Differences between mouse and rat myocardial contractile responsiveness to calcium

Genetically altered mice have become an increasingly important tool for the study of mechanisms of cardiac function, and therefore it is vital to characterize the basic contractile properties of the mouse heart. As a first approach to this goal, we first optimized perfusion conditions and characterized the effect of incremental left ventricular balloon inflation on end-diastolic, systolic and developed pressures in the isovolumically-contracting mouse heart. Under constant loading conditions, we determined developed pressure in response to changing perfusate calcium (1.25, 2.5, 3.75 and 5.0 mM) and perfusate temperature (30 and 37 degrees C). We then compared the intrinsic inotropic responsiveness to changes in extracellular calcium of left ventricular myocardium from mouse to that from the rat. In the baseline state (1.25 mM extracellular calcium; [Ca2+]o), both isometric contraction duration and normalized active force at the peak of the active force-length relationship (Lmax) were less in mouse than in rat myocardium. Under isotonic conditions, temporal parameters of shortening and the relative shortening were less in mouse vs rat myocardium. Increasing [Ca2+]o from 1.25 to 2.5 mM markedly increased active isometric force and rate of force development (+dF/dt) in the mouse. However, rat myocardium responded to a lesser extent. Under isotonic conditions, peak shortening and the rate of shortening also increased to a greater extent in mouse relative to rat myocardium. Increasing the bath calcium concentration to 5.0 mM increased isometric force and +dF/dt further in the rat but not the mouse, suggesting that two species operate at different points on the force vs [Ca2+]o relationship. We conclude that mouse myocardium exhibits increased sensitivity to changes in [Ca2+]o within the physiologic range in comparison to rat. These differences do not appear to be due to differences in loading conditions. The data suggest that differences in inotropic responsiveness to calcium may reflect intrinsic differences in myocardial calcium sensitivity between species.     

Some determinants of quality and yield in the isolation of adult heart cells from rat

We investigated the effect of changes in perfusate substrate and Ca content on the quality and yield of isolated adult rat heart cells. When 1 mM Ca was added to the recirculating perfusate 15 min after collagenase addition, the ATP level of cells in the heart 15 min later, and their morphology in histological section, was no different from when no Ca was added back. The cells subsequently isolated were of similar yield, but a greater percentage were rod-shaped, compared with cells isolated without Ca restoration to the perfusate. Increased yield could be obtained by including substrates in the perfusate in addition to glucose. Either fatty acids or amino acids were effective. We conclude that: (1) all cells in the heart are Ca tolerant at the end of enzyme perfusion; (2) the presence of substrates in addition to glucose can help cells survive the isolation process.     

Inhibition of hypoxic pulmonary vasoconstriction by antagonists of store-operated Ca2+ and nonselective cation channels

Previous studies indicated that acute hypoxia increased intracellular Ca(2+) concentration ([Ca(2+)](i)), Ca(2+) influx, and capacitative Ca(2+) entry (CCE) through store-operated Ca(2+) channels (SOCC) in smooth muscle cells from distal pulmonary arteries (PASMC), which are thought to be a major locus of hypoxic pulmonary vasoconstriction (HPV). Moreover, these effects were blocked by Ca(2+)-free conditions and antagonists of SOCC and nonselective cation channels (NSCC). To test the hypothesis that in vivo HPV requires CCE, we measured the effects of SOCC/NSCC antagonists (SKF-96365, NiCl(2), and LaCl(3)) on pulmonary arterial pressor responses to 2% O(2) and high-KCl concentrations in isolated rat lungs. At concentrations that blocked CCE and [Ca(2+)](i) responses to hypoxia in PASMC, SKF-96365 and NiCl(2) prevented and reversed HPV but did not alter pressor responses to KCl. At 10 microM, LaCl(3) had similar effects, but higher concentrations (30 and 100 microM) caused vasoconstriction during normoxia and potentiated HPV, indicating actions other than SOCC blockade. Ca(2+)-free perfusate and the voltage-operated Ca(2+) channel (VOCC) antagonist nifedipine were potent inhibitors of pressor responses to both hypoxia and KCl. We conclude that HPV required influx of Ca(2+) through both SOCC and VOCC. This dual requirement and virtual abolition of HPV by either SOCC or VOCC antagonists suggests that neither channel provided enough Ca(2+) on its own to trigger PASMC contraction and/or that during hypoxia, SOCC-dependent depolarization caused secondary activation of VOCC.     

