Perfusate Calcium Concentration Research Articles

A Langendorff-like system to quantify cardiac pump function in adult zebrafish.

ABSTRACTZebrafish are increasingly used as a vertebrate model to study human cardiovascular disorders. Although heart structure and function are readily visualized in zebrafish embryos because of their optical transparency, the lack of effective tools for evaluating the hearts of older, nontransparent fish has been a major limiting factor. The recent development of high-frequency echocardiography has been an important advance for in vivo cardiac assessment, but it necessitates anesthesia and has limited ability to study acute interventions. We report the development of an alternative experimental ex vivo technique for quantifying heart size and function that resembles the Langendorff heart preparations that have been widely used in mammalian models. Dissected adult zebrafish hearts were perfused with a calcium-containing buffer, and a beat frequency was maintained with electrical stimulation. The impact of pacing frequency, flow rate and perfusate calcium concentration on ventricular performance (including end-diastolic and end-systolic volumes, ejection fraction, radial strain, and maximal velocities of shortening and relaxation) were evaluated and optimal conditions defined. We determined the effects of age on heart function in wild-type male and female zebrafish, and successfully detected hypercontractile and hypocontractile responses after adrenergic stimulation or doxorubicin treatment, respectively. Good correlations were found between indices of cardiac contractility obtained with high-frequency echocardiography and with the ex vivo technique in a subset of fish studied with both methods. The ex vivo beating heart preparation is a valuable addition to the cardiac function tool kit that will expand the use of adult zebrafish for cardiovascular research.

Focal but reversible diastolic sheet dysfunction reflects regional calcium mishandling in dystrophicmdxmouse hearts

Cardiac dysfunction is a primary cause of patient mortality in Duchenne muscular dystrophy, potentially related to elevated cytosolic calcium. However, the regional versus global functional consequences of cellular calcium mishandling have not been defined in the whole heart. Here we sought for the first time to elucidate potential regional dependencies between calcium mishandling and myocardial fiber/sheet function as a manifestation of dystrophin-deficient (mdx) cardiomyopathy. Isolated-perfused hearts from 16-mo-old mdx (N = 10) and wild-type (WT; N = 10) were arrested sequentially in diastole and systole for diffusion tensor MRI quantification of myocardial sheet architecture and function. When compared with WT hearts, mdx hearts exhibited normal systolic sheet architecture but a lower diastolic sheet angle magnitude (|β|) in the basal region. The regional diastolic sheet dysfunction was normalized by reducing perfusate calcium concentrations. Optical mapping of calcium transients in isolated hearts (3 mdx and 4 WT) revealed a stretch-inducible regional defect of intracellular calcium reuptake, reflected by a 25% increase of decay times (T(50)) and decay constants, at the base of mdx hearts. The basal region of mdx hearts also exhibited greater fibrosis than did the apex, which matched the regional sheet dysfunction. We conclude that myocardial diastolic sheet dysfunction is observed initially in basal segments along with calcium mishandling, ultimately culminating in increased fibrosis. The preservation of relatively normal calcium reuptake and diastolic/systolic sheet mechanics throughout the rest of the heart, together with the rapid reversibility of functional defects by reducing cytosolic calcium, points to the significance of regional mechanical factors in the progression of the disease.

Influence of perfusate calcium concentration on the inotropic insulin effect in isolated guinea pig and rat hearts.

Conflicting data on the inotropic effect of insulin are present in the literature suggesting a positive inotropic property or no inotropic effect or even a negative influence. To clarify the reason for these diverging findings, dose-response curves of insulin have been performed in isolated working rat and guinea pig hearts perfused with Krebs-Henseleit buffer containing 0.9, 1.25, 2.5, and 5 mM Ca(2+) at 37 degrees C. At 1.25 mM [Ca(2+)], insulin (8 to 16 IU) regularly improved the inotropic state. LVdP/dt(max) increased significantly from 1,900 to 2,300 mm Hg/s (+21 %) in guinea pig hearts and from 3,197 to 4,345 mm Hg/s (+36 %) in rat hearts. LVEDP did not change significantly. Myocardial oxygen consumption increased parallel with contractility. Heart rate was not influenced in either species. Coronary flow increased by 16.5 % in guinea pig hearts, but decreased in rat hearts by 13.6 % (p < 0.05 each). With 0.9 mM [Ca(2+)] the positive inotropic effect of insulin did not further augment. At 2.5 mM [Ca(2+)] insulin exhibited in both species no significant change of LVdP/dt(max) but very high insulin doses depressed the heart. At 5 mM [Ca(2+)] insulin depressed the heart significantly already at lower concentrations. At 31 degrees C and 1.25 mM [Ca(2+)] the positive inotropic insulin effect was preserved. We conclude that the positive inotropic insulin effect in rat and guinea pig hearts depends on the extracellular [Ca(2+)], i.e., is maximal around 1.25 mM [Ca(2+)] and is reduced or absent at higher [Ca(2+)] or may even become negative.

