Diabetes and thyroid hormones affect connexin-43 and PKC-epsilon expression in rat heart atria.

We have examined the changes of intercellular electrical coupling protein connexin-43 (Cx43) and of PKC-epsilon in heart atria of diabetic rats and/or after the treatment with triiodothyronine (T(3)). Diabetes was induced in Wistar-Kyoto rats by streptozotocin (50 mg/kg, i.v.) and atria were examined after 5 (acute stage) and 10 (chronic stage) weeks. T(3) (10 microg/100 g/day) was applied via a gastric tube for the last 10 days prior to the end of the experiments to non-diabetic and to the half of diabetic rats. Expression and phosphorylated status of Cx43, as well as expression of PKC-epsilon, were analyzed by Western blots using mouse monoclonal anti-Cx43 and rabbit polyclonal anti-PKC-epsilon antibodies. We found that the Cx43 expression was significantly increased after the treatment with T(3) and in the acute diabetes. Both in diabetes and after T(3) treatment the phosphorylation of Cx43 isoforms was markedly suppressed compared to the non-diabetic and T(3)-untreated controls. Such a down-regulation was less pronounced in diabetic rats after the T(3)-treatment. The expression of atrial PKC-epsilon was increased in diabetic rats. This increase was suppressed after T(3) administration and the expression was decreased in T(3)-treated non-diabetic rats. We suggest that the reduced Cx43 phosphorylation in diabetic and hyperthyroid rats can deteriorate a cell-to-cell coupling and consequently facilitate a development of atrial tachyarrhythmia in diabetic or hyperthyroid animals.

Phosphatase is responsible for run down, and probably G protein-mediated inhibition of inwardly rectifying K+ currents in guinea pig chromaffin cells.

The mechanism of G protein-mediated inhibition of an inwardly rectifying K+ current (IIR) in adrenal chromaffin cells was investigated using the whole-cell version of the patch clamp technique. In case of recording with use of ATP-containing patch solution, the IIR was well maintained; otherwise, it ran down within 15 min. This run down was not prevented by replacement with adenylyl-imidodiphosphate, a nonhydrolysable analogue of ATP, but was markedly reduced by the addition to the ATP-free solution of 1 microM calyculin A, a specific inhibitor of serine/threonine phosphatase 1 (PP1) and 2A (PP2A). The addition of alkaline phosphatase to the ATP-containing solution facilitated run down of the current, and application of 100 microM H-7, a general kinase inhibitor, reversibly suppressed IIR. These results taken together suggest that inwardly rectifying K+ channels are under the influence of kinase and phosphatase without external signals. Infusion of nonhydrolysable analogues of GTP, guanosine-5'-O-(3-thiophosphate) (GTP gamma S) or guanylyl-imidodiphosphate, through the pipette produced little inward current at -55 mV, but completely inhibited IIR within approximately 5 or 6 min in all cells tested in the presence of 12 microM Mg2+ inside the cell. In contrast, infusion of aluminum fluoride (AlF) complex, another GTP binding (G) protein activator, consistently produced large inward currents, but did not alter IIR noticeably for 15 min in 17% of the cells tested. In the other cells, the inhibition of IIR developed slowly after long latent periods. This inhibitory potency of AlF was not enhanced by an increase in Mg2+ concentrations. Subtraction of the current-voltage relationship before from that noted during the generation of inward current by AlF complex revealed that the inward current diminished progressively with hyperpolarizations, as is the case with a nonselective cation current (INS) induced by a muscarinic agonist. Thus, AlF complex seems to be potent with the generation of INS, but not with IIR inhibition. The addition of 3 microM calyculin A significantly retarded the IIR inhibition by GTP gamma S, whereas that of 1 microM okadaic acid, another inhibitor of PPI and PP2A, markedly prevented the decline of IIR by AIF complex. Our observations suggest that the low potency of AlF complex in inhibiting IIR may be due to interference with phosphatase activity and that the activation of G protein suppresses IIR, probably by enhancing the apparent activity of phosphatase, which may explain run down of the current.

Modulation of cardiac connexin-43 by omega-3 fatty acid ethyl-ester supplementation demonstrated in spontaneously diabetic rats.

