardiomyocyte death. 6 Most of the studies on ␦-sarcoglycandeficient animals have been conducted on skeletal and cardiac muscles. The few studies on smooth muscle concerned the vasospasm of coronary arteries, but there are no data on the peripheral vessels. Sarcoglycans are transmembrane components of the dystrophin-glycoprotein complex, which links the cytoskeleton to the extracellular matrix.7 At the cellular level, disruption of the dystrophinglycoprotein complex leads to increased permeability to divalent cations through channel-blocker-sensitive pathways and entry of calcium via nonspecific cation channels. 8 -11 The mechanisms of this enhanced Ca 2ϩ influx are not fully understood, but changes in the activity of several Ca 2ϩ channels have been described in dystrophin-deficient myocytes.12-15 Dystrophin, through PDZ domain-containing adaptor proteins known as syntrophins, can link the cytoskeleton to various membrane proteins carrying a PDZ domain, including ion channels. 16 This cytoskeleton-ion channel interaction contributes to receptor/channel localization and to the regulation of voltage-, ligand-, and storeoperated ion channels. Indeed, restoration of functional dystrophin-sarcoglycan complex formation by gene transfer of minidystrophin or ␦-sarcoglycan normalizes ion channel function in dystroph...

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The present study demonstrates that B-type Ca2+ channels observed in rat ventricular myocytes markedly reacted to agents known to affect the ion-motive plasma membrane Ca2+-ATPase (PMCA) pump. Chlorpromazine (CPZ)-activated B-type Ca2+ channels were completely blocked by internal application of PMCA pump inhibitors, namely La3+ (100 microm), eosin (10 microm) and AIF(3) (100 microm). Calmodulin (50 U/ml), the main endogenous positive regulator of PMCA, was unable to activate but significantly reduced CPZ-activated B-type channel activity. In the same manner, ATP (1 and 4 mm), the main energizing substrate of PMCA, was able to reversibly and significantly reduce this activity in a dose-dependent manner. Interestingly, anti-PMCA antibody 5F10, but not anti-Na/K ATPase antibody (used as a negative control) induced a marked Ba2+-conducting channel activity that shared the same characteristics with that of CPZ-activated B-type channels. 5F10-Activated channels were mostly selective towards Ba2+ , mainly had three observed conductance levels (23, 47 and 85 pS), were observed with a frequency of about 1 out of 5 membrane patches and were completely blocked by 10 microm eosin. These results suggest that B-type Ca2+ channels are some form of the PMCA pump.

After the thrombus formation in cardiac cavities or coronaries, the serine protease thrombin is produced and can therefore reach the myocardial tissue by the active process of extravasation and binds to the G protein-coupled protease-activated receptor-1 (PAR1) expressed in human myocardium. The role of PAR1 was investigated in the thrombin effect on sodium current (I Na ). I Na was recorded in freshly isolated human atrial myocytes by the whole-cell patch-clamp method. Action potentials (AP) were recorded in guinea pig ventricular tissue by the conventional glass microelectrode technique. Thrombinactivated PAR1 induced a tetrodotoxin-blocked persistent sodium current, I NaP , in a concentration-dependent manner with an apparent EC 50 of 28 U/ml. The PAR1 agonist peptide SFLLR-NH 2 (50 M) was able to mimic PAR1-thrombin action, whereas PAR1 antagonists N 3 -cyclopropyl-7-((4-(1-methylethyl)-phenyl)methyl)-7H-pyrrolo(3,2-f)quinazoline-1,3-diamine (SCH 203099; 10 M) and 1-(3,5-di-tert-butyl-4-hydroxy-phenyl)-2-[3-(3-ethyl-3-hydroxy-pentyl)-2-imino-2,3-dihydro-imidazol-1-yl]-ethanone (ER 112787) (1 M), completely inhibited it. The activated PAR1 involves the calcium-independent phospholipase-A 2 signaling pathway because two inhibitors of this cascade, bromoenol lactone (50 M) and haloenol lactone suicide substrate (50 M), block PAR1-thrombin-induced I NaP . As a consequence of I NaP activation, in guinea pig right ventricle papillary muscle, action potential duration (APD) were significantly increased by 20% and 15% under the respective action of 32 U/ml thrombin and 50 M SFLLR-NH 2 , and these increases in APD were prevented by 1 M tetrodotoxin or markedly reduced by application of 1 M SCH 203099 or ER 112787. Thrombin, through PAR1 activation, increases persistent component of the Na ϩ current resulting in an uncontrolled sodium influx into the cardiomyocyte, which can contribute to cellular injuries observed during cardiac ischemia.

Episodic release of GnRH is essential for reproductive function. In vitro studies have established that this episodic release is an endogenous property of GnRH neurons and that GnRH secretory pulses are associated with synchronization of GnRH neuron activity. The cellular mechanisms by which GnRH neurons synchronize remain largely unknown. There is no clear evidence of physical coupling of GnRH neurons through gap junctions to explain episodic synchronization. However, coupling of glial cells through gap junctions has been shown to regulate neuron activity in their microenvironment. The present study investigated whether glial cell communication through gap junctions plays a role in GnRH neuron activity and secretion in the mouse. Our findings show that Glial Fibrillary Acidic Protein-expressing glial cells located in the median eminence in close vicinity to GnRH fibers expressed Gja1 encoding connexin-43. To study the impact of glial-gap junction coupling on GnRH neuron activity, an in vitro model of primary cultures from mouse embryo nasal placodes was used. In this model, GnRH neurons possess a glial microenvironment and were able to release GnRH in an episodic manner. Our findings show that in vitro glial cells forming the microenvironment of GnRH neurons expressed connexin-43 and displayed functional gap junctions. Pharmacological blockade of the gap junctions with 50 μM 18-α-glycyrrhetinic acid decreased GnRH secretion by reducing pulse frequency and amplitude, suppressed neuronal synchronization and drastically reduced spontaneous electrical activity, all these effects were reversed upon 18-α-glycyrrhetinic acid washout.