Effects of endothelin-1 on K(+) currents from rat ventricular myocytes.

It has been suggested that the positive inotropic effect of the vasoactive peptide hormone, endothelin-1 (ET-1), involves inhibition of cardiac K(+) currents. In order to identify the K(+) currents modulated by ET-1, the outward K(+) currents of isolated rat ventricular myocytes were investigated using whole-cell patch-clamp recording techniques. Outward currents were elicited by depolarisation to +40 mV for 200 ms from the holding potential of -60 mV. Currents activated rapidly, reaching a peak (I(pk)) of 1310 +/- 115 pA and subsequently inactivating to an outward current level of 1063 +/- 122 pA at the end of the voltage-pulse (I(late)) (n = 11). ET-1 (20 nM) reduced I(pk) by 247.6 +/- 60.7 pA (n = 11, P < 0.01) and reduced I(late) by 323.2 +/- 43.9 pA (P < 0.001). The effects of ET-1 were abolished in the presence of the nonselective ET receptor antagonist, PD 142893 (10 microM, n = 5). Outward currents were considerably reduced and the effects of ET-1 were not observed when K(+) was replaced with Cs(+) in the experimental solutions; this indicates that ET-1 modulated K(+)-selective currents. A double-pulse protocol was used to investigate the inactivation of the currents. The voltage-dependent inactivation of the currents from potentials positive to -80 mV was fitted by a Boltzmann equation revealing the existence of an inactivating transient outward component (I(to)) and a noninactivating steady-state component (I(ss)). ET-1 markedly inhibited I(ss) by 43.0 +/- 3.8% (P < 0.001, n = 7) and shifted the voltage-dependent inactivation of I(to) by +3.3 +/- 1.2 mV (P < 0.05). Although ET-1 had little effect on the onset of inactivation of the currents elicited from a conditioning potential of -70 mV, the time-independent noninactivating component of the currents was markedly inhibited. In conclusion, the predominant effect of ET-1 was to inhibit a noninactivating steady-state background K(+) current (I(ss)). These results are consistent with the hypothesis that I(ss) inhibition contributes to the inotropic effects of ET-1.     

Indo-1 binding to protein in permeabilized ventricular myocytes alters its spectral and Ca binding properties.

We have examined the binding of the fluorescent Ca indicator indo-1 to cellular protein in permeabilized ventricular myocytes and also to soluble and particulate myocyte protein. Using either a filtration technique or equilibrium dialysis, and conditions similar to those in a cardiac myocyte patch clamped with 100 microM indo-1 in the patch pipette, we found that 72% of the total indo-1 was bound to myocyte protein at a protein concentration of 100 mg/ml. This corresponds to a binding of 3.8 +/- 0.5 nmol indo-1/mg protein. Separation of the myocyte protein into a soluble and a particulate fraction showed that 63% of the bound indo-1 was bound to soluble protein, corresponding to a binding of 3.22 +/- 0.99 nmol/mg, whereas 37% of the bound indo-1 was bound to particulate protein (0.85 +/- 0.14 nmol/mg) at a low [Ca] (pCa approximately 9). Binding of indo-1 in permeabilized myocytes was approximately 60% higher at a saturating Ca concentration (pCa = 3), than under Ca free conditions (1 mM EGTA). Simultaneous measurements of free [Ca] with a Ca selective electrode and indo-1 fluorescence showed that, the dissociation constant (Kd) for Ca was increased 4-5 fold in the presence of permeabilized myocytes as compared to the value obtained in vitro. In agreement with the binding experiments we estimate that the true Kd and the apparent Kd (using ratiometric measurements) for Ca binding to indo-1 are increased approximately four fold, at a myocyte protein concentration of 100 mg/ml.     