Altered Creatine Kinase Enzyme Kinetics in Diabetic Cardiomyopathy. A31P NMR Magnetization Transfer Study of the Intact Beating Rat Heart

M. Spindler, K. W. Saupe, R. Tian, S. Ahmed, M. A. Matlib and J. S. Ingwall. Altered Creatine Kinase Enzyme Kinetics in Diabetic Cardiomyopathy. A31P NMR Magnetization Transfer Study of the Intact Beating Rat Heart.Journal of Molecular and Cellular Cardiology (1999) 31, 2175–2189. To determine whether the decreased contractile performance in diabetic hearts is associated with a reduced energy reserve due to decreased creatine kinase (CK) activity, we measured total CK activity (Vmax) in vitro and CK reaction velocity in vivo using31P NMR spectroscopy in isolated perfused rat hearts after 4 and 6 weeks of diabetes. After 4 weeks of diabetes, Vmaxdecreased by 22% with a larger decrease of CK MB than of CK MM and mitochondrial-CK isoenzymes. There was no further decrease in these parameters after 6 weeks of diabetes. Isovolumic contractile performance of 4 and 6 week diabetic hearts, estimated as rate-pressure product under identical perfusion and loading conditions (EDP set at 6–8 mmHg), was only 50% of that of control. ATP, PCr and total creatine concentrations were not different in control and 4 or 6 weeks diabetic rat hearts. After 4 weeks of diabetes, CK reaction velocity decreased by 22%. This was in proportion to the decline of Vmaxand therefore predicted by the rate equation for the CK reaction. However, the further decline in the CK reaction velocity after 6 weeks of diabetes (45%) was greater than that predicted from the CK rate equation (17% decrease), and cannot be explained by substrate control of the enzyme. When hearts were inotropically stimulated by increasing perfusate calcium concentration, CK reaction velocity increased slightly (15%) in both control and diabetic hearts, thereby maintaining a constant ATP concentration. We conclude that in the diabetic myocardium, the CK reaction velocity decreases but does not limit the availability of high-energy phosphates for contraction over the range of workloads studied. We also conclude that a mechanism(s) in addition to substrate control regulates CK reaction velocity in the 6 week diabetic hearts.

Acceleration of widespread adenoviral gene transfer to intact rabbit hearts by coronary perfusion with low calcium and serotonin.

Previous attempts at adenoviral gene transfer to the intact heart have been limited by the requirement for prolonged exposure to high virus concentrations. In an ex vivo coronary perfusion model of intact adult rabbit hearts, we previously reported gene transfer to 96% of cardiac myocytes after a 60 min exposure to 1.6 x 10(9) p.f.u./ml Ad beta gal, a recombinant adenovirus encoding beta-galactosidase. Here we sought to decrease the virus exposure time by enhancing microvascular permeability to increase the efficiency of adenoviral gene transfer. Baseline perfusion with 1.0 x 10(8) p.f.u./ml Ad beta gal in normal Krebs solution (1 mM calcium) caused infection of 22% of myocytes at 30 min and 40% at 60 and 120 min. Increasing the virus concentration, decreasing perfusate calcium concentration, or pretreating with serotonin or bradykinin in Krebs solution or L-NAME in heparinized rabbit blood significantly decreased the necessary exposure time. Under optimal conditions of serotonin pretreatment, 50 mumol/l perfusate calcium, and a virus concentration of 1.6 x 10(9) p.f.u./ml, 2 min of coronary perfusion sufficed to produce near-total infection. This profound enhancement of infection parameters has important implications for in vivo myocardial gene transfer, where a similar strategy could facilitate gene therapy for common myocardial disorders.

