Enkephalins have a direct positive inotropic effect on cultured cardiac myocytes.

Enkephalins have peripheral vascular effects, and enkephalinergic innervation of the heart has been reported. To determine whether enkephalins have direct effects on myocardium, we studied the effects of [D-Ala2, Met5]enkephalinamide and [D-Ala2, D-Leu5]enkephalin on amplitude of contraction (measured with an optical-video system) in spontaneously beating monolayer cultures of chicken embryo ventricular cells, a preparation devoid of intact neural elements. [D-Ala2, Met5]enkephalinamide and [D-Ala2, D-Leu5]enkephalin as well as [Met5]- and [Leu5]enkephalin increased contractility in a concentration-dependent manner. The enkephalin-induced maximal contractile effects were 28% and 30% above control, with EC50 values of 0.53 and 0.17 microM for [D-Ala2, Met5]enkephalinamide and [D-Ala2, D-Leu5]enkephalin, respectively. The positive inotropic effect was antagonized by naloxone but not by propranolol, phentolamine, diphenhydramine, or cimetidine. Naloxone alone had no effect on contractility at a concentration (0.1 microM) that blocked positive inotropic effects of [D-Ala2, Met5]enkephalinamide and [D-Ala2, D-Leu5]enkephalin. To demonstrate the presence of opiate receptors, we studied [3H]naloxone binding in homogenates of cultured chicken embryo ventricular cells. Analysis of binding curves under equilibrium conditions indicated that [3H]naloxone bound specifically to membranes of cultured heart cells with KD = 18.5 +/- 5.4 nM and Bmax = 46.8 +/- 11.7 fmol/mg of protein. We conclude that enkephalins exert a direct positive inotropic effect on cultured heart cells, increasing contractile state via specific opiate receptors.     

Endocardial endothelium mediates positive inotropic response to alpha 1-adrenoceptor agonist in mammalian heart

Although the positive inotropic response to alpha 1-agonists has been well documented, the mechanism is incompletely understood. In isolated papillary muscle of cat and rabbit, low physiological concentrations of phenylephrine (PE) (10(-9)-10(-7) M; 35 degrees C) under beta-blockade (propranolol 10(-6) M) and in physiological [Ca2+]o (1.25 mM), significantly increased peak twitch tension (TT) with typical unchanged or slightly prolonged twitch contraction duration. This typical response necessitated the presence of an intact endocardial endothelium (EE) since no inotropic response was observed at or below 10(-7) M PE in muscles where the EE had been damaged by 1 s exposure to 1% Triton X-100 prior to the first PE addition. After Triton-induced EE damage prior to the first PE addition, a direct myocardial inotropic response could be demonstrated, but this response required higher concentrations of PE (10(-6)-10(-3) M) and was significantly less pronounced than in muscles with intact EE. At elevated (2.5 mM) or high (7.5 mM) Ca2+, a concentration dependent inotropic response to PE could be induced at the higher concentrations of PE (10(-6) M or higher), but the inotropic response to low concentrations of PE (10(-7) M or below) was absent even in the presence of an intact EE. In papillary muscles with intact EE at physiological [Ca2+]o of 1.25 mM, the higher concentrations of PE (10(-6) M or higher) destroyed the EE. Indeed, after washing with fresh bathing solution following prolonged exposure to these high concentrations of PE, the baseline twitches resembled twitches in muscles where the EE had been experimentally damaged. Selective destruction of the EE by PE in these conditions with no morphological damage to the subjacent myocardium was confirmed on electron microscopy. A second concentration-response curve to PE was significantly depressed, shifted to the right and with no response at or below 10(-7) M PE. Subsequent successive curves were not significantly different from the second one. At the higher Ca2+ (2.5 mM or 7.5 mM), neither functional nor morphological evidence of damage to the EE was observed regardless of the PE concentration and exposure time; in some muscles EE damage did occur at high Ca2+, however, but only after multiple exposures to high PE each followed by abundant wash. At all concentrations of PE, the alpha 1-antagonist prazosin (10(-5) M) blocked the inotropic response regardless of [Ca2+]o and prevented morphological endothelial damage at 1.25 mM Ca2+.     