Contractile regulation by overexpressed ETA requires intact T tubules in adult rat ventricular myocytes

Endothelin (ET)-1 regulates the contractility and growth of the heart by binding G protein-coupled receptors of the ET type A receptor (ET(A))/ET type B (ET(B)) receptor family. ET(A), the predominant ET-1 receptor subtype in myocardium, is thought to localize preferentially within cardiac T tubules, but the consequences of mislocalization are not fully understood. Here we examined the effects of the overexpression of ET(A) in conjunction with T-tubule loss in cultured adult rat ventricular myocytes. In adult myocytes cultured for 3 to 4 days, the normally robust positive inotropic effect (PIE) of ET-1 was lost in parallel with T-tubule degeneration and a decline in ET(A) protein levels. In these T tubule-compromised myocytes, an overexpression of ET(A) using an adenoviral vector did not rescue the responsiveness to ET-1, despite the robust expression in the surface sarcolemma. The inclusion of the actin polymerization inhibitor cytochalasin D (CD) during culture prevented gross morphological changes including a loss of T tubules and a rounding of intercalated discs, but CD alone did not rescue the responsiveness to ET-1 or prevent ET(A) downregulation. The rescue of a normal PIE in 3- to 4-day cultured myocytes required both an increased expression of ET(A) and intact T tubules (preserved with CD). Therefore, the activation of ET(A) localized in T tubules was associated with a strong PIE, whereas the activation of ET(A) in surface sarcolemma was not. The results provide insight into the pathological cardiac conditions in which ET(A) is upregulated and T-tubule morphology is altered.     

Impulse conduction and gap junctional remodelling by endothelin-1 in cultured neonatal rat ventricular myocytes

Endothelin-1 (ET-1) is an important contributor to ventricular hypertrophy and failure, which are associated with arrhythmogenesis and sudden death. To elucidate the mechanism(s) underlying the arrhythmogenic effects of ET-1 we tested the hypothesis that long-term (24 hrs) exposure to ET-1 impairs impulse conduction in cultures of neonatal rat ventricular myocytes (NRVM). NRVM were seeded on micro-electrode-arrays (MEAs, Multi Channel Systems, Reutlingen, Germany) and exposed to 50 nM ET-1 for 24 hrs. Hypertrophy was assessed by morphological and molecular methods. Consecutive recordings of paced activation times from the same cultures were conducted at baseline and after 3, 6 and 24 hrs, and activation maps for each time period constructed. Gap junctional Cx43 expression was assessed using Western blot and confocal microscopy of immunofluorescence staining using anti-Cx43 antibodies. ET-1 caused hypertrophy as indicated by a 70% increase in mRNA for atrial natriuretic peptide ( P < 0.05), and increased cell areas ( P < 0.05) compared to control. ET-1 also caused a time-dependent decrease in conduction velocity that was evident after 3 hrs of exposure to ET-1, and was augmented at 24 hrs, compared to controls ( P < 0.01). ET-1 increased total Cx43 protein by ∼40% ( P < 0.05) without affecting non- phosphorylated Cx43 (NP-Cx43) protein expression. Quantitative confocal microscopy showed a ∼30% decrease in the Cx43 immunofluorescence per field in the ET-1 group ( P < 0.05) and a reduced field stain intensity ( P < 0.05), compared to controls. ET-1-induced hypertrophy was accompanied by reduction in conduction velocity and gap junctional remodelling. The reduction in conduction velocity may play a role in ET-1 induced susceptibility to arrhythmogenesis.     

血管紧张素II诱导培养的成年大鼠心肌细胞凋亡

研究血管紧张素Ⅱ（ＡｎｇⅡ）诱导培养的成年大鼠心肌细胞（ＡＲＶＭｓ）凋亡。酶灌流消化法分离培养ＡＲＶＭｓ,不同处理后,光镜观察形态改变,琼脂糖凝胶电泳定性分析ＤＮＡ降解程度。结果发现培养的ＡＲＶＭｓ经ＡｎｇⅡ１０μｍｏｌ／Ｌ处理４８ｈ后,大部分细胞变圆,胞浆浓缩;电泳显示核酸断裂片段“梯形”结构,上述改变在７２ｈ更为明显。上述作用可被氯沙坦、维拉帕米和ｓｔａｕｒｏｓｐｏｒｉｎｅ所取消。这表明Ａｎｇ     

Negative inotropic effects of angiotensin II, endothelin-1 and phenylephrine in indo-1 loaded adult mouse ventricular myocytes

