Adenosine-sensitive afterdepolarizations and triggered activity in guinea pig ventricular myocytes.

This study examines the cellular basis and specificity of the effects of adenosine on early afterdepolarizations (EADs), delayed afterdepolarizations (DADs), and triggered activity (TA) induced by various drugs with different mechanisms of action. Membrane potential and currents were measured in isolated guinea pig ventricular myocytes. Adenosine (10-100 microM) significantly (p less than 0.05) reduced the amplitude of DADs and suppressed TA induced by isoproterenol (10-50 nM) and forskolin (1 microM) but not those induced by dibutyryl cAMP (1 microM), ouabain (1-5 microM), and 7.2 mM [Ca2+]o. Adenosine also abolished EADs and TA induced by isoproterenol. In contrast, adenosine failed to abolish EADs and TA induced by quinidine (3 microM) or those that occurred spontaneously (i.e., in the absence of drugs). Transient inward current (ITi) was induced on repolarization after 2-second-long single depolarizing voltage steps or after 12-second-long trains of 300-msec depolarizing pulses. Concomitant with the attenuation of DADs, adenosine suppressed ITi caused by isoproterenol and forskolin but not those induced by ouabain, dibutyryl cAMP, and elevated [Ca2+]o. The amplitude of ITi was dependent on the magnitude of the activating voltage step, but the suppression of ITi by adenosine was not. The selective A1-adenosine receptor antagonist N-0861 (9-methyladenine derivative) antagonized the effects of adenosine on afterdepolarizations, ITi, and TA. In myocytes from guinea pigs treated with pertussis toxin, adenosine failed to attenuate DADs and ITi or abolish TA induced by isoproterenol or forskolin. In parallel experiments, isoproterenol (10 nM) raised cellular cAMP from 5.7 +/- 0.2 to 8.1 +/- 0.1 pmol and the selective A1 receptor agonist cyclopentyladenosine (5 microM) reduced it to 6.5 +/- 0.2 pmol (p less than 0.05). Thus, adenosine specifically attenuates afterdepolarizations and abolishes TA by suppressing ITiS that are associated with stimulation of adenylate cyclase via a pertussis toxin-sensitive A1 receptor-mediated action. In conclusion, the response of TA to adenosine may identify a mechanism of afterdepolarization related to stimulation of adenylate cyclase.     

Effect of forskolin, isoproterenol and IBMX on angiotensin converting enzyme and cyclic AMP production by cultured bovine endothelial cells

The production of angiotensin converting enzyme (ACE) is known to be increased by glucocorticoids, thyroid hormones and converting enzyme inhibitors. We have recently reported that active cAMP analogues also stimulate production of the enzyme. The effect of stimulation of adenylate cyclase in cultured endothelial cells or of phosphodiesterase inhibition on ACE production was therefore evaluated. The phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX) (10(-4) M), produced 10.5 +/- 1.3 and 1.3 +/- 0.1 (P less than 0.01 and P greater than 0.1) fold increases in extracellular and cellular cAMP levels and a 1.55 +/- 0.10 (P less than 0.0001) fold increase in ACE accumulation. The adenylate cyclase stimulator, forskolin (0.01-10 microM), acutely stimulated cellular cAMP accumulation in a dose-dependent manner, reaching a 2.8 +/- 0.1-fold increase at 10 microM. After 48 h exposure to 10 microM forskolin, significant increases in cellular (1.90 +/- 0.38-fold increase, P less than 0.0001) and extracellular cAMP (2.35 +/- 0.26-fold increase, P less than 0.0001) were also observed but ACE accumulation was unchanged (108 +/- 10% of control, P greater than 0.5). The beta-adrenoceptor agonist, isoproterenol (1-1000 nM), acutely stimulated cellular cAMP accumulation, with a threshold effect at 10 nM, an ED50 of approximately 30 nM, and a plateau effect of 2.0 +/- 0.13-fold increase by 100 nM. After 48 h exposure to isoproterenol (1 microM), extracellular cAMP levels were increased significantly (1.68 +/- 0.33-fold increase, P less than 0.01) but ACE production was slightly inhibited (83 +/- 7% of control, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of growth in low potassium medium or ouabain on membrane Na,K-ATPase, cation transport, and contractility in cultured chick heart cells