Mechanisms for aldosterone and spironolactone-induced positive inotropic actions in the rat heart

Previously, we reported that aldosterone and spironolactone have inotropic effects in the isolated perfused heart. To address the mechanisms underlying these inotropic effects, we examined the effects of aldosterone and spironolactone on isolated cardiac myocyte shortening, intracellular calcium ([Ca+2]i), pHi, and calcium-dependent actinomyosin ATPase activity. Aldosterone significantly increased shortening in cardiac myocytes (8.0+/-1.0 versus 16.0+/-1.3%, P<0.01) but neither diastolic [Ca+2]i (61.0+/-1.1 versus 66.0+/-4.4 nmol/L) nor peak systolic [Ca+2]i (302+/-11 versus 304+/-17 nmol/L) was affected. Spironolactone-increased shortening was also not coupled with changes in peak systolic calcium; however, diastolic calcium was significantly increased by spironolactone. Aldosterone, but not spironolactone, increased pHi from 7.23+/-0.03 to 7.59+/-0.02 (P<0.01); this was completely blocked by coadministration of 100 micromol/L of ethyl-isopropyl amiloride (EIPA), an inhibitor of the Na+/H+ exchanger (P<0.01). Consistent with this finding, aldosterone increased cytosolic sodium concentration ([Na+]i) from 9.2+/-0.15 to 11.4+/-0.2 mmol/L and produced a leftward shift in the pCa ATPase curve (pCa=5.82+/-0.02 versus 6.35+/-0.02, P<0.01) without affecting maximal myosin ATPase activity. Conversely, spironolactone, but not aldosterone, significantly increases maximal actomyosin ATPase activity (837+/-59 versus 355+/-52 nmol inorganic phosphate (P(i)) x min(-1) x g tissue(-1)). Collectively, these data strongly suggest that the inotropic actions of aldosterone and spironolactone are caused by different mechanisms of action. Aldosterone appeared to increase inotropy primarily through increased cytosolic pH, whereas spironolactone increased myosin ATPase calcium sensitivity and diastolic calcium concentration.     

Positive inotropic effect of insulin-like growth factor-1 on normal and failing cardiac myocytes.

OBJECTIVE: The acute administration of growth hormone (GH) or insulin-like growth factor-1 (IGF-1) improves cardiac performance, possibly contributing to the beneficial effects of GH therapy on heart failure (HF). GH can induce the production of IGF-1 and thus the actions of GH may be mediated through its IGF-1 induction. However, these effects have not yet been demonstrated in failing hearts and the cellular basis of GH or IGF-1-induced inotropic effects remains unknown. We examined the direct effects of GH and IGF-1 on the contractile function and intracellular calcium ([Ca2+]i) homeostasis in normal and failing myocytes. METHODS: To determine whether GH and IGF-1 have a direct effect on myocardial contractility and whether the GH/IGF-1-induced effect was the results of changes in Ca2+ activation, cell shortening and [Ca2+]i transient were simultaneously measured in the left ventricular myocyte preparations, isolated from normal and rapid pacing-induced HF dogs. RESULTS: Basal shortening of HF myocytes was reduced by 64% (p < 0.01). In normal and HF myocytes, GH (0.4-40 x 10(-3) IU/ml) had no effect on either cell shortening or [Ca2+]i transients. In normal myocytes, IGF-1 exerted a positive inotropic effect in a time- and dose-dependent manner (25-500 ng/ml), associated with a parallel increase of [Ca2+]i transient amplitude. IGF-1 increased the shortening magnitude in normal (121 +/- 5% increase from baseline, p < 0.05) and HF (118 +/- 4% increase from baseline, p < 0.05) myocytes. It also increased [Ca2+]i transient amplitude in normal and HF cells by 124 +/- 4 and 125 +/- 7%, respectively. The percent increase of cell shortening and [Ca2+]i transient amplitude was comparable between normal and HF myocytes. Furthermore, IGF-1 did not shift the trajectory of the relaxation phase in the phase-plane plots of cell length vs. [Ca2+]i, indicating that it did not change myofilament Ca2+ sensitivity. CONCLUSIONS: In both normal and HF conditions, IGF-1 exerted an acute direct positive inotropic effect in adult cardiac myocytes by increasing the availability of [Ca2+]i to the myofilaments, possibly explaining the beneficial effect of GH on HF.     