Previous data suggest that type 1 diabetes mellitus leads to the deterioration of myocardial intercellular communication mediated by connexin-43 (Cx43) channels. We therefore aimed to explore Cx43, PKC signaling and ultrastructure in non-treated and omega-3 fatty acid (omega-3) treated spontaneously diabetic Goto-Kakizaki (GK) rats considered as type 2 diabetes model. Four-week-old GK and non-diabetic Wistar-Clea rats were fed omega-3 (200 mg/kg/day) for 2 months and compared with untreated rats. Real-time PCR and immunoblotting were performed to determine Cx43, PKC-epsilon and PKC-delta expression. In situ Cx43 was examined by immunohistochemistry and subcellular alterations by electron microscopy. Omega-3 intake reduced blood glucose, triglycerides, and cholesterol in diabetic rats and this was associated with improved integrity of cardiomyocytes and capillaries in the heart. Myocardial Cx43 mRNA and protein levels were higher in diabetic versus non-diabetic rats and were further enhanced by omega-3. The ratio of phosphorylated (functional) to non-phosphorylated Cx43 was lower in diabetic compared to non-diabetic rats but was increased by omega-3, in part due to up-regulation of PKC-epsilon. In addition, pro-apoptotic PKC-delta expression was decreased. In conclusion, spontaneously diabetic rats at an early stage of disease benefit from omega-3 intake due to its hypoglycemic effect, upregulation of myocardial Cx43, and preservation of cardiovascular ultrastructure. These findings indicates that supplementation of omega-3 may be beneficial also in the management of diabetes in humans.

Store-operated Ca2+ entry uncoupled with ryanodine receptor and junctional membrane complex in heart muscle cells.

Store-operated Ca2+ entry (SOCE) is the Ca2+ influx that is activated on depletion of intracellular Ca2+ stores. Although SOCE is found in a variety of cell types, its activation mechanism and molecular identity remain to be clarified. Current experimental results suggest that SOCE channels are activated by direct coupling with Ca2+ release channels on depleted stores. Here we report SOCE in cardiac myocytes, that was prominently sensitive to Zn2+ but resistant to inhibitors for voltage-dependent Ca2+ channels and Na+/Ca2+ exchangers. The SOCE activity may be developmentally regulated, because the SOCE was easily detected during embryonic and neonatal stages but not in mature myocytes from adult hearts. In cardiac myocytes, ryanodine receptor type 2 (RyR-2) is thought to be the sole Ca2+ release channel on the intracellular store, and junctophilin type 2 (JP-2) contributes to formation of the junctional complex between the cell surface and store membranes. Using the knockout mice, we also examined possible involvement of the Ca2+ release channel and junctional membrane complex in cardiac SOCE. Apparently normal SOCE activities were retained in mutant myocytes lacking RyR-2 or JP-2, suggesting that neither the Ca2+ release channel nor junctional membrane complex is involved in activation of cardiac SOCE.

Effect of sphingosylphosphorylcholine on the single channel gating properties of the cardiac ryanodine receptor.

The effects of the lysosphingolipid, sphingosylphosphorylcholine (SPC), on the cardiac ryanodine receptor (RyR) were examined. The open probability of cardiac RyR incorporated in lipid bilayers was decreased by cytoplasmic, but not lumenal side application of micromolar concentrations of SPC. Modification of channel function was characterized by the appearance of a long-lived closed state in addition to the brief channel closings observed in the presence and absence of SPC. Open channel kinetics and ion conduction properties, however, were not altered by this compound. These results suggest that SPC, a putative second messenger derived from sphingomyelin, may regulate Ca(2+) release from the sarcoplasmic reticulum by modifying the gating kinetics of the RyR.

Unique topology of the internal repeats in the cardiac Na+/Ca2+ exchanger.

Hydropathy analysis predicts 11 transmembrane helices in the cardiac Na+/Ca2+ exchanger. Using cysteine susceptibility analysis and epitope tagging, we here studied the membrane topology of the exchanger, in particular of the highly conserved internal alpha-1 and alpha-2 repeats. Unexpectedly, we found that the connecting loop in the alpha-1 repeat forms a re-entrant membrane loop with both ends facing the extracellular side and one residue (Asn-125) being accessible from the inside and that the region containing the alpha-2 repeat is mostly accessible from the cytoplasm. Together with other data, we propose that the exchanger may consist of nine transmembrane helices.

Mechanism of activation of nonselective cation channels by putative M4 muscarinic receptor in guinea-pig chromaffin cells.