Effects of acetate on energy metabolism and function in the isolated perfused rat heart

Impairment of cardiac contractile function is an important component of acetate associated hypotension during hemodialysis treatments. We examined the effect of acetate on cardiac energy metabolism using the isovolumic isolated perfused heart model. In this preparation, acetate (10 M) caused decreases in tissue ATP concentrations (12.3 +/- 0.8 vs. 15.6 +/- 1.0 micromol/g dry at 30 min, P < 0.05) as well as marked impairment of systolic function (dpdt = 863 +/- 135 vs. 1288 +/- 166 mm Hg/second at 30 min, P < 0.05). Although altering perfusate calcium concentrations (0.6, 1.2 and 2.4 mM) affected physiological responses to acetate (5 and 10 mM), the reductions in tissue ATP concentrations were similar. In isolated heart mitochondria, acetate (100 microM -10 mM) selectively impaired octanoate and palmityl carnitine supported State 3 respiration in a dose dependent fashion (P < 0.01), but did not affect respiration when succinate, pyruvate/malate or malate/glutamate was used as substrate. We suggest that high concentrations of acetate selectively impair fatty acid metabolism in heart issue. This in turn leads to decreases in ATP production and tissue ATP concentrations that ultimately result in impaired contractile function. As this occurs at relatively low concentrations of acetate, this finding may be relevant to other parenterally-administered acetate containing fluids.

NMR spectroscopy studies of severe hypokalemia in isolated rat hearts.

Effects of acute tissue potassium depletion on cellular energy metabolism are poorly understood. To examine this issue, we performed the following studies in an isovolumic isolated perfused heart preparation. Perfusion of isolated hearts with media lacking potassium (K = 0 mmol/l) for 30 min resulted in ventricular fibrillation, rapid decreases in creatine phosphate (PCr) and ATP, and increases in Pi. During reinstitution of normal perfusate potassium, hearts did not resume normal contractions, and no increases in tissue ATP were observed. However, some normalization of PCr and Pi were noted during reinstitution of normal perfusate. Perfusion with media containing K = 2 mmol/l caused significant but less dramatic decreases in tissue ATP concentrations than perfusion with media containing K = 0 mmol/l. Reduction of perfusate calcium from 1.2 (normal) to 0.6 mmol/l in media containing K = 0 mmol/l attenuated the fall in ATP seen with media containing K = 0 mmol/l. Conversely, increasing perfusate calcium to 2.4 mmol/l in media containing K = 2 mmol/l markedly worsened the fall in tissue ATP seen in media containing K = 2 mmol/l. In this subgroup (K = 2 mmol/l, Ca = 2.4 mmol/l), ventricular fibrillation developed approximately one-half of the time. However, no differences in the rate of ATP fall were observed between those hearts that fibrillated and those that did not. During perfusion with media containing K = 0 mmol/l, nuclear magnetic resonance (NMR)-visible tissue potassium concentrations fell rapidly and dramatically. Significant but less severe reductions in NMR-visible potassium were seen during perfusion with media containing K = 2 mmol/l. With K = 2 mmol/l perfusate, the rate of cellular potassium loss was influenced by perfusate calcium concentration. When cardiac mitochondria were examined after perfusion with media containing K = 0 mmol/l, evidence for calcium loading as well as respiratory dysfunction was noted. These data indicate that reductions in perfusate potassium caused dramatic reductions in tissue ATP and NMR-visible potassium concentrations. The abnormal energy metabolism that results from acute cellular potassium depletion appears to be due, at least in part, to impaired energy production by cardiac mitochondria that become calcium loaded.

Impact of Isradipine on Contractile Performance, Metabolism, and Coronary Resistance Studied in Isolated Rat Hearts

In anesthetized dogs, isradipine has been reported to induce peripheral vasodilation and increase cardiac output (CO) and myocardial contractility, whereas myocardial oxygen consumption (MVO2) decreases, suggesting that isradipine may increase overall metabolic efficiency of the ventricle of intact animals. Whether isradipine has any direct myocardial effects that could cause intrinsic increase in metabolic efficiency or whether the observation relates to favorable isradipine-induced changes in hemodynamic loading conditions is not known. Therefore, we determined the direct myocardial effects of isradipine on contractile strength and metabolic efficiency in isolated rat heart. Isolated crystalloid perfused rat hearts were instrumented for measurement of ventricular pressure, volume, and MVO2. Isradipine decreased developed pressure (DP) and MVO2 in a concentration-dependent manner; at 32 nM irradipine, both quantities were approximately 70% of their control values. Isradipine caused a downward shift of the end-systolic pressure-volume relation (ESPVR) and in the relation between ventricular work (indexed by pressure-volume area, PVA) and MVO2, indicating that for any given amount of total mechanical work performed, the rat heart consumed less O2 during administration of isradipine than under control conditions. However, the magnitude of the downward shift of this relation was nearly identical to that observed in a separate group of hearts in which we decreased contractility by decreasing the perfusate calcium concentration. Thus, isradipine does not appear to have a contractility-independent effect on myocardial efficiency.