EDG1 receptor stimulation leads to cardiac hypertrophy in rat neonatal myocytes

Sphingosine 1 phosphate (S1P), an aminophospholipid, acts extracellularly as a ligand via the specific G protein-coupled receptors of the endothelial differentiation gene (EDG) 1, 3, 5, 6 and 8 receptors family and intracellularly as a second messenger in various cellular types. The aim of this work was to investigate biological activity of S1P in cardiomyocytes with respect to related sphingolipids. S1P was applied for 48 h on rat neonatal cardiomyocytes at 10 nM, 100 nM and 1 microM. S1P induced a concentration-dependent cellular hypertrophy evidenced by an increase in cell size, [3H]-phenylalanine incorporation, protein content and Brain Natriuretic Peptide (BNP) secretion. Among the lipids tested S1P exhibits the lower EC50 (67 nM) followed by dihydro-S1P (107 nM) and sphingosylphosphorylcholine (1.6 microM). The effect of S1P could be related to a stimulation of the EDG1 receptor since we showed that the EDG1 receptor is predominantly expressed at the mRNA and protein levels in rat cardiomyocytes and that specific anti-EDG1 antibodies inhibited the hypertrophic effect induced by S1P. Furthermore the expression level of most other EDG receptors for S1P appeared very low in cardiac myocytes. S1P (100 nM) increased the phosphorylation of p42/44MAPK, p38MAPK, JNK, Akt and p70(S6K), this effect being reversed by inhibitors of their respective phosphorylation which also rescue the hypertrophic phenotype. Finally, S1P stimulated actin stress fibre formation reverted by the Rho inhibitor, the C3 exoenzyme. Altogether, our results show that S1P induces cardiomyocyte hypertrophy mainly via the EDG1 receptor and subsequently via Gi through ERKs, p38 MAPK, JNK, PI3K and via Rho pathway.     

Evidence for the existence of inositol tetrakisphosphate in mammalian heart. Effect of alpha 1-adrenoceptor stimulation

The time course of the effects of phenylephrine (10 mumol/l) on force of contraction and on inositol phosphates in electrically driven left auricles from rat hearts labeled with [3H]inositol was studied. All experiments were performed in the presence of propranolol (1 mumol/l) and LiCl (10 mmol/l). Products measured after separation with high-performance liquid chromatography were inositol 1-phosphate (1-IP1), inositol 1,4-bisphosphate (1,4-IP2), inositol 1,3,4-trisphosphate (1,3,4,-IP3), inositol 1,4,5-trisphosphate (1,4,5-IP3), and inositol 1,3,4,5-tetrakisphosphate (1,3,4,5-IP4). All inositol phosphates increased after stimulation with phenylephrine. 1,4,5-IP3 was the first compound to rise maximally within 30 seconds; this rise was followed by an increase in 1,3,4,5-IP4 and 1,4-IP2 beginning within 2 minutes. The increase in 1,3,4-IP3 and 1-IP1 was slower and did not reach steady state within 15 minutes. The positive inotropic effect of phenylephrine was maximal after 5 minutes. It is concluded that the increase in the presumed second messengers 1,4,5-IP3 and 1,3,4,5-IP4 coincides with the positive inotropic effect after alpha 1-adrenoceptor stimulation. Since the increase in 1,4,5-IP3 precedes the increase in force of contraction, 1,4,5-IP3 may initiate the positive inotropic effect of alpha 1-adrenoceptor agonists and 1,3,4,5-IP4 maintains the increase in force of contraction.     

p38 Mitogen-activated protein kinase mediates a negative inotropic effect in cardiac myocytes