Angiotensin II (Ang II). endothelin-1 (ET-1) and phenylephrine are receptor agonists that share the signal transduction acting through acceleration of phosphoinositide hydrolysis in the heart. Because the regulation of myocardial contractility induced by these receptor agonists shows a wide range of species-dependent variation among experimental animals, we carried out experiments to elucidate the mechanism of contractile regulation induced by these agents in mice which are employed currently more as transgenic models. Effects of Ang II, ET-1 and phenylephrine on cell shortening and Ca2+ transients were investigated in single ventricular myocytes loaded with indo-1/AM. Ang II (10(-8), 10(-7) M), ET-1 (10(-10), 10(-9) M) and phenylephrine (10(-6), 10(-5) M in the presence of the beta-adrenoceptor antagonist timolol) decreased the cell shortening [Ang II: 58.4+/-9.03 (n = 8), 50.3+/-11.90% (n = 6); ET-1: 48.4+/-8.27, 31.2+/-6.45% (n = 5); phenylephrine: 45.7+/-11.60, 28.7+/-5.89% (n = 5)]. By contrast, the amplitude of Ca2+ transients was not significantly influenced by these agonists. The selective protein kinase C inhibitor chelerythrine at 10(-6) M significantly inhibited the decrease in cell shortening induced by these receptor agonists. These results indicate that Ang II, ET-1 and phenylephrine elicit a negative inotropic effect with insignificant alteration of Ca2+ transients, which may be mainly mediated by activation of protein kinase C in mouse ventricular cardiomyocytes.     

Matrix metalloproteinases mediate beta-adrenergic receptor-stimulated apoptosis in adult rat ventricular myocytes

Changes in the synthesis and activity of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are associated with myocardial remodeling. Here we measured the expression and activity of MMPs and TIMPs, and tested the hypothesis that increased MMP activity plays a proapoptotic role in -adrenergic receptor (-AR)-stimulated apoptosis of adult rat ventricular myocytes (ARVMs). -AR stimulation (isoproterenol, 24 h) increased mRNA levels of MMP-2 and TIMP-1 while it decreased TIMP-2 mRNA levels as analyzed by real-time PCR. Western blot analysis, immunocytochemical analysis, in-gel zymography, and MMP-2 activity assay confirmed -AR-stimulated increases in MMP-2 protein levels and activity. Inhibition of MMPs using GM-6001 (a broad-spectrum inhibitor of MMPs), SB3CT (inhibitor of MMP-2), and purified TIMP-2 inhibited -AR-stimulated apoptosis as determined by TdT-mediated dUTP nick end labeling staining. Treatment with active MMP-2 alone increased the number of apoptotic cells. This increase in MMP-2-mediated apoptosis was inhibited by GM-6001 and SB3CT pretreatment. Coimmunoprecipitation studies indicated increased physical association of MMP-2 with ₁-integrins after -AR stimulation. Inhibition of MMP-2 using SB3CT or stimulation of ₁-integrin signaling using laminin inhibited the increased association of MMP-2 with ₁-integrins. -AR stimulation increased poly-ADP-ribose-polymerase cleavage, which was inhibited by inhibition of MMP-2. These data suggest the following: 1) -AR stimulation increases MMP-2 expression and activity and inhibits TIMP-2 expression; 2) inhibition of MMPs, most likely MMP-2, inhibits -AR-stimulated apoptosis; and 3) the apoptotic effects of MMP-2 may be mediated, at least in part, via its interaction with ₁ integrins and poly-ADP-ribose-polymerase cleavage. integrins; poly-ADP-ribose-polymerase     

Gs and Gi coupling of adrenomedullin in adult rat ventricular myocytes

Adrenomedullin (ADM) acts as an autocrine or a paracrine factor in the regulation of cardiac function. The intracellular mechanisms involved in the direct effect of ADM on adult rat ventricular myocytes (ARVMs) are still to be elucidated. In ARVMs from normal rats, ADM produced an initial (< 30 min) increase in cell shortening and Ca2+ transients and a marked decrease in both on prolonged incubation (> 1 h). Both effects were sensitive to ADM antagonist ADM-(22-52). Treatment with SQ-22536, an inhibitor of adenylate cyclase, blocked the positive inotropic effect of ADM and potentiated its negative inotropic effect. The negative inotropic effect was sensitive to inhibition by pertussis toxin (PTX), an inhibitor of Gi proteins and KT-5720, an inhibitor of PKA. The observations suggest a switch from Gs-coupled to PTX-sensitive, PKA-dependent Gi coupling by ADM in ARVMs. The ADM-mediated Gi-signaling system involves cAMP-dependent pathways because SQ-22536 further increased the negative inotropic actions of ADM. Also, because ADM is overproduced by ARVMs in our rat model of septic shock, ARVMs from LPS-treated rats were subjected to treatment with ADM-(22-52) and PTX. The decrease in cell shortening and Ca2+ transients in LPS-treated ARVMs could be reversed back with ADM-(22-52) and PTX. This indicates that ADM plays a role in mediating the negative inotropic effect in LPS-treated ARVM through the activation of Gi signaling. This study delineates the intracellular pathways involved in ADM-mediated direct inotropic effects on ARVMs and also suggests a role of ADM in sepsis.     