1. Mechanisms involved in the generation of nonselective cation currents (INS) by muscarinic agonists in the chromaffin cell were investigated by the perforated patch method. 2. Bath application of muscarine (0.1-30 microM) produced an inward INS with or without a transient outward current at -40 mV, whereas oxotremorine (0.06-60 microM) induced INS alone. Rectangular hyperbolas with EC50s of 2.01 and 0.21 microM were fitted to muscarine- and oxotremorine-induced INSS, respectively, and the maximal amplitude of the former was about 3.4 times larger than that of the latter. 3. In 36% of the cells exposed to Ca(2+)-free solution, muscarine INS was suppressed, being 53% of control 20 min after the perfusion, and in four cells that were incubated with Ca(2+)-free solution for 2 h or more, the INS averaged 44% of that induced subsequently in normal solution. In contrast, muscarine INS was enhanced by about 30% when A-23187 was added to normal solution. 4. W-7 and W-5, calmodulin-related agents, were almost equally potent in inhibiting muscarine INS, whereas compound 5, a potent inhibitor of calmodulin-dependent kinase II (CaM kinase II), produced no evident inhibition. 5. HA1004, a weak kinase C inhibitor, induced a reversible suppression of muscarine INS with an IC50 of 163 microM, whereas H-8, another kinase inhibitor, produced an even small degree of inhibition. Administration of phorbol 12, 13-dibutyrate did not mimic muscarinic stimulation of NS channels; rather, it led to a progressive inhibition of INS and this inhibition was almost complete within 20 min. An inactive phorbol ester had no such effect. 6. The muscarinic antagonists, pirenzepine and AF-DX 116, shifted the dose-response curve for the muscarine INs to the right in a parallel manner. The KDS for pirenzepine and AF-DX 116 were estimated to be 13 nM (95% confidence interval, 11-16 nM) and 365 nM (283-470 nM), respectively.7. These results suggest that muscarine efficiently produces INS, probably through binding to the M4 subtype, that intracellular Ca2+ has a facilitating, but not an essential role in the generation of INs, and that neither CaM kinase II nor protein kinase C is involved.

Fluctuation analysis of nonselective cation currents induced by AIF complex in guinea-pig chromaffin cells.

Properties of aluminium fluoride (AIF) complex-activated nonselective cation (NS) channels in guinea-pig chromaffin cells were investigated using the patch clamp technique. As the membrane potential was hyperpolarized from the holding potential of -55 mV, the AIF-induced nonselective cation current (INS) diminished progressively. With hyperpolarizations to -100 mV or more negative potentials, the AIF.INS almost instantaneously disappeared. The apparent unit conductance of AIF INS was estimated to be 3 pS by fluctuation analysis. The open state probability of AIF-activated NS channels became large with a decrease in concentration of free Mg2+ ions inside the cell and was less than 0.5 at 12 microM Mg2+. It is concluded that NS channels in the chromaffin cell apparently differ from those in smooth muscle cells.

ADP indirectly supports activation of non-selective cation channels by AlF complex in guinea-pig chromaffin cells.

Infusion of the G protein activator, AlF complex, into guinea-pig adrenal chromaffin cells produced an inward non-selective cation current (INS) at -55 mV. Under metabolically suppressed conditions, this current was abolished by removal of ATP from the AlF-containing pipette solution. ADP could substitute for ATP in generation of the current. This supporting action of ADP was eliminated by addition to the AlF solution of the ATP-consuming system (hexokinase and 2-deoxyglucose). A similar generation of INS occurred when AMP and phosphocreatine were simultaneously added to AlF solution, but not when either of the agents was added separately. These results suggest that under conditions of whole-cell current recordings, the machinery for ATP production is preserved and that ADP indirectly supports generation of INS by the AlF complex.

Mg2+-dependent phosphatase as an inhibitory mediator of the nonselective cation current induced by aluminum fluoride in guinea-pig chromaffin cells.

Internal administration of the G protein activator, guanosine-5'-o-(3-thiotriphosphate) (GTP gamma S) or aluminum fluoride (AIF) complex, produced an inward nonselective cation current (INS) at -55 mV. This current was rapidly diminished under conditions of high intracellular Mg2+ ([Mg2+] = 979 microM), the half decay time (T1/2) being 80 to 100 s. As [Mg2+] in AlF solutions decreased from 400 to 12 microM, the maximum amplitude of AlF-induced INS became larger and the current was diminished more slowly. The AlF INS in the presence of 12 microM Mg2+ reversed polarity at about +9 mV, irrespective of the extent of decline. Bath application of muscarine produced a sustained INS in the absence of AlF complex, but in its presence, the overall current comprising a spontaneously developed INS and muscarine-induced INS was rapidly diminished. Addition of vanadate (0.5 mM) to 979 microM Mg2+ -containing AlF solution mimicked the effects of low Mg2+ solution. Inversely, addition of alkaline phosphatase (40 units/ml) to 12 microM Mg2+ AlF solution reproduced the effects of high Mg2+ solution. It is suggested that AlF complex deactivates INS through facilitating an apparent activity of Mg2+ -dependent phosphatase.