p38 Mitogen-activated protein kinase (MAPK) is one of the most ancient signaling molecules and is involved in multiple cellular processes, including cell proliferation, cell growth, and cell death. In the heart, enhanced activation of p38 MAPK is associated with ischemia/reperfusion injury and the onset of heart failure. In the present study, we investigated the function of p38 MAPK in regulating cardiac contractility and its underlying mechanisms. In cultured adult rat cardiomyocytes, activation of p38 MAPK by adenoviral gene transfer of an activated mutant of its upstream kinase, MKK3bE, led to a significant reduction in baseline contractility, compared with uninfected cells or those infected with a control adenoviral vector (Adv-beta-galactosidase). The inhibitory effect of MKK3bE on contractility was largely prevented by coexpressing a dominant-negative mutant of p38 MAPK or treating cells with a p38 MAPK inhibitor, SB203580. Conversely, inhibition of endogenous p38 MAPK activity by SB203580 rapidly and reversibly enhanced cell contractility in a dose-dependent manner, without altering L-type Ca(2+) currents or Ca(2+)(i) transients. MKK3bE-induced p38 activation had no significant effect on pH(i), whereas SB203580 had a minor effect to elevate pH(i). Furthermore, activation of p38 MAPK was unable to increase troponin I phosphorylation. Thus, we conclude that the negative inotropic effect of p38 MAPK is mediated by decreasing myofilament response to Ca(2+), rather than by altering Ca(2+)(i) homeostasis and that the reduced myofilament Ca(2+) sensitivity is unlikely attributable to troponin I phosphorylation or alterations in pH(i). These findings reveal a novel function of p38 MAPK and shed a new light on our understanding of the coincidence of p38 MAPK activation and the onset of heart failure.     

Introduction: Mechanisms of positive inotropic effects

Summary This review deals with the principal mechanisms which are known to play a role in positive inotropism: 1) The myoplasmic Ca²⁺ concentration may be increased by increases in cyclic AMP. Beside receptor-mediated stimulation (isoprenaline) or direct stimulation (forskolin) of the adenylate cyclase, the cyclic AMP may be increased by phosphodiesterase inhibition; 2) Cyclic AMP-independent activation of Ca²⁺ channels can be brought about by alpha-adrenergic agents (phenylephrine) or so-called calcium agonists; 3) Only a small increase in myoplasmic Na⁺ concentration can greatly enhance the force of contraction by an increase in the intracellular Ca²⁺ concentration. This is possible by inhibition of the Na⁺/K⁺-ATPase (glycosides) or by prolongation of the open state of Na⁺ channels (DPI 201-106); 4) A direct inhibition of the Na⁺/Ca²⁺ exchange has been discussed for amiloride; 5) A prolongation of the action potential induced by K⁺ channel-inhibiting agents such as 4-amino-pyridine may increase the myoplasmic Ca²⁺ concentration by a prolongation of the slow Ca²⁺ inward current; 6) An increased Ca²⁺ sensitivity of the contractile proteins has been demonstrated for a number of compounds in vitro; the contribution of such an effect to the overall positive inotropism is unknown because a calcium sensitizer without any effects on calcium or sodium movements is not yet available.     

alpha1-adrenoceptor stimulation potentiates L-type Ca2+ current through Ca2+/calmodulin-dependent PK II (CaMKII) activation in rat ventricular myocytes

alpha1-Adrenoceptor stimulation (alpha1ARS) modulates cardiac muscle contraction under physiological conditions by means of changes in Ca2+ current through L-type channels (ICa,L) and Ca2+ sensitivity of the myofilaments. However, the cellular mechanisms of alpha1ARS are not fully clarified. In this study, we investigated the role of Ca2+/calmodulin-dependent PK II (CaMKII) in the regulation of ICa,L during alpha1ARS in isolated adult rat ventricular myocytes by using the perforated patch-clamp technique. CaMKII inhibition with 0.5 microM KN-93 abolished the potentiation in ICa,L observed during alpha1ARS by 10 microM phenylephrine. In the presence of PKC inhibitor (10 microM chelerythrine), the potentiation of ICa,L by phenylephrine also disappeared. In Western immunoblotting analysis, phenylephrine (> or =1 microM) increased the amount of autophosphorylated CaMKII (active CaMKII) significantly, and this increase was abolished by CaMKII inhibition or PKC inhibition. Also, we investigated changes in the subcellular localization of active CaMKII by using immunofluorescence microscopy and immunoelectron microscopy. Before alpha1ARS, active CaMKII was exclusively located just beneath the plasmalemma. However, after alpha1ARS, active CaMKII was localized close to transverse tubules, where most of L-type Ca2+ channels are located. From these results, we propose that CaMKII, which exists near transverse tubules, is activated and phosphorylated by alpha1ARS and that CaMKII activation directly potentiates ICa,L in rat ventricular myocytes.     

Effect of positive inotropic agents on myosin isozyme population and mechanical activity of cultured rat heart myocytes.