Lipopolysaccharide induces apoptosis in adult rat ventricular myocytes via cardiac AT(1) receptors

Lipopolysaccharide (LPS) from gram-negative bacteria circulates in acute, subacute, and chronic conditions. It was hypothesized that LPS directly induces cardiac apoptosis. In adult rat ventricular myocytes (isolated with depyrogenated digestive enzymes to minimize tolerance), LPS (10 ng/ml) decreased the ratio of Bcl-2 to Bax at 12 h; increased caspase-3 activity at 16 h; and increased annexin V, propidium iodide, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining at 24 h. Apoptosis was blocked by the caspase inhibitor benzyloxycarbonyl-valine-alanine-aspartate fluoromethylketone (Z-VAD-fmk), captopril, and angiotensin II type 1 receptor (AT(1)) inhibitor (losartan), but not by inhibitors of AT(2) receptors (PD-123319), tumor necrosis factor-alpha (TNFRII:Fc), or nitric oxide (N(G)-monomethyl-L-arginine). Angiotensin II (100 nmol/l) induced apoptosis similar to LPS without additive effects. LPS in vivo (1 mg/kg iv) increased apoptosis in left ventricular myocytes for 1-3 days, which dissipated after 1-2 wk. Losartan (23 mg. kg(-1). day(-1) in drinking water for 3 days) blocked LPS-induced in vivo apoptosis. In conclusion, low levels of LPS induce cardiac apoptosis in vitro and in vivo by activating AT(1) receptors in myocytes.     

A mathematical model of action potential heterogeneity in adult rat left ventricular myocytes.

Mathematical models were developed to reconstruct the action potentials (AP) recorded in epicardial and endocardial myocytes isolated from the adult rat left ventricle. The main goal was to obtain additional insight into the ionic mechanisms responsible for the transmural AP heterogeneity. The simulation results support the hypothesis that the smaller density and the slower reactivation kinetics of the Ca(2+)-independent transient outward K(+) current (I(t)) in the endocardial myocytes can account for the longer action potential duration (APD), and more prominent rate dependence in that cell type. The larger density of the Na(+) current (I(Na)) in the endocardial myocytes results in a faster upstroke (dV/dt(max)). This, in addition to the smaller magnitude of I(t), is responsible for the larger peak overshoot of the simulated endocardial AP. The prolonged APD in the endocardial cell also leads to an enhanced amplitude of the sustained K(+) current (I(ss)), and a larger influx of Ca(2+) ions via the L-type Ca(2+) current (I(CaL)). The latter results in an increased sarcoplasmic reticulum (SR) load, which is mainly responsible for the higher peak systolic value of the Ca(2+) transient [Ca(2+)](i), and the resultant increase in the Na(+)-Ca(2+) exchanger (I(NaCa)) activity, associated with the simulated endocardial AP. In combination, these calculations provide novel, quantitative insights into the repolarization process and its naturally occurring transmural variations in the rat left ventricle.     

Structural adaptation in adult rabbit ventricular myocytes

The mechanism of induction of cardiomyocyte (CM) dedifferentiation, as seen in chronic hibernating myocardium, is largely unknown. Recently, a cellular model was proposed consisting of long-term cocultures of adult rabbit CMs and cardiac fibroblasts in which typical structural characteristics of hibernation-like dedifferentiation could be induced. Only CMs in close contact with fibroblasts underwent these changes. In this study, we further investigated the characteristics of the fibroblast-CM interaction to seek for triggers and phenomena involved in CM dedifferentiation. Adult rabbit CMs were cocultured with cardiac or 3T3 fibroblasts. Heterocellular interactions and the structural adaptation of the CMs were quantified and studied with vital microscopy and electron microscopy. Immunocytochemical analysis of several adhesion molecules, i.e., N-cadherin, vinculin, β1-integrin, and desmoplakin, were examined. Upon contact with CMs, fibroblasts attached firmly and pulled the former cells, resulting in anisotropic stretch. Quantification of the attachment sites revealed a predominant binding of the fibroblast to the distal ends of the CM in d 1 cocultures and a shift towards the lateral sides of the CMs on d 2 of coculture, suggesting a redistribution of CM membrane proteins. Immunocytochemical analysis of cell adhesion proteins showed that these were upregulated at the heterocellular contact sites. Addition of autologous and nonautologous fibroblasts to the CM culture similarly induced a progressive and accelerated structural adaptation of the CM. Dynamic passive stretch invoked by the fibroblasts and/or intercellular communication involving cell adhesion molecule expression at the interaction sites may play an important role in the induction of hibernation-like dedifferentiation of the cocultured adult rabbit CMs. These authors contributed equally to this study.     