Growth of cultured cells in low potassium medium has been shown to result in an increase in the number of Na,K-ATPase sites. This phenomenon and its physiological and pharmacological consequences were examined in spontaneously beating monolayers of cultured chick heart cells. Growth of cells in 1 mM extracellular potassium, 2 microM ouabain, or 1 microM veratridine for 48 hours caused 60%, 40%, or 20% increases, respectively, in the total number of specific ouabain binding sites measurable in intact cells. Acute exposure of control cells grown in 4 mM to 1 mM extracellular potassium caused elevation of steady state [Na+]i by 37%, while 1 microM veratridine exposure increased [Na+]i by 12%. After 48 hours of growth in 1 mM extracellular potassium, intracellular sodium concentrations declined to near-control levels. In cells grown in low extracellular potassium and then equilibrated with 4 mM potassium for 30 minutes, the positive inotropic effects of 1 mM extracellular potassium and 0.3 microM isoproterenol, expressed as a percent of contractile response to 3.6 mM calcium, were 40 +/- 6% and 37 +/- 5% (means +/- SEM), respectively, in low potassium-grown cells, compared with 63 +/- 8% and 35 +/- 4% in control cells. Growth of cells in low potassium shifted the concentration-effect curve for ouabain to the right. The rapid component of calcium uptake in zero extracellular sodium was significantly lower in low potassium-grown cells than in control cells after equilibration in 1 mM extracellular potassium for 30 minutes. These findings demonstrate that prolonged exposure of cultured heart cells to 1 mM extracellular potassium or ouabain causes induction of additional functional sarcolemmal sodium pump sites. The increased levels of intracellular sodium caused by these interventions appear to be an important determinant of sodium pump site density. The reduced contractile response of cells grown in 1 mM extracellular potassium and ouabain (but not isoproterenol) supports the view that elevated intracellular sodium due to Na,K-ATPase inhibition mediates the positive inotropic response to low extracellular potassium and ouabain, probably via augmented transsarcolemmal sodium-calcium exchange. In addition, our results support a mechanism of inotropic action of digitalis glycosides based on inhibition of the sodium pump rather than altered calcium binding properties of sarcolemmal sites due to cardiac glycoside binding to Na,K-ATPase.     

Compartmentation of cAMP in adult canine ventricular myocytes. Relation to single-cell free Ca2+ transients.

Isolated adult canine ventricular myocytes were used to study the role of compartmentation of cAMP in the diverse functional responses to various drugs that elevate cAMP. Myocytes presented with the beta-agonist isoproterenol accumulated cAMP with a half maximally effective concentration (EC50) of 3.55 x 10(-8) M. Approximately 45% of the total cAMP was recovered in the particulate fraction of digitonin-lysed myocytes under these conditions. With phosphodiesterase inhibition (10 microM isobutylmethylxanthine), isoproterenol-stimulated cAMP production was up to 3.4-fold greater, but the proportion of total cAMP residing in the particulate fraction declined to less than 20%. Similar results were obtained with forskolin, a direct activator of adenylate cyclase. Treatment with isoproterenol shortened the duration at 50% maximum peak height (T 1/2) and increased the peak fluorescence ratio of electrically triggered single-cell free Ca2+ transients in fura-2-loaded canine myocytes. Isoproterenol dose-response curves gave EC50 values of 1.7 x 10(-9) and 4.4 x 10(-9) M for effects on T 1/2 and peak height, respectively. Alterations in peak height and T 1/2 of Ca2+ transients also showed a dose dependency on isobutylmethylxanthine and forskolin. Comparison of myocyte cAMP content with the corresponding changes in free Ca2+ transients demonstrated a close correlation between particulate cAMP and the extent of shortening or increase in peak height of the fura-2 Ca2+ transients (r = 0.92 for each). However, when these two parameters were plotted as a function of total cAMP, the resulting curves were nonlinear and divergent for each agent tested. The results support the hypothesis that differences in responses to agents that augment cAMP can be explained in part by compartmentation of cAMP. Furthermore, Ca2+ mobilization seems to be most affected by cAMP located in the particulate compartment of canine cardiac myocytes.     

Glucagon-like peptide-1 increases cAMP but fails to augment contraction in adult rat cardiac myocytes