End-systolic pressure-volume and MVO2-pressure-volume area relations of isolated rat hearts.

We tested the utility of a standard isolated, crystalloid-perfused, isovolumic rat heart preparation for studying ventricular metabolism in terms of the myocardial oxygen consumption-pressure-volume area (MVO2-PVA) relations. The end-systolic pressure-volume relations (ESPVRs) determined between volumes of 0.15 and 0.65 ml were fit equally well by linear and nonlinear regression analysis within the data range but predicted widely differing volume-intercept (Vo) values. Linear regression analysis of the ESPVRs provided a mean slope (Ees) of 419 +/- 186 mmHg.g.ml-1 and Vo of 0 +/- 0.12 ml, respectively (n = 6). The MVO2-PVA relations were linear with a slope and MVO2 intercept of 1.30 +/- 0.31 x 10(-5) ml O2.mmHg-1.ml-1 and 0.38 +/- 0.09 x 10(-3) ml O2-beat-1.g-1, respectively. These MVO2-PVA parameters were not significantly different from those obtained when nonlinear regression analysis was applied to the ESPVR. Decreasing perfusate calcium concentration ([Ca2+]) (n = 7) resulted in a downward shift in the ESPVR, a decrease in the MVO2-PVA intercept (0.52 +/- 0.26 vs. 0.34 +/- 0.20 x 10(-3) ml O2.beat-1.g-1, P less than 0.01), and no significant change in the MVO2-PVA slope (1.33 +/- 0.47 vs. 1.57 +/- 0.69 x 10(-5) ml O2.mmHg-1.ml-1, NS). We conclude that this preparation may be a useful alternative to more expensive preparations for selected experiments in cardiac energetics.

Contractile dysfunction during metabolic acidosis: role of impaired energy metabolism

To investigate the mechanisms by which acidosis depresses cardiac function, a Langendorff isolated perfused rat heart preparation was studied using 31P magnetic resonance spectroscopy. Isolated hearts were subjected to normal perfusion conditions or experimental manipulations simulating severe metabolic acidosis, substrate depletion, impairment of oxidative metabolism, or low perfusate calcium concentrations. All maneuvers resulted in marked reductions in oxygen consumption and the force of myocardial contraction (dP/dt). Metabolic acidosis had bioenergetic changes suggestive of impaired energy production, specifically, increases in Pi and decreases in phosphocreatine concentrations, which did not occur in hearts subjected to low perfusate calcium concentrations. In acidotic perfusions as well as substrate depletion and impairment of oxidative metabolism, the change in dP/dt correlated best with the change in the intracellular concentration of monovalent Pi (P(im)) (r = 0.70, P less than 0.01), whereas in hearts subjected to a low perfusate calcium concentration, there was no relationship between dP/dt and the change in Pim concentrations. More detailed analysis of the time course of the metabolic and physiological changes with metabolic acidosis revealed a discordance between changes in Pim and the decreases in dP/dt during the first 20 min of the induction of acidosis and the first 10 min of recovery from acidosis. These data suggest that metabolic acidosis has a major direct effect on energy metabolism in this model. Moreover, impairment of oxidative metabolism in concert with decreases in intracellular pH may be important in the contractile failure associated with prolonged metabolic acidosis.

Altered responsiveness of hypertrophied rat hearts to alpha- and beta-adrenergic stimulation

Inotropic responsiveness to α- and β-adrenergic agents was examined in pressure-overload hypertrophied rat hearts. Pressure overload was induced in rats by abdominal aortic constriction. Three weeks post-constriction, hearts were isolated and perfused with buffer containing various concentrations of (1) calcium (2) isoproterenol (3) forskolin, or (4) phenylephrine. The change in rate of left ventricular pressure development ( Δ + dP dt ) with increasing perfusate calcium concentrations was comparable in hypertrophied hearts of aortic-constricted rats (AC) and hearts of sham-operated rats (SO). However, with isoproterenol or forskolin stimulation, inotropic responsiveness ( Δ + dP dt ) was 50% lower in hypertrophied hearts of AC. This was associated with significantly lower tissue cAMP levels. Beta-adrenoceptor number and affinity were unchanged in the hypertrophied myocardium. Maximum inotropic responsiveness to phenylephrine was also lower in hypertrophied hearts and was associated with reduced α-adrenoceptor numbers. The data suggest that altered inotropic responsiveness to α-adrenergic stimulation may, in part, be due to reduced cardiac α-adrenoceptor density. However, post-receptor mechanisms including alterations in cAMP metabolism may contribute to the reduced responsiveness to β-adrenergic stimulation in hypertrophied hearts of AC.