To examine whether catecholamines have a direct effect on myosin heavy chain expression of heart myocytes or whether they act via an altered work load, myocytes from neonatal rat hearts were cultured in thyroid hormone-free media containing various positive inotropic and chronotropic agents. The velocity and frequency of contraction of the myocytes were monitored using an optoelectronic system. After 3-5 days of culture, myosin isozyme populations, cellular cAMP content, and 2-deoxy-D-glucose uptake of the myocytes were determined. Compared with myocytes cultured in the absence of inotropic agents (32.6 +/- 3.5% V1), the proportion of myosin V1 was significantly (p less than 0.05) increased in the case of 1 microM isoproterenol (48.2 +/- 5.9% V1), 1 microM forskolin (57.1 +/- 11.7% V1), and 1 mM dibutyryl cAMP (79.1 +/- 2.0% V1). Dibutyryl cAMP increased V1 to a similar level as 30 nM triiodothyronine did (70.2 +/- 13.0% V1). Only a small increase was observed in myocytes cultured in the presence of 10 microM phenylephrine (40.4 +/- 8.4% V1), 10 microM ouabain (40.6 +/- 11.9% V1), or 10 microM Bay K 8644 (40.7 +/- 11.7% V1). The agents with a marked effect on myosin heavy chain expression resulted in a higher cAMP content; isoproterenol and forskolin also stimulated 2-deoxy-D-glucose uptake. All agents resulted in a higher velocity of contraction; with the exception of ouabain, frequency of contraction was also increased. A change in Ca2+ concentration in the medium from 1.3 to 2.4 mM resulted in a small increase in V1 (40.7 +/- 5.2% V1) but had the same effect on contraction velocity as dibutyryl cAMP did. Furthermore, 10 nM isoproterenol also increased V1 in myocytes that were arrested with 10 microM verapamil. The increase in V1 in the case of dibutyryl cAMP, isoproterenol, and forskolin is thus most probably not a correlate of the increased mechanical activity but of the high cellular cAMP content.     

iPLA2 inhibitor blocks negative inotropic effect of HIV gp120 on cardiac myocytes

Recent improvements in survival from AIDS have been accompanied by an increased recognition of the potential importance of other manifestations of HIV infection, including cardiomyopathy. Mechanisms responsible for HIV cardiomyopathy are unknown, but may include direct effects of HIV proteins on the heart. We previously provided support for direct effects of HIV proteins by demonstrating a negative inotropic effect of the HIV coat protein, gp120, on isolated adult rat ventricular myocytes (ARVM). We now report that this negative inotropic effect of HIV gp120 is mediated by a signaling pathway involving p38 MAP kinase, iPLA2 and troponin I. Exposure of ARVM to HIV gp120 resulted in maximal activation of iPLA2 by 60 min as reflected in hydrolysis of arachidonyl thiophosphatidylcholine that was completely blocked by the iPLA2 inhibitor, bromoenol lactone (BEL) or the p38 MAP kinase inhibitor, SB203580. The negative inotropic effect of gp120 was blocked by BEL, as well as SB203580. BEL did not block gp120 stimulated phosphorylation of p38 MAP kinase itself, and/or its downstream effectors, ATF2 or MAPKAP2. However, BEL did block gp120-stimulated phosphorylation of troponin I. Thus, the negative inotropic effect of HIV gp120 requires activation of p38 MAP kinase and iPLA2; as well as troponin I phosphorylation. Activation of this novel p38 MAP kinase-iPLA2-troponin I signaling pathway may contribute to HIV cardiomyopathy.     

Central nervous alpha1-adrenoceptor stimulation induces duodenal luminal release of melatonin

Intracerebroventricular (i.c.v.) infusion of the alpha1-adrenoceptor agonist phenylephrine elicits vagal and sympathetic neural stimulation of the bicarbonate secretion by the duodenal mucosa. Melatonin originating from mucosal enterochromaffin cells (EC cells) has been proposed to mediate this centrally elicited stimulation. However, the release of intestinal melatonin has not been studied. Rats were anesthetized with thiobarbiturate, a 12-mm segment of duodenum with intact blood supply was cannulated in situ and bicarbonate secretion titrated by pH-stat. The mean arterial blood pressure was continuously recorded. Melatonin in the duodenal luminal perfusate was determined by high-performance liquid chromatography with electrochemical detection. Intracerebroventricular infusion of phenylephrine (12.2 microM/kg/hr) induced more than 10-fold increase in release of melatonin into the duodenal lumen and an increase in HCO secretion from 7.6 +/- 0.5 to 18.6 +/- 2.1 microEq/cm/hr. The melatonin receptor (MT2 > MT1) antagonist luzindole (600 nM/kg, i.v.) almost abolished the marked rise in bicarbonate secretion induced by i.c.v. phenylephrine but, in contrast, did not affect the release of melatonin. These results strongly suggest that release of melatonin from the mucosa mediates the duodenal secretory response to centrally elicited neural stimulation.     