The gut hormone, glucagon-like peptide-1 (GLP-1), which is secreted in nanomolar amounts in response to nutrients in the intestinal lumen, exerts cAMP/protein kinase A-mediated insulinotropic actions in target endocrine tissues, but its actions in heart cells are unknown. GLP-1 (10 nmol/L) increased intracellular cAMP (from 5.7+/-0.5 to 13.1+/-0.12 pmol/mg protein) in rat cardiac myocytes. The effects of cAMP-doubling concentrations of both GLP-1 and isoproterenol (ISO, 10 nmol/L) on contraction amplitude, intracellular Ca(2+) transient (CaT), and pH(i) in indo-1 and seminaphthorhodafluor (SNARF)-1 loaded myocytes were compared. Whereas ISO caused a characteristic increase (above baseline) in contraction amplitude (160+/-34%) and CaT (70+/-5%), GLP-1 induced a significant decrease in contraction amplitude (-27+/-5%) with no change in the CaT after 20 minutes. Neither pertussis toxin treatment nor exposure to the cGMP-stimulated phosphodiesterase (PDE2) inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine or the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine nor the phosphatase inhibitors okadaic acid or calyculin A unmasked an ISO-mimicking response of GLP-1. In SNARF-1-loaded myocytes, however, both ISO and GLP-1 caused an intracellular acidosis (DeltapH(i) -0.09+/-0.02 and -0.08+/-0.03, respectively). The specific GLP-1 antagonist exendin 9-39 and the cAMP inhibitory analog Rp-8CPT-cAMPS inhibited both the GLP-1-induced intracellular acidosis and the negative contractile effect. We conclude that in contrast to beta-adrenergic signaling, GLP-1 increases cAMP but fails to augment contraction, suggesting the existence of functionally distinct adenylyl cyclase/cAMP/protein kinase A compartments, possibly determined by unique receptor signaling microdomains that are not controlled by pertussis toxin-sensitive G proteins or by enhanced local PDE or phosphatase activation. Furthermore, GLP-1 elicits a cAMP-dependent modest negative inotropic effect produced by a decrease in myofilament-Ca(2+) responsiveness probably resulting from intracellular acidification.     

Transregulation of adenylyl-cyclase-coupled inhibitory receptors in heart failure enhances anti-adrenergic effects on adult rat cardiomyocytes

OBJECTIVE: The aim was to study the L-type calcium current (ICa,L) in cardiac myocytes as a possible target of insulin in the regulation of cardiac function. METHOD: Using the whole-cell configuration of the patch-clamp technique, we investigated the stimulation of ICa,L by insulin in isolated rat ventricular myocytes. RESULTS: The stimulation of ICa,L by insulin was dose-dependent (EC50 = 33 nM) and reversible. Maximum stimulation of ICa,L over basal ICa,L was 86 +/- 11% (n = 25) at 1 microM insulin. Insulin (1 microM) shifted the current-voltage relationship and potential-dependent availability of ICa,L to more negative potentials by about 3.5 and 1.5 mV, respectively. The maximum conductance of ICa,L was increased by 1 microM insulin, from 26 +/- 4 to 39 +/- 5 nS (n = 11). Isoproterenol (100 nM), which stimulated ICa,L by 156 +/- 23% (n = 10) over basal ICa,L, acted faster than insulin. The half-maximum stimulation of ICa,L by isoproterenol and insulin was reached after 44 +/- 5 and 80 +/- 9 s, respectively. Insulin and isoproterenol responses were not additive. Insulin (1 microM) and isoproterenol (100 nM) stimulation of ICa,L was inhibited by Rp-cAMPS (1 mM) to 12 +/- 3 and 32 +/- 4%, respectively. Insulin (1 microM) increased cAMP content in rat cardiomyocytes by about two-fold. Insulin-like growth factor-1 (IGF-1; 5 microM) increased ICa,L by only 5.9 +/- 0.9% (n = 6). CONCLUSIONS: Our data show that insulin stimulates the L-type calcium current in isolated rat ventricular myocytes in a dose-dependent and reversible manner and suggest that this effect is mediated by insulin receptors and the cAMP-dependent protein kinase.     

Inotropic responses to isoproterenol and phosphodiesterase inhibitors in intact guinea pig hearts: comparison of cyclic AMP levels and phosphorylation of sarcoplasmic reticulum and myofibrillar proteins

The influence of selective (milrinone: 10, 50, 100 microM) and nonselective phosphodiesterase (isobutylmethylxanthine: 0.1, 10, 100 microM) inhibitors and beta-adrenergic stimulation (isoproterenol: 0.01, 0.1 microM) on phospholamban and myofibrillar protein phosphorylation was studied in guinea pig hearts perfused with [32P]orthophosphate. Changes in protein phosphorylation were compared to alterations in tissue cyclic AMP (cAMP) levels and positive inotropic effects induced by these agents. Isoproterenol (0.01 microM), milrinone (50 microM), and isobutylmethylxanthine (100 microM) all produced similar, twofold increases in dP/dt and -dP/dt but only stimulation with isobutylmethylxanthine and isoproterenol was associated with significant increases in phospholamban phosphorylation. At these equipotent doses, the effects of isobutylmethylxanthine were associated with higher increases (3.1-fold) in cAMP than those observed with isoproterenol (twofold). Milrinone (50 microM) produced a 2.5-fold increase in cAMP levels but failed to change phospholamban phosphorylation. Higher doses of milrinone (100 microM) resulted in relatively high (4.1-fold) cAMP levels, and this was associated with increased (1.5-fold) phosphorylation of phospholamban. Phosphorylation of troponin I was significantly increased at 0.01 microM and 0.1 microM isoproterenol, while phosphorylation of C protein was observed only at 0.1 microM isoproterenol. Isobutylmethylxanthine and milrinone did not significantly increase phosphorylation of either troponin I or C protein at any of the doses studied. These findings indicate that cardiotonic agents acting via the cAMP pathway may produce similar inotropic responses at different levels of cAMP and phosphorylation of sarcoplasmic reticulum and myofibrillar proteins.     