Phosphatidylinositol hydrolysis is involved in production of Ca(2+)-dependent currents, but not non-selective cation currents, by muscarine in chromaffin cells.

Whether phosphatidylinositol hydrolysis and a subsequent Ca2+ mobilization are responsible for muscarine-induced transient outward currents (IO) and non-selective cation currents (INS) in the guinea-pig chromaffin cell was investigated using the perforated patch method. IO, but not INS, failed to be reproduced in Ca(2+)-free solution and was markedly reduced by prior exposure to caffeine under Ca(2+)-free conditions or by addition to normal solution of cyclopiazonic acid (CPA), a Ca2+ ATPase inhibitor. Application of CPA in Ca(2+)-free solution, however, suppressed INS by about 50% in 73% of the cells tested. Bath application of 1.5 mM neomycin, a phospholipase C inhibitor, induced the time-dependent decline of IO with near abolition at 20 min or less, whereas it produced a time-independent decrease of INS and an inwardly rectifying K+ current. INS in the presence or absence of neomycin was well fitted to rectangular hyperbolas with the same ED50 of 2.17 microM, but with a 33% smaller maximum amplitude in the former, indicating a non-competitive inhibition by neomycin. We conclude that, while phosphatidylinositol hydrolysis mediates the production of IO, it does not mediate that of INS by muscarine.

Are the antiarrhythmic-defibrillating effects of D-sotalol due to or despite the prolongation of the action potential duration?

These results support our hypothesis that class III compounds, with a positive inotropic effect, increase intercellular coupling and synchronization, mainly by preventing intracellular Ca overload. They act as defibrillating compound, similar to cAMP and adrenaline, most probably due to their so called sympathomimetic effect. In our opinion, their cardioprotective effects, resembling cardioversion, are not related to their ability to prolong APD and ERP. Moreover, we suggest that any compound that possesses these sympathomimetic effects, but without inducing the arrhythmogenic prolongation of APD, may exhibit a potent, safety and more efficient antiarrhythmic - defibrillating ability.

The potentiation of carbachol-induced transient contractions in guinea-pig stomach muscles by a low temperature.

A transient contraction in guinea-pig stomach muscles was induced by carbachol (CCh, 10(-6) M) in a Ca(2+)-free Krebs solution. This CCh-induced transient contraction (CITC) was remarkable from 22 to 26 degrees C, but not so at other temperatures in most preparations. When a second CCh (10(-6) M) was applied to the tissue with rinsing off CCh 5 min after an application of the first CCh for 10 min, no CITC was observed. The peak amplitude of the CITC increased with the CCh concentration dose-dependently, and that of CITC (by 10(-6) M CCh) declined exponentially over time after washing the tissue with a Ca(2+)-free Krebs solution. The decline time constant increased from 4.8 +/- 1.2 min (mean +/- SEM; n = 5) at 35 degrees C to 13.2 +/- 2.5 min (mean +/- SEM; n = 5) at 22 degrees C. Furthermore, the falling phase of CITC (by 10(-6) M CCh) also showed an exponential decay of 7.4 +/- 0.2 s (mean +/- SEM; n = 6) at 35 degrees C in the time constant. This time constant increased to 22.3 +/- 0.3 s (mean +/- SEM; n = 6) at 22 degrees C. From the above results, it is concluded that CITC may be due to the Ca(2+)-release from an intracellular store site while released Ca2+ may be immediately excluded to an extracellular space by the Ca2+ pump of the plasma membrane, but the Ca2+ pump activity may be reduced and in addition the stored Ca2+ is kept for long time by a low temperature, which thus results in an increase in the amplitude and a prolongation of the falling phase of CITC.

The long-lasting contraction induced by transmural stimulation in the longitudinal muscle of the guinea-pig gastric corpus.

The mechanisms involved in the long-lasting contraction induced by transmural stimulation were investigated in the isolated longitudinal muscle of the guinea-pig gastric corpus. Transmural stimulation induced an initial rapid contraction, a subsequent relaxation and a long-lasting contraction which was mimicked closely by the responses to noradrenaline and ATP. The rapid contraction was blocked by tetrodotoxin or by atropine, and the subsequent relaxation was also blocked by tetrodotoxin, but not by atropine. The long-lasting contractions were not inhibited by atropine, phentolamine, and propranolol. However, some of those were partially inhibited by tetrodotoxin and guanethidine, and others were enhanced by tetrodotoxin. The noradrenaline- but not ATP-induced long-lasting contraction was blocked by phentolamine. Furthermore, indomethacin completely abolished the long-lasting contractions induced by transmural stimulation, noradrenaline, and ATP, but did not significantly affect the response to prostaglandin E2. The results indicate that the long-lasting contraction induced by transmural stimulation is mediated through prostaglandins release which may be triggered by neuro-transmitters or by endogenous substances.