Beta-arrestin2-mediated inotropic effects of the angiotensin II type 1A receptor in isolated cardiac myocytes

The G protein-coupled receptor kinases (GRKs) and beta-arrestins, families of molecules essential to the desensitization of G protein-dependent signaling via seven-transmembrane receptors (7TMRs), have been recently shown to also transduce G protein-independent signals from receptors. However, the physiologic consequences of this G protein-independent, GRK/beta-arrestin-dependent signaling are largely unknown. Here, we establish that GRK/beta-arrestin-mediated signal transduction via the angiotensin II (ANG) type 1A receptor (AT(1A)R) results in positive inotropic and lusitropic effects in isolated adult mouse cardiomyocytes. We used the "biased" AT(1A)R agonist [Sar(1), Ile(4), Ile(8)]-angiotensin II (SII), which is unable to stimulate G(alpha)q-mediated signaling, but which has previously been shown to promote beta-arrestin interaction with the AT(1A)R. Cardiomyocytes from WT, but not AT(1A)R-deficient knockout (KO) mice, exhibited positive inotropic and lusitropic responses to both ANG and SII. Responses of WT cardiomyocytes to ANG were dramatically reduced by protein kinase C (PKC) inhibition, whereas those to SII were unaffected. In contrast, cardiomyocytes from beta-arrestin2 KO and GRK6 KO mice failed to respond to SII, but displayed preserved responses to ANG. Cardiomyocytes from GRK2 heterozygous knockout mice (GRK2(+/-)) exhibited augmented responses to SII in comparison to ANG, whereas those from GRK5 KO mice did not differ from those from WT mice. These findings indicate the existence of independent G(alpha)q/PKC- and GRK6/beta-arrestin2-dependent mechanisms by which stimulation of the AT(1A)R can modulate cardiomyocyte function, and which can be differentially activated by selective receptor ligands. Such ligands may have potential as a novel class of therapeutic agents.     

Methionine-induced positive inotropic effect in rat heart: possible role of phospholipid N-methylation

Perfusion of isolated rat heart with L-methionine produced a positive inotropic effect that was temporally preceded, as well as accompanied, by an increase of methyl group incorporation into N-methylated phospholipids of the myocardium. Maximal increase in contractile force development was associated with maximal methyl group incorporation. Both parameters showed a dose-related dependence on methionine and correlated positively (r = 0.965) upon regression analysis of the data. The presence of adenosine, L-homocysteine thiolactone and erythro-9-(2-hydroxy-3-nonyl) adenine in the perfusion medium inhibited the positive inotropic effect as well as the incorporation of methyl groups into phospholipids. Cycloleucine, an inhibitor of S-adenosylmethionine synthetase, also reduced the increase in contractility by methionine. Methionine-induced positive inotropic effect could be modulated by varying Ca2+ concentration in the perfusate and was inhibited by ryanodine, a blocker of sarcoplasmic reticular Ca2+ release. These observations indicate that L-methionine may serve as a powerful positive inotropic agent and suggest that phospholipid N-methylation plays an important role in functional activity of rat heart.     