Effects of endothelin-1 on Ca2+ signaling in guinea-pig ventricular myocytes: role of protein kinase C

The effects of ET-1 on contraction, Ca2+ transient and L-type Ca2+ current (ICa.L) were studied in single cells isolated from ventricles of guinea-pig hearts. The aim of our study was to elucidate the mechanism of the positive inotropic effect during endothelin receptor stimulation by focusing on the role of PKC. ET-1 at concentrations of 5 and 10 nM produced a biphasic pattern of inotropism: a first decrease in contraction by 34.4 +/- 2.5% of the control followed by a sustained increase in contraction by 66.6 +/- 8.4% (mean +/- SEM, n = 9). The Ca2+ transient decreased by 13.5 +/- 1.0% during the negative inotropic phase, while it increased by 58.1 +/- 8.4% (n = 10) during the positive inotropic phase. Using the whole-cell voltage-clamp technique with conventional microelectrodes, the application of ET-1 (5 nM) increased the ICa.L by 32.6 +/- 5.1% (n = 10), which was preceded by a short-lived decrease in ICa.L. Incubation of myocytes with pertussis toxin (PTX, at 2 micrograms/ml for > 3 h at 35 degrees C) failed to block the ET-1-induced enhancement of ICa.L. The increases in contraction, Ca2+ transient, and ICa.L by ET-1 were inhibited by pretreatment with 5-N-methyl-N-isobutyl amiloride (MIA; 10 microM), an amiloride analog, and a novel selective Na+/H+ exchange inhibitor HOE694 (10 microM). To determine whether activation of protein kinase C (PKC) is responsible for the enhancement of ICa.L by ET-1, we tested a PKC inhibitor, GF109203X, and found that it does exert an inhibitory effect on the ET-1-induced ICa.L increase. Our study suggests that during ET receptor stimulation an increase in ICa.L due to stimulation of Na+/H+ exchange via PKC activation causes an increase in Ca2+ transients and thereby in the contractile force of the ventricular myocytes.     

Involvement of cyclin D activity in left ventricle hypertrophy in vivo and in vitro

OBJECTIVE: The goal of this study was to determine if the properties of the transient outward potassium (I(to)), TTX-resistant sodium (I(Na)) and L-type calcium (I(Ca)) currents are altered during changes in cardiac cell shape. METHODS: Ventricular myocytes were isolated from 3- to 4-day-old neonatal rats and cultured on either non-aligned or aligned collagen thin gels. In contrast to the flat, stellar-shaped myocytes obtained when the cells are plated on non-aligned collagen gels, myocytes plated on aligned gels display an elongated, rod-like shape. Ion channel expression was measured using the whole-cell arrangement of the patch clamp technique and Western blot analysis. RESULTS: Peak values for I(to), I(Na) and I(Ca) were 9+/-1, 71+/-13 and 7+/-1 pA/pF, respectively, in the flat cells, and increased to 21+/-2, 190+/-26 and 13+/-1 pA/pF, respectively, in the aligned cells. Application of forskolin (2 microM) and 3-isobutyl-1-methylxanthine (100 microM) resulted in a 101+/-18% increase in I(Ca) in the flat cells, but increased the current by only 43+/-9% in the aligned cells. Internal dialysis of the myocytes with cAMP strongly increased the peak I(Ca) in the flat cells, but caused no significant change in the aligned cells. While both basal and forskolin-stimulated levels of cAMP were the same in the two cell morphologies, the expression of the calcium channel alpha(1C) subunit was increased in the aligned cells. CONCLUSIONS: The expression and regulatory properties of voltage-gated calcium channels are modified during changes in neonatal rat myocyte shape.     

Mechanisms of vasodilation induced by NKH477, a water-soluble forskolin derivative, in smooth muscle of the porcine coronary artery.