Factors involved in the susceptibility of spontaneously hypertensive rats to low K+-induced arrhythmias.

Disorders of intracellular Ca2+ homeostasis and intercellular coupling are thought to be crucial in the initiation and maintenance of malignant arrhythmias. The aim of this study was to investigate possible arrhythmogenic factors in spontaneously hypertensive rats (SHR) as well as their susceptibility to low K+-related arrhythmias. The experiments were performed on isolated hearts of 13 weeks-old SHR and age-matched Wistar Kyoto rats (WKY). Equilibration of the heart by Langendorff perfusion with oxygenated, 37 degrees C warm, standard Krebs solution at a constant pressure was followed by perfusion with low K+ solution for 60 min, unless sustained ventricular fibrillation occurred earlier. Electrocardiogram and epicardial monophasic action potentials (MAPs) were continuously monitored for incidence of arrhythmias and action potential changes. Myocardial tissue was taken for ultrastructural analysis and immunodetection of the main gap junction protein, connexin-43. The results showed that hypertrophic hearts of SHR exhibited prolongation of MAPs and a decrease in phosphorylation of connexin-43. Moreover, they were more prone to low K+-induced early after-depolarisations and ventricular premature beats as well as to connexin-43 and ultrastructural alterations than WKY rats. Consequently, the incidence of ventricular tachycardia (70% vs. 50%) and both transient (50% vs. 25%) and sustained (60% vs. 25%) ventricular fibrillation was higher in SHR than WKY rats. The results suggest that both prolongation of MAP and connexin-43 alterations are important arrhythmogenic factors facilitating arrhythmias in the setting of Ca2+ disorders due to hypokalaemia.

Thyroid hormones suppress epsilon-PKC signalling, down-regulate connexin-43 and increase lethal arrhythmia susceptibility in non-diabetic and diabetic rat hearts.

We examined whether thyroid hormones affect myocardial epsilon-PKC signalling, downstream target substrate, connexin-43 (Cx43) and arrhythmogenesis in non-diabetic and diabetic rats. Diabetes was induced by a single streptozotocin injection (50mg/kg, i.v.). Triiodothyronine (T(3)) was applied by gavage (1microg/kg of body weight for 10 days) to 4 weeks and 9 weeks diabetic and age-matched non-diabetic rats. Western blot analysis of Cx43 and epsilon-PKC, immunofluorescence of Cx43, ultrastructure of cardiomyocytes and myocardial conduction velocity were performed. Isolated perfused heart preparation was used to test ventricular fibrillation susceptibility. T(3) significantly decreased epsilon-PKC expression in non-diabetic and suppressed in diabetic rat heart ventricles. Decline of epsilon-PKC signalling was associated with decrease of Cx43 phosphorylation in diabetic and to a greater extent in non-diabetic rat hearts. However, conduction velocity was significantly decreased in diabetic while enhanced due to T(3) and increased in non-diabetic T(3)-treated rat heart ventricles compared to non-treated. T(3)-induced down-regulation of Cx43 was associated with increased cardiac propensity to ventricular fibrillation. Findings indicate that activation of epsilon-PKC signalling linked with phosphorylation of Cx43 is one of the mechanisms involved in the adaptation of the heart to hyperglycemia. Suppression of epsilon-PKC and Cx43 phosphorylation by T(3) abolish benefit of adaptation rendering the heart prone to lethal arrhythmias.

Cell-to-cell coupling studied by diffusional methods in myocardial cells.

The diffusion of large molecular substances from cell to cell in multicellular and enzymatically isolated cell pairs is described. Permeability of the gap junctional membrane to these molecules and the critical diffusing diameter of the myocardial gap junctional channel are discussed.

Effects of insulin on mammalian cardiac muscle.

Insulin increases the contractile force of myocardium with hyperpolarization and a faster rate of fall of the action-potential plateau. This phenomenon is augmented inthe reduced-K+ or ouabain solution. It is suggested that the insulin-induced positive inotropism can be explained by facilitation of inward movement of Ca2+, which possibly is associated with an increase in outward movement of K+.