Intracellular sodium and the positive inotropic effect of veratridine and cardiac glycoside in sheep Purkinje fibers

Veratridine is a sodium channel toxin that exerts a powerful positive inotropic effect and prolongs the action potential duration in the heart. To determine the basis of the inotropic action of veratridine and to examine the effects of dissimilar methods of raising intracellular sodium activity on contractility, we measured twitch tension and intracellular sodium activity using sodium-sensitive microelectrodes in stimulated sheep Purkinje fibers exposed to veratridine and in voltage-clamped fibers exposed to veratridine and cardiac glycoside. In stimulated fibers, veratridine (0.1-1 microM) produced coincident increases in intracellular sodium activity, action potential duration, and tension. In voltage-clamped fibers, veratridine (1-2 microM) and acetylstrophanthidin (0.1 microM) raised intracellular sodium activity and tension to a comparable degree. Tetrodotoxin (10 microM) abolished the mechanical, electrophysiological, and ionic changes produced by veratridine. The relationship between intracellular sodium activity and tension in voltage-clamped fibers (n = 6) was indistinguishable for veratridine and acetylstrophanthidin and could be fitted either with a linear function with slopes of 122.8% and 124.2%, respectively, or with a power function with slopes of 4.60 and 4.54, respectively, where the slope represents the exponential power of intracellular sodium activity to which tension is proportional. These results indicate that the positive inotropic action of veratridine is entirely accounted for by accumulation of intracellular sodium, which increases intracellular calcium available for contraction by sodium-calcium exchange. This study is the first direct demonstration that veratridine or any other sodium channel toxin affects intracellular sodium activity and suggests that the inotropic potency of veratridine and cardiac glycoside rely on the same mechanism, namely, elevation of intracellular sodium.     

Protective effect of eicosapentaenoic acid on ouabain toxicity in neonatal rat cardiac myocytes.

Isolated neonatal cardiac myocytes have been utilized as a model for the study of cardiac arrhythmogenic factors. The myocytes respond to the toxic effects of a potent cardiac glycoside, ouabain at 0.1 mM, by an increase in their spontaneous beating rate and a reduction in amplitude of contractions resulting within minutes in a lethal state of contracture. Incubating the isolated myocytes for 3-5 days in culture medium enriched with 5 microM arachidonic acid [20:4 (n-6)] had no effect on the development of lethal contracture after subsequent exposure to 0.1 mM ouabain. By contrast, incubating the myocytes for 3-5 days with 5 microM eicosapentaenoic acid [20:5 (n-3)] completely prevented the toxic effects of ouabain at 0.1 mM. There were no measurable differences in the degree to which ouabain inhibited Na,K-ATPase activity by comparing the control with the arachidonic acid- or the eicosapentaenoic acid-enriched myocytes. No differences in bumetanide-inhibitable 86Rb flux were observed between the three preparations. However, measurements with fura-2 of cytosolic free calcium levels indicated that control and arachidonic acid-enriched myocytes developed toxic cytosolic calcium concentrations of 845 +/- 29 and 757 +/- 64 nM, respectively, on exposure to 0.1 mM ouabain, whereas in eicosapentaenoic acid-enriched myocytes, physiologic calcium levels (214 +/- 29 nM) were preserved. Incubating the myocytes with eicosapentaenoic acid (5 microM) for 3-5 days resulted in a small reduction of arachidonic acid and a small but significant increase of eicosapentaenoic acid in membrane phospholipids of the myocytes.     

Inhibition of protein phosphatase 1 induces apoptosis in neonatal rat cardiac myocytes: role of adrenergic receptor stimulation

The mechanisms that regulate cardiac myocyte apoptosis are not well understood. To study the role of protein phosphatase 1 (PP1) and 2A (PP2A) in apoptosis, we exposed cultured neonatal rat cardiac myocytes to the phosphatase inhibitor okadaic acid (OA). Exposure (18 h) to 100 nM OA (a concentration which inhibits both PP1 and PP2A) decreased the number of adherent cells, caused genomic DNA fragmentation, and increased the percentage of apoptotic cells. These effects did not occur at a lower concentration of OA (1 nM) which is relatively specific for PP2A. Stimulation of α₁- or β-adrenergic receptors with norepinephrine (NE) in the presence of propranolol or prazosin partially blocked OA-induced apoptosis as measured by flow cytometry. Likewise, stimulation of adenylyl cyclase with forskolin reduced OA-induced apoptosis. Conversely, inhibition of protein kinase A with H89 or protein kinase C with chelerethrine potentiated OA-induced apoptosis. OA increased caspase-3 activity, and this effect was reduced by NE. Thus, inhibition of PP1 stimulates apoptosis in NRVM and stimulation of adrenergic receptors protects against OA-induced apoptosis. Received: 28 March 2000 Returned for revision: 2 May 2000 Revision received: 19 May 2000 Accepted: 22 May 2000