To study the mechanism of vasodilation induced by 6-(3-dimethylaminopropionyl) forskolin (NKH477), a water-soluble forskolin derivative, its effects on the acetylcholine (ACh)-induced contraction of muscle strips of porcine coronary artery were examined. [Ca2+]i, isometric force, and cellular concentrations of cAMP and inositol 1,4,5-trisphosphate were measured. NKH477 (0.1-1.0 microM), isoproterenol (0.01-0.1 microM), or forskolin (0.1-1.0 microM) increased cAMP and attenuated the contraction induced by 128 mM K+ or 10 microM ACh in a concentration-dependent manner. These agents, at concentrations up to 0.3 microM, did not change the amount of cGMP. NKH477 (0.1 microM) attenuated the contraction induced by 128 mM K+ without corresponding changes in the evoked [Ca2+]i responses. ACh (10 microM) produced a large phasic increase followed by a small tonic increase in [Ca2+]i and produced a sustained contraction. The ACh-induced phasic increase in [Ca2+]i, but not the tonic increase, disappeared after application of 0.1 microM ionomycin. NKH477 (0.1 microM) attenuated both the increase in [Ca2+]i and the force induced by 10 microM ACh in muscle strips that were not treated with ionomycin and inhibited the ACh-induced contraction without corresponding changes in [Ca2+]i in ionomycin-treated muscle strips. These results suggest that NKH477 inhibits ACh-induced Ca2+ mobilization through its action on ionomycin-sensitive storage sites. In ionomycin-treated and 128 mM K(+)-treated muscle strips, 0.1 microM NKH477 shifted the [Ca2+]i-force relation to the right in the presence or absence of 10 microM ACh. In beta-escin-skinned smooth muscle strips, 0.1 microM NKH477 shifted the pCa-force relation to the right but had no effects on Ca(2+)-independent contraction. We conclude that in smooth muscle of porcine coronary artery, NKH477 inhibits ACh-induced contraction by both attenuating ACh-induced Ca2+ mobilization and reducing the sensitivity of the contractile machinery to Ca2+, possibly by activating cAMP-dependent mechanisms.     

Intracellular beta-blockade: overexpression of Galpha(i2) depresses the beta-adrenergic response in intact myocardium

OBJECTIVE: Increased levels of inhibitory G proteins have been observed in heart failure, but their physiological relevance in mediating the reduced beta-adrenergic response is largely unknown. METHODS: To evaluate the functional consequences of Galpha(i2) overexpression, we studied myocardial contraction in intact isometric contracting cardiac rabbit trabeculae and isolated myocytes after adenovirus-mediated gene transfer of Galpha(i2). RESULTS: Neither Galpha(i2) nor lacZ (control) overexpression altered baseline contractile force. After 72 h of continuous contractions, developed force (F(dev)) increased after addition of 1 microM isoproterenol by 28.5+/-9.7 mN/mm(2) in the control group, which was unchanged from the initial response at t=0 h (23.7+/-3.8 mN/mm(2)). In sharp contrast, in preparations transfected with AdGalpha(i2), the response to isoproterenol was significantly attenuated (5.9+/-2.0 vs. 27.6+/-4.2 mN/mm(2), t=72 vs. 0 h, respectively, P<0.01). In a primary culture of transfected isolated myocytes from a nearly identical baseline, isoproterenol increased cell shortening by 3.1+/-0.6% in the lacZ transfected myocytes, but only by 1.3+/-0.5% in Galpha(i2) transfected myocytes (t=72 h, P<0.01). In Galpha(i2) transfected myocytes, pertussis toxin restored beta-adrenergic responsiveness, indicating specificity of attenuation by the transgene. CONCLUSIONS: Overexpression of Galpha(i2) attenuates the positive inotropic effects of beta-adrenergic stimulation in myocardium. In addition, the method we developed allows investigation of a causal link between altered protein expression and subsequent alterations in contractile function in a physiological relevant in vitro manner.     