Perfusate calcium concentration, inotropic state and contractile stability during prolonged perfusion of the isolated working rabbit heart: T. Murashita, D.J. Hearse, R.D. Kempsford. Cardiovascular Research, The Rayne Institute, St. Thomas' Hospital, London SE1 7EH, UK

Perfusate calcium concentration, inotropic state and contractile stability during prolonged perfusion of the isolated working rabbit heart: T. Murashita, D.J. Hearse, R.D. Kempsford. Cardiovascular Research, The Rayne Institute, St. Thomas' Hospital, London SE1 7EH, UK

Successful five-day perfusion preservation of the canine kidney.

Over 20 years ago, successful 3-day-perfusion preservation of canine kidneys was obtained. Since then, consistent 5-day preservation has not been reported. In this study, we investigated how the perfusate calcium concentration affected both mitochondrial function and posttransplant viability in dog kidneys preserved for 5 days. Dog kidneys were preserved by machine perfusion (5 degrees C) using a hydroxyethyl starch-gluconate solution that contained either 0.0, 0.5, 1.5, or 5.0 mM calcium. Mitochondria isolated from preserved kidneys has a loss of respiratory control when either 0.0, 1.5, or 5.0 mM calcium were present. However, the use of a perfusate with 0.5 mM calcium preserved the mitochondrial function at levels equivalent to controls for 5 days. Transplantation of kidneys preserved for 5 days with 0.0 or 1.5 mM calcium yielded poor survival (0% and 17%, respectively). The use of a 0.5-mM calcium perfusate increased posttransplant survival to 63% (5 of 8 transplanted). Donor pretreatment of kidneys with chlorpromazine (2.5 mg/kg i.v.) did not improve the function of mitochondria isolated from preserved kidneys but did increase survival in the 1.5-mM calcium group to 67% (4 of 6 transplanted) and in the 0.5 mM calcium group to 100% (7 of 7 transplanted). This is the first report to document consistently successful 5-day preservation of canine kidneys and clearly shows the importance of the perfusate calcium concentration in long-term kidney preservation. The specific mechanism by which calcium or chlorpromazine exert their effect is not known, but it is apparent that excessively high or low concentrations of calcium are damaging to the preserved organ, and an optimal calcium concentration combined with metabolic inhibition of calcium-dependent pathways can significantly improve the function of organs preserved for extended time periods.

The effect of perfusate calcium concentration on ultrastructural changes in 5-day hypothermically preserved canine kidneys

The effect of perfusate calcium concentration on ultrastructural changes in 5-day hypothermically preserved canine kidneys

Stimulation of glycogenolysis and vasoconstriction in the perfused rat liver by the thromboxane A2 analogue U-46619.

Infusion of the thromboxane A2 analogue U-46619 into isolated perfused rat livers resulted in dose-dependent increases in glucose output and portal vein pressure, indicative of constriction of the hepatic vasculature. At low concentrations, e.g. less than or equal to 42 ng/ml, glucose output occurred only during agonist infusion; whereas at concentrations greater than or equal to 63 ng/ml, a peak of glucose output also was observed upon termination of agonist infusion coincident with relief of hepatic vasoconstriction. Effluent perfusate lactate/pyruvate and beta-hydroxybutyrate/acetoacetate ratios increased significantly in response to U-46619 infusion. Hepatic oxygen consumption increased at low U-46619 concentrations (less than or equal to 20 ng/ml) and became biphasic with a transient spike of increased consumption followed by a prolonged decrease in consumption at higher concentrations. Increased glucose output in response to 42 ng/ml U-46619 was associated with a rapid activation of glycogen phosphorylase, slight increases in tissue ADP levels, and no increase in cAMP. At 1000 ng/ml, U-46619 activation of glycogen phosphorylase was accompanied by significant increases in tissue levels of AMP and ADP, decreases in ATP, and slight increases in cAMP. In isolated hepatocytes, U-46619 did not stimulate glucose output or activate glycogen phosphorylase. Reducing the perfusate calcium concentration from 1.25 to 0.05 mM resulted in a marked reduction of the glycogenolytic response to U-46619 (42 ng/ml) with no efflux of calcium from the liver. U-46619-induced glucose output and vasoconstriction displayed a similar dose dependence upon the perfusate calcium concentration. Thus, U-46619 exerts a potent agonist effect on glycogenolysis and vasoconstriction in the perfused rat liver. The present findings support the concept that U-46619 stimulates hepatic glycogenolysis indirectly via vasoconstriction-induced hypoxia within the liver.