Down-regulation and recovery of endothelin-1 binding and signaling in rat cardiomyocytes.

Down-regulation and recovery of endothelin (ET) receptors and of ET-dependent phosphoinositide-specific phospholipase C (PI-PLC) signaling was examined in cultured cardiomyocytes from neonatal rats. Three hours treatment with 5 nM ET-1 decreased surface receptors to 30%, and transduction to 19%, of their respective time-zero values. After extensive washing and a 3 h recovery period surface receptors returned to 74% of the time-zero value, with concomitant recovery of signal transduction to 75% of the time-zero value. The recovery of PI-PLC signaling in these cells is in contrast with a previous report, but consistent with recovery of the receptor complement.     

Post-translational modifications of tubulin and microtubule stability in adult rat ventricular myocytes and immortalized HL-1 cardiomyocytes

Little is known about the subcellular distribution and the dynamics of tubulins in adult cardiac myocytes although both are modified during cardiac hypertrophy and heart failure. Using confocal microscopy, we examined post-translational modifications of tubulin in fully differentiated ventricular myocytes isolated from adult rat hearts, as well as in immortalized and dividing HL-1 cardiomyocytes. Detyrosinated Glu-alpha-tubulin was the most abundant post-translationally modified tubulin found in ventricular myocytes, while acetylated- and delta2-alpha-tubulins were found in lower amounts or absent. In contrast, dividing HL-1 cardiomyocytes exhibited high levels of tyrosinated or acetylated alpha-tubulins. A mild nocodazole treatment (0.1 microM, 1 h) disrupted microtubules in HL-1 myocytes, but not in adult ventricular myocytes. A stronger treatment (10 microM, 2 h) was required to disassemble tubulins in adult myocytes. Glu-alpha-tubulin containing microtubules were more resistant to nocodazole treatment in HL-1 cardiomyocytes than in ventricular myocytes. Endogenous activation of the cAMP pathway with the forskolin analog L858051 (20 microM) or the beta-adrenergic agonist isoprenaline (10 microM) disrupted the most labile microtubules in HL-1 cardiomyocytes. In contrast, isoprenaline (10 microM), cholera toxin (200 ng/ml, a G(S)-protein activator), L858051 (20 microM) or forskolin (10 microM) had no effect on the microtubule network in ventricular myocytes. In addition, intracellular Ca2+ accumulation induced either by thapsigargin (2 microM) or caffeine (10 mM) did not modify microtubule stability in ventricular myocytes. Our data demonstrate the unique stability of the microtubule network in adult cardiac myocytes. We speculate that microtubule stability is required to support cellular integrity during cardiac contraction.     

Characterization of calcium-dependent forms of protein kinase C in adult rat ventricular myocytes