Tumor necrosis factor-alpha decreases the phosphorylation levels of phospholamban and troponin I in spontaneously beating rat neonatal cardiac myocytes

The tumor necrosis factor (TNF) alpha level is elevated in patients with advanced heart failure, and the phosphorylation of contractile regulatory proteins is reduced in the human heart. We hypothesized that TNFalpha affects the phosphorylation of proteins involved in regulating contraction; phospholamban (PLB), myosin light chain 2 (MLC2) and troponin I (TnI). Spontaneously beating rat neonatal cardiac myocytes, prelabelled with [32P]orthophosphate, were treated with TNFalpha for 30 min, and stimulated with isoproterenol for 5 min. 32P-labelled myofibrillar proteins were isolated by 15% SDS-PAGE. Baseline phosphorylation levels of PLB, TnI and an unknown 23kDa phosphoprotein were decreased by TNFalpha in a dose-dependent manner. Moreover, TNFalpha attenuated the phosphorylation levels of PLB and TnI increased by a concentration of 0.01 microM isoproterenol, but not by 1 microM of isoproterenol. Although TNFalpha had no effect on the cAMP content or cAMP-dependent protein kinase activity in the presence or absence of isoproterenol, an inverse relationship was observed between the concentration of TNFalpha and the cGMP content in cardiac myocytes, and treatment with TNFalpha resulted in a concentration-dependent increase in type 2A protein phosphatase activity. The observation that TNFalpha decreases phosphorylation levels of PLB and TnI in cardiac myocytes suggests that the reduction of these protein phosphorylation levels is partially responsible for alterations of intracellular Ca2+-cycling and the force of contraction in TNF alpha-treated cardiac myocytes. Furthermore, TNFalpha reduces myocyte contraction and protein phosphorylation states possibly via cAMP-independent mechanisms, at least in part, by the activation of type 2A protein phosphatase.     

Influence of pyruvate on economy of contraction in isolated rabbit myocardium

BACKGROUND: Treatment of acute heart failure frequently requires positive-inotropic stimulation. However, there is still no inotropic agent available, which combines a favourable haemodynamic profile with low expenditure for energy metabolism. Pyruvate exhibits positive inotropic effects in vitro and in patients with heart failure. The effect on myocardial energy metabolism however remains unclear, but is meaningful in light of a clinical application. AIMS AND METHODS: We investigated the influence of pyruvate on contractility and oxygen consumption in isolated isometric contracting rabbit myocardium compared to beta-adrenergic stimulation with isoproterenol. RESULTS: Pyruvate (30 mM) increased developed force from 18.7+/-4.1 to 50.8+/-12.1 mN/mm2 (n=10, p<0.01). Force-time integral (FTI) increased by 329%, oxygen consumption assessed by diffusion-microelectrode technique increased from 2.86+/-0.30 mlO2/min*100 g to 6.28+/-1.28 mlO2/min*100 g (n=7, p<0.05). Economy of myocardial contraction calculated as the ratio of total FTI to oxygen consumption remained unchanged. In contrast, while isoproterenol (10 microM) produced a comparable increase in developed force from 21.4+/-8.3 to 67.3+/-15 mN/mm2 (n=7, p<0.01), FTI increased only by 260% and MVO2 increased from 2.96+/-0.43 to 6.12+/-1.01 mlO2/min*100 g (n=7, p<0.01); thus, economy decreased by 23% (n=7, p<0.05). CONCLUSION: Pyruvate does not impair economy of myocardial contraction while isoproterenol decreases economy. Regarding energy expenditure, pyruvate appears superior to isoproterenol for the purpose of positive inotropic stimulation.     

Investigation of signaling pathways that mediate the inotropic effect of urotensin-II in human heart

OBJECTIVE: This study investigated signaling pathways that may contribute to the potent positive inotropic effect of human urotensin-II (hU-II) in human isolated right atrial trabeculae obtained from patients with coronary artery disease. METHODS: Trabeculae were set up in tissue baths and stimulated to contract at 1 Hz. Tissues were incubated with 20 nM hU-II with or without phorbol 12-myristate 13-acetate (PMA, 10 microM) to desensitize PKC, the PKC inhibitor chelerythrine (10 microM), 10 microM 4alpha-phorbol that does not desensitize PKC, the myosin light chain kinase inhibitor wortmannin (50 nM, 10 microM), or the Rho kinase inhibitor Y-27632 (0.1-10 microM). Activated RhoA was determined by affinity immunoprecipitation, and phosphorylation of signaling proteins was determined by SDS-PAGE. RESULTS: hU-II caused a potent positive inotropic response in atrial trabeculae, and this was concomitant with increased phosphorylation of regulatory myosin light chain (MLC-2, 1.8+/-0.4-fold, P<0.05, n=6) and PKCalpha/betaII (1.4+/-0.2-fold compared to non-stimulated controls, P<0.05, n=7). Pretreatment of tissues with PMA caused a marked reduction in the inotropic effect of hU-II, but did not affect hU-II-mediated phosphorylation of MLC-2. The inotropic response was inhibited by chelerythrine, but not 4alpha-phorbol or wortmannin. Although Y-27632 also reduced the positive inotropic response to hU-II, this was associated with a marked reduction in basal force of contraction. RhoA.GTP was immunoprecipitated in tissues pretreated with or without hU-II, with findings showing no detectable activation of RhoA in the agonist stimulated tissues. CONCLUSIONS: The findings indicated that hU-II increased force of contraction in human heart via a PKC-dependent mechanism and increased phosphorylation of MLC-2, although this was independent of PKC. The positive inotropic effect was independent of myosin light chain kinase and RhoA-Rho kinase signaling pathways.     