Aortic pressure as a determinant of cardiac protein degradation.

Mechanical parameters and intracellular mediators that may control protein degradation were studied in isolated rat hearts subjected to increased aortic pressure. Elevation of aortic pressure from 60 to 120 mmHg in Langendorff preparations provided glucose or pyruvate as substrate decreased the rate of protein degradation during the second hour of perfusion. Intracellular contents of ATP or creatine phosphate or the creatine phosphate/creatine ratio did not indicate that energy depletion accounted for these effects. When ventricular pressure development was prevented by ventricular draining, and hearts were arrested with tetrodotoxin, protein degradation still decreased as aortic pressure was raised. The effect of elevated aortic pressure on proteolysis was unchanged when perfusate calcium concentrations were 0.6, 3.0, or 5.1 mM, or when indomethacin or meclofenamate was added to the perfusion buffer. These results provided no evidence to indicate that intraventricular pressure development or cardiac contraction was responsible for the inhibitory effect of increased aortic pressure on protein degradation. Instead, they suggested that stretch of the ventricular wall, as a consequence of increased aortic pressure, could be the mechanical parameter most closely related to the restraint on proteolysis. No evidence was obtained that the lower rate of degradation depended on energy or calcium availability or prostaglandin synthesis.

Platelet-activating factor-mediated vasoconstriction and glycogenolysis in the perfused rat liver.

Infusion of platelet-activating factor (alkyl acetylglycerophosphocholine (AGEPC] into isolated perfused rat livers caused a dose-dependent, transient increase in portal vein pressure, indicative of constriction of the hepatic vasculature. A close correlation was observed between the changes in portal pressure and concomitant transient increases in hepatic glucose output. The two processes displayed similar dose dependence and were attenuated to a similar extent by reducing the perfusate calcium concentration. Reducing the perfusate free calcium concentration to 1 nM by co-infusion of EGTA did not abolish completely the hepatic responses to AGEPC. Verapamil inhibited both the hemodynamic and glycogenolytic responses to AGEPC in a dose-dependent fashion; the IC50 was approximately 10 microM at an AGEPC concentration of 6.6 X 10(-11) M. Also, both responses displayed similar degrees of tachyphylaxis in response to repeated short infusions of AGEPC. Measurement of glycogen phosphorylase a in extracts from freeze-clamped livers demonstrated a rapid increase in phosphorylase a in response to infusion of AGEPC. A small but significant increase in whole tissue ADP was found in response to AGEPC (2 X 10(-8) M); cAMP levels were not changed by AGEPC infusion. It is concluded that glycogenolysis in the perfused liver in response to AGEPC may be a result of the hemodynamic effects of AGEPC, rather than a direct effect of the phospholipid mediator on the hepatocyte.

Cardiotoxicity of lead at various perfusate calcium concentrations: Functional and metabolic responses of the perfused rat heart