The presence and subcellular localization of the Ca2+-dependent protein kinase C (PKC) isoforms and were investigated in freshly isolated adult rat cardiac ventricular myocytes. PKC activity was measured in cytosolic and particulate fractions prepared from control myocytes and those treated with either phorbol ester (phorbol 12-myristate 13-acetate, PMA) or a permeant synthetic diacylglycerol analog (1-oleoyl-2-acetylglycerol, OAG) in the absence or presence of an inhibitor of diacylglycerol kinase activity, compound R59022. Preliminary studies detected no Ca2+-/phospholipid-dependent histone kinase activity in either subcellular fraction. To reproducibly observe Ca2+-/phospholipid-dependent protein kinase activity, partial purification using a MonoQ HR 5/5 column and the presence of the peptide inhibitor of the cAMP-dependent protein kinase were essential. MonoQ chromatography of cytosolic and particulate fractions resulted in three peaks of Ca2+/phospholipid-dependent protein kinase activity. In the cytosolic fraction a large peak of activity eluted at 230-300 mM NaCl. Isoform-specific antisera indicated both PKC and PKC were present. In the particulate fraction two peak of Ca2+-/phospholipid-dependent protein kinase activity, both containing PKCa immunoreactivity, were observed. The larger peak eluted at 230-300 mM NaCl. In addition, a peak eluting at lower salt concentrations contained a Ca2+-/phospholipid-independent histone kinase activity. This peak of kinase activity contained PKC immunoreactive bands of 80- and 50-kDa. The 80-kDa band was the holoenzyme of PKC whereas the band of lower molecular mass was likely a proteolytic fragment. In both cytosolic and particulate fractions, the peak of kinase activity eluting at 230-300 mM NaCl contained PKC in the form of an 80-kDa doublet; this suggested the presence of autophosphorylated PKC. Incubation of the myocytes with PMA, but not OAG, resulted in translocation of PKC from the cytosolic to the particulate fraction. Curiously, a transient decrease in PKC activity was observed in both subcellular fractions following treatment with either OAG or ethanol (1%). Results from this study show that freshly isolated adult rat cardiac ventricular myocytes contain both PKC and PKC, and that these isoforms translocate to the particulate fraction in response to treatment with PMA, but not OAG. (Mol Cell Biochem 166: 11-23, 1997)     

Rate of diastolic Ca release from the sarcoplasmic reticulum of intact rabbit and rat ventricular myocytes.

The sarcoplasmic reticulum (SR) of cardiac myocytes loses Ca during rest. In the present study, we estimated the rest-dependent unidirectional Ca efflux from the SR in intact rabbit and rat ventricular myocytes. We determined the time course of depletion of the SR Ca content (assessed as the amount of Ca released by caffeine) after inhibition of the SR Ca-ATPase by thapsigargin. Before rest intervals in Na-containing, Ca-free solution, a 3-min preperfusion with 0Na,0Ca solution was performed to deplete Nai but keep the SR Ca content constant. The decrease in Nai should stimulate Ca efflux via Na/Ca exchange when Nao is reintroduced. Thapsigargin treatment was limited to the last 2 min of preperfusion with 0Na,0Ca solution to minimize SR Ca loss before addition of Na, while attaining complete block of the SR Ca pump. Total SR Ca content was estimated from the [Ca]i transient evoked by caffeine, taking into account passive cellular Ca buffering. The time constants for SR Ca loss after thapsigargin were 385 and 355 s, whereas the pre-rest SR Ca content was estimated to be 106 and 114 microM (mumol/l nonmitochondrial cell volume) in rabbit and rat myocytes, respectively. The unidirectional Ca efflux from the SR was similar in the two cell types (rabbit: 0.27 microM s-1; rat: 0.32 microM s-1). These values are also comparable with that estimated from elementary Ca release events ("Ca sparks," 0.2-0.8 microM s-1). Thus, resting leak of Ca from SR may be primarily via occasional openings of SR Ca release channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha-adrenergic agonist and endothelin-1 induced intracellular Ca response in the presence of a Ca entry blocker in cultured rat ventricular myocytes

Previously we demonstrated that stimulation of cultured neonatal rat ventricular myocytes by either α₁-adrenergic agonist or endothelin-1 resulted in a rapid formation of total inositolphosphates, although the levels of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate did not rise significantly. The aim of this study was to examine whether stimulation by α₁-adrenergic agonist and endothelin-1 could still elicit phosphatidylinositol cycle mediated intracellular Ca²⁺ mobilization in these cells. The intracellular free Ca²⁺ concentration ([Ca ²⁺]i) was measured by single cell imaging dual wavelength fluorescence microscopy in Fura-2-loaded cardiomyocytes. The interference of agonist induced [Ca²⁺]; responses by the beat to beat variation of [Ca²⁺]i was prevented by arresting the cells with the Ca²⁺ entry blocker diltiazem (10 μM). The [Ca²⁺]i response (expressed as % of baseline ratio of fluorescence intensities of Fura-2 at 340 nm and 380 nm excitation wavelength), induced by phenylephrine (10⁻⁴ M) and endothelin-1 (10⁻⁸ M) was small, up to 20% of baseline after 9–20 min. In contrast, Ca²⁺-influx induced by incubation in Na⁺-free buffer caused a steep increase of [Ca²⁺]; up to 150% of baseline after 30 s.Analysis of single cells following stimulation with phenylephrine or endothelin-1 showed heterogeneity with respect to a rise in [Ca²⁺. However, if rapid Ca²⁺-influx was induced by incubation in Na⁺-free buffer, [Ca²⁺]i responses in individual myocytes occurred homogeneously.It is concluded that the α₁-adrenergic agonist and endothelin-1induced [Ca²⁺]i responses are delayed in time, small and quite heterogeneous among cells. The findings are in agreement with earlier observations which revealed no detectable overall increase of the Ca²⁺ releasing inositolphosphates under these conditions and suggest that other second messengers, such as 1,2-diacylglycerol, are involved in the agonist mediated Ca²⁺ signals.     