Concentration-response curves of positive inotropic agents before and after ouabain pretreatment

Current therapy for congestive heart failure (diuretics, digitalis, vasodilators) may be insufficient. Addition of a second positive inotropic substance to digitalised patients has been previously shown to increase cardiac index and decrease vascular resistance. To test the hypothesis that the positive inotropy of ouabain can be increased by other inotropic agents, the following studies were performed. Firstly, concentration-response curves of positive inotropic agents (ouabain, dobutamine, dopamine, orciprenaline, phenylephrine, theophylline, amrinone, sulmazole and histamine) were measured in contracting left atria and papillary muscles from cat and guinea pig hearts. The maximal increase in force of contraction was similar for all compounds except histamine and phenylephrine which gave decreased effects in guinea pig heart muscle. Secondly, these positive inotropic agents were added to the contracting heart muscles after maximal inotropy without toxicity of a ouabain concentration which gave more than 90% of the maximal increase in force of contraction. In guinea pig left atria, dobutamine was the only compound to give a significant, although transient, increase in force of contraction above the maximal ouabain response. Theophylline (2 X 10(-4) mol X litre-1, EC25) produced significant decreases in force of contraction. In papillary muscles, low concentrations of all positive inotropic compounds, except amrinone, significantly increased force of contraction after a submaximal ouabain concentration. However, the maximal increase in force of contraction after combined addition of ouabain and a second inotropic agent was not different from the maximal increase with ouabain, dobutamine or dopamine alone. Addition of higher concentrations of the second inotropic agents after ouabain pretreatment led to a markedly increased incidence of toxicity with only transient positive inotropic effects. These results indicate that any haemodynamic improvement observed in adequately digitalised patients after combined positive inotropic therapy is unlikely to result from directly additive inotropic effects but is probably a result of other cardiovascular effects such as vasodilatation.     

Stimulation by calcitonin gene-related peptide of atrial natriuretic peptide secretion in vitro and its mechanism of action

Calcitonin gene-related peptide (CGRP) is present in nerve fibers within atrial tissue, raising the possibility that CGRP release may influence atrial natriuretic peptide (ANP) secretion. We, therefore, examined the effect of CGRP on immunoreactive ANP (ANP-IR) secretion. Isolated rat left atria paced at 2 Hz were superfused with 0.1 microM CGRP. A biphasic 2-fold increase in ANP-IR secretion occurred in response to CGRP. We next examined the mechanism of CGRP-stimulated secretion. The biphasic ANP-IR secretory response to CGRP was similar to that induced by superfusion with the beta-adrenergic agonist isoproterenol and (Bu)2cAMP, but distinct from that of the non-cAMP dependent stimuli phenylephrine, ouabain, and Bay K 8644. Superfusion with 0.1 microM CGRP for 4 min with 100 microM isobutylmethylxanthine increased atrial cAMP content from 4.29 +/- 1.21 to 10.32 +/- 2.14 pmol/mg atrial weight (P less than 0.001). Atria were next superfused with methacholine, an inhibitor of adenylyl cyclase activation. The addition of 0.1 microM isoproterenol or 0.1 microM CGRP to the superfusate containing 10 microM methacholine failed to stimulate ANP-IR secretion and lowered cAMP accumulation by 70%. Superfusion with 10 microM atropine negated the inhibitory effects of methacholine. We conclude that 1) CGRP stimulates ANP-IR secretion in vitro; and 2) CGRP-stimulated secretion appears to be mediated by cAMP. Thus, ANP-IR secretion may be modulated by atrial nerve fibers containing CGRP in vivo.     