Equilibrated rat hearts were perfused for 60 min with a standard crystalloid buffer containing either 0.9, 1.8, 3.5, or 5.0 m m Ca with or without added lead (0.3 and 30 μ m). Contractile tension ( T), rate of tension development ( dT dt ), electrocardiographic (EKG), His bundle electrographic (HBE) indices, heart rate (HR), preejection period (PEP), and coronary flow rate (CFR) were recorded as a function of perfusion time. Endpoint analyses of myocardial phosphatic metabolites were performed on heart perchloric acid extracts by standard phosphorus-31 nuclear magnetic resonance (P-31 NMR) spectroscopic techniques. The contractile activity and glycerol 3-phosphorylcholine content of the myocardium were found to vary directly as a function of the perfusate calcium concentration; however, except for a prolonged atrioventricular-His bundle conduction time detected in hearts treated with 0.9 m m Ca, the variable perfusate calcium concentrations were devoid of any other significant physiologic and metabolic effects. In contrast, perfusion of control equilibrated hearts with 30 μ m lead significantly attenuated the positive, and exacerbated the negative, inotropic responses to elevated, and low perfusate calcium concentrations, respectively. Moreover, a secondary, time-dependent decline in myocardial contractile strength was also observed in response to this lead concentration, which was progressively more pronounced with each increment in the perfusate calcium concentration. The preejection period of ventricular systole was also prolonged in response to 30 μ m lead; however, this effect was less pronounced at higher perfusate calcium concentrations. Hearts perfused with 30 μ m lead were also characterized by significant prolongation in atrioventricular node and His bundle conduction time, reduced coronary flow rate, and decreased heart rate, irrespective of the perfusate calcium concentration. Hearts treated with 0.3 μ m lead exhibited functional properties that were diminished, but still comparable to control hearts. Analysis of myocardial phosphatic metabolite amounts following 60 min of perfusion revealed a significant lead-induced reduction in the energy status of the heart. The combination of 5.0 m m calcium with either 0.3 or 30 μ m lead resulted in significant disturbances in phosphoglyceride, glycolytic, and high-energy phosphate pathways. These findings suggest that the cardiotoxic actions of lead are linked to complex mechanisms that are partially related to an interference with calcium-dependent cellular processes. The observed negative inotropic effects of lead (0.3 or 30 μ m) with 5.0 m m calcium indicate that the actions of lead may be enhanced under conditions of increased metabolic demand.

Role of extracellular calcium and calcium efflux in the activation of hepatic glycogenolysis by calcium-dependent hormones.

The role of extracellular calcium in the glycogenolytic effects of calcium-dependent hormones was examined in a rat liver perfusion system. Decreasing the perfusate CaCl2 concentration resulted in a concentration-dependent inhibition of glucose output by maximal concentrations of vasopressin (20 nM) and angiotensin II (10 nM), but not of glucagon (1.4 nM), cyclic AMP (100 microM), dibutyryl cyclic AMP (10 microM) or phenylephrine (5 microM). However, the effect of phenylephrine was inhibited when livers were perfused with CaCl2-free perfusate containing 0.5 mM EGTA in a duration-dependent manner. These effects were exerted through the inhibition of the maximal response of each hormone, and were associated with a parallel decrease in phosphorylase activation but not with changes in tissue cyclic AMP concentrations. When livers were preloaded with 45Ca for 45 min and then washed for either 15 min or 45 min, these hormones elicited a rapid and transient 45Ca efflux regardless of the perfusate calcium concentration. The sequential perfusion of two hormones resulted in the loss of 45Ca efflux by the second hormone. These results suggest that the glycogenolytic effects of vasopressin and angiotensin II depend on the extracellular calcium and that of phenylephrine primarily on the cellular calcium. It was also demonstrated that these calcium-dependent hormones mobilize calcium from the same pools. However, the mobilization of cellular calcium does not necessarily correlate directly with the glycogenolytic actions of vasopressin and angiotensin II.

Dependence of protein synthesis on aortic pressure and calcium availability.

Increased aortic pressure accelerated protein synthesis in control-beating and arrested-drained hearts supplied with either glucose or pyruvate. Elevation of perfusion pressure from 60 to 120 mm Hg increased oxygen consumption in control-beating but not in arrested-drained preparations. Energy availability, as assessed by adenylate energy charge or creatine phosphate/creatine ratio, or both, was increased in arrested-drained hearts supplied with glucose and perfused at 60 and 120 mm Hg aortic pressure. In control-beating or arrested-drained hearts supplied with pyruvate, energy availability was not improved by elevation of aortic pressure from 60 to 120 mm Hg. An increase of perfusate calcium concentration from 0.5 to 5.0 mM in control-beating Langendorff preparations supplied with glucose and perfused at an aortic pressure of 90 mm Hg doubled oxygen consumption and decreased energy availability, but had no effect on the rate of protein synthesis. In arrested-drained hearts supplied with either glucose or pyruvate and calcium concentrations ranging from 0.5 to 5.0 mM, the rates at 120 mm Hg aortic pressure were 11-25% higher than at 60 mm Hg. These findings provide no evidence to implicate increased oxidative metabolism, energy availability, or extracellular calcium concentration as important factors in the mechanism that accounts for the effects of increased aortic pressure on protein synthesis.