Cyclic AMP metabolism in intact rat ventricular cardiac myocytes: Interaction of carbachol with isoproterenol and 3-isobutyl-1-methylxanthine

Experiments were carried out to elucidate the characteristics of regulation of cyclic AMP levels in intact myocardial cells. For this purpose, the influence of isoproterenol, a nonselective cyclic nucleotide phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) and carbachol on cyclic AMP levels was investigated in isolated rat cardiac myocytes. The extent of cyclic AMP accumulation induced by isoproterenol was much less than that produced by IBMX: submaximal concentrations of isoproterenol and IBMX elevated the cyclic AMP level 2.4- and 4.8-fold of the control level, respectively. Both agents in combination increased the cyclic AMP level markedly 48-fold. Carbachol inhibited the cyclic AMP accumulation induced by isoproterenol, IBMX and their combination by 30%, 60% and 80% of the respective response. The extent of inhibition produced by carbachol of the cyclic AMP accumulation induced by IBMX + isoproterenol was smaller than that caused by propranolol, and carbachol produced only a marginal additional inhibitory action to that of propranolol, implying that carbachol does not affect the process of cyclic AMP degradation. The present findings indicate that in intact cardiac myocytes the rate of cyclic AMP degradation catalyzed by PDE may be a crucial process of cyclic AMP turnover. This view is supported by the observations that the inhibitory action of carbachol on the effect of isoproterenol was less than that on the effect of IBMX, and that the inhibitory action of carbachol was markedly enhanced by the simultaneous presence of IBMX.     

Characterization of two distinct depolarization-activated K+ currents in isolated adult rat ventricular myocytes

Depolarization-activated outward K+ currents in isolated adult rat ventricular myocytes were characterized using the whole-cell variation of the patch-clamp recording technique. During brief depolarizations to potentials positive to -40 mV, Ca(2+)-independent outward K+ currents in these cells rise to a transient peak, followed by a slower decay to an apparent plateau. The analyses completed here reveal that the observed outward current waveforms result from the activation of two kinetically distinct voltage-dependent K+ currents: one that activates and inactivates rapidly, and one that activates and inactivates slowly, on membrane depolarization. These currents are referred to here as Ito (transient outward) and IK (delayed rectifier), respectively, because their properties are similar (although not identical) to these K+ current types in other cells. Although the voltage dependences of Ito and IK activation are similar, Ito activates approximately 10-fold and inactivates approximately 30-fold more rapidly than IK at all test potentials. In the composite current waveforms measured during brief depolarizations, therefore, the peak current predominantly reflects Ito, whereas IK is the primary determinant of the plateau. There are also marked differences in the voltage dependences of steady-state inactivation of these two K+ currents: IK undergoes steady-state inactivation at all potentials positive to -120 mV, and is 50% inactivated at -69 mV; Ito, in contrast, is insensitive to steady-state inactivation at membrane potentials negative to -50 mV. In addition, Ito recovers from steady-state inactivation faster than IK: at -90 mV, for example, approximately 70% recovery from the inactivation produced at -20 mV is observed within 20 ms for Ito; IK recovers approximately 25-fold more slowly. The pharmacological properties of Ito and IK are also distinct: 4-aminopyridine preferentially attenuates Ito, and tetraethylammonium suppresses predominantly IK. The voltage- and time-dependent properties of these currents are interpreted here in terms of a model in which Ito underlies the initial, rapid repolarization phase of the action potential (AP), and IK is responsible for the slower phase of AP repolarization back to the resting membrane potential, in adult rat ventricular myocytes.