Isoproterenol sensitivity of isolated cardiac myocytes from rats with monocrotaline-induced right-sided hypertrophy and heart failure

Rats treated with the alkaloid monocrotaline developed right ventricular hypertrophy with a left:right ventricle weight ratio of 1.35 +/- 0.10 (mean +/- s.e.m., n = 25) compared with 3.83 +/- 0.40 (n = 14) in diet-matched controls (P less than 0.001). Urine volume and sodium content were reduced and body water increased consistent with heart failure. In 10 out of 26 treated rats pleural, pericardial or peritoneal effusions were present. Urine norepinephrine content was significantly raised (P less than 0.02) but epinephrine was unchanged. Plasma norepinephrine levels were raised though not significantly. Myocytes isolated from the right ventricle had a reduced myosin Ca2+-activated ATPase (P less than 0.05) activity and a shift towards slower V2 and V3 myosin isoforms. There was no decrease in maximum contraction amplitude with calcium or isoproterenol in either left or right ventricular cells of treated rats. Right ventricular cells from treated rats showed a reduced rate of contraction in maximum isoproterenol (P less than 0.05) and a significant rightward shift in PD2 (P less than 0.05) representing a two-fold increase in EC50 for isoproterenol compared with right ventricular cells from control animals. There was no shift in EC50 for isoproterenol in left ventricle cells. In parallel experiments, myocytes isolated from both ventricles of rats treated with isoproterenol for one week showed a rightward shift of more than 50-fold in the isoproterenol concentration-response curve and a depressed response to maximum isoproterenol. In the rat monocrotaline model of right-sided cardiac hypertrophy and failure, changes in sensitivity to beta-adrenoceptor agonists are slight, and present only in the right ventricle. The lack of change in the left ventricle seems to suggest that this functional desensitisation is not a consequence of raised circulating catecholamines.     

Effects of cAMP, furosemide, and ouabain on transtracheal tetracycline and chloramphenicol influx in the rat

Using an in vitro perfusion model of the rat trachea, we studied the effects of dibutyryl cAMP (10(-3)M), terbutaline sulfate (1.7 X 10(-4)M), furosemide (10(-3)M), and ouabain (10(-3)M) on net transtracheal tetracycline and chloramphenicol influx. Submucosal cAMP and terbutaline increased transepithelial potential difference and short-circuit current and net tetracycline influx from control values of 0.496 +/- 0.066 (mean +/- SEM, n = 9) and 0.451 +/- 0.077 nM/cm2 h (n = 7) by 51 and 29%, respectively. Submucosal furosemide and ouabain decreased transepithelial potential difference, short-circuit current, and net tetracycline influx from control values by 22 and 29%, respectively. Neither cAMP, terbutaline, furosemide, nor ouabain had any effect on net chloramphenicol influx. These data suggest that drugs used in conjunction with antibiotics in patients with obstructive pulmonary disease may alter delivery of certain antibiotics into the airway lumen.     

Dual effect of serotonin on growth of bovine pulmonary artery smooth muscle cells in culture.

We have previously reported that serotonin (5-hydroxytryptamine [5HT]) alters cultured bovine pulmonary artery smooth muscle cell (SMC) configuration through two different regulatory mechanisms. We now report that 5HT also regulates SMC growth through these same two mechanisms--a stimulatory event initiated intracellularly and inhibition of growth resulting from a cell surface action. 5HT (1 microM) plus 0.1 mM iproniazid (a 5HT metabolic inhibitor) produced a severalfold stimulation of DNA synthesis (as measured by [3H]thymidine incorporation) of SMCs after a 17-24-hour incubation with only a slight elevation of cellular cAMP. This stimulatory effect responded synergistically with other growth factors including platelet-derived growth factor, fibroblast growth factor, and epidermal growth factor and was effectively reversed by 5HT uptake inhibition. It was not produced by 5-hydroxyindoleacetic acid, a metabolite of 5HT. In the presence of 1 microM 5HT plus 0.1 mM isobutylmethylxanthine (IBMX), cAMP was elevated eightfold, dendritic formation occurred, and [3H]thymidine labeling of SMCs was inhibited. Inhibition of labeling by [3H]thymidine was mimicked by other agents that elevated cellular cAMP (10 microM histamine, 1 microM isoproterenol plus 0.1 mM IBMX, and 10 microM forskolin) and by 1 mM dibutyryl cAMP. This inhibitory effect was not blocked by either inhibition of 5HT uptake or 5HT-receptor antagonists ketanserin (5HT2); methiothepin, spiperone, and mianserin (5HT1/5HT2); and 3-tropanyl-indole-3-carboxylate and 3-tropanyl-3,5-dichlorobenzoate (5HT3). However, similar to 5HT, the 5HT1A agonist, (+/-)-8-hydroxy-(+/-)-2-dipropylamino-8-hydroxy-1,2,3, 4-tetrahydronaphthalenehydrobromide, in association with IBMX, produced an elevation in cAMP and inhibition of labeling by [3H]thymidine. 5HT, in the presence of either iproniazid or IBMX, did not alter [Ca2+]i, indicating that [Ca2+]i was not a signal for either of these actions.(ABSTRACT TRUNCATED AT 250 WORDS)