Calcium and renin release: inhibition of low sodium-induced renin secretion by high calcium concentration in rat kidney perfusion

The effects of changes in calcium on renin secretion have been studied in the isolated perfused rat kidney. Perfusion with free calcium buffer significantly decreases renin secretion as compared with control experiments (Ca++: 2.5 mM/l). Other calcium concentrations (1.25 mM/l) and 5 mM/l) do not affect basal renin secretion. When the renin release is previously increased by low sodium concentration (Na+: 110 mM/l) however, perfusion with high calcium buffer (Ca++: 5 mM/l) significantly inhibits this stimulation.     

Properties and regulation of a trans-plasma membrane redox system of perfused rat heart

Ferricyanide was reduced to ferrocyanide by the perfused rat heart at a linear rate of 78 nmol/min per g of heart (non-recirculating mode). Ferricyanide was not taken up by the heart and ferrocyanide oxidation was minimal (3 nmol/min per g of heart). Perfusate samples from hearts perfused without ferricyanide did not reduce ferricyanide. A single high-affinity site (apparent K_m=22 μM) appeared to be responsible for the reduction. Perfusion of the heart with physiological medium containing 0.5 mM ferricyanide did not alter contractility, biochemical parameters or energy status of the heart. Perfusate flow rate and perfusate oxygen concentration exerted opposing effects on the rate of ferricyanide reduction. A net decreased reduction rate resulted from a decreased perfusion flow rate. Thus, the rate of supply of ferricyanide dominated over the stimulatory effect of oxygen restriction; the latter effect only becoming apparent when the oxygen concentration was lowered at a high perfusate flow rate. Whereas glucose (5 mM) increased the rate of ferricyanide reduction, pyruvate (2 mM), acetate (2 mM), lactate (2 mM) and 3-hydroxybutyrate (2 mM) each had no effect. Insulin (3 nM), glucagon (0.5 μM), dibutyryl cyclic AMP (0.1 mM) and the β-adrenergic agonist ritodrine (10 μM) also had no effect, however the α₁-adrenergic agonist, methoxamine (10 μM), produced a net increase in the rate of ferricyanide reduction. It is concluded that a trans-plasma membrane electron efflux occurs in perfused rat heart that is sensitive to oxygen supply, glucose, perfusion flow rate, and the α-adrenergic agonist methoxamine.     

The effect of lanthanum on the mechanical function of the isolated perfused rat heart at different extracellular calcium concentrations.

The effects of 50 microM lanthanum (La3+) on the contractile force, rate and coronary flow of rat hearts perfused with solutions containing 2.5, 5, 7.5 mM calcium (Ca2+) have been investigated. La3+ produced a rapid and marked decrease in contractile force within 1-3 min ("early La(3+)-effect"). The inhibition of contractility by La3+ was reduced progressively when the Ca2+ ion concentration in the perfusion fluid was raised from 2.5 to 7.5 mM. However, after 10-80 min of La3+ perfusion the contractile force was increased significantly ("late La(3+)-effect"). Elevation of Ca2+ during exposure to La3+ increased its effect. During the late La(3+)-effect, a marked decrease in heart rate and a significant increase in time to reach peak tension, time for half relaxation and twitch duration was observed. High concentrations of perfusate Ca2+ decreased the chronotropic response to La3+, in contrast, elevated Ca2+ potentiated La(3+)-induced increase in time to reach peak tension, time for half relaxation and twitch duration. La3+ produced a significant decrease in coronary flow. High Ca2+ augmented the decrease coronary flow. The findings indicate that La3+ may produce marked effects on myocardial function. High extracellular Ca2+ reduces the La(3+)-induced initial decrease in force of contraction, but potentiates the late increase in contractile force by La3+. Elevated external Ca2+ also increases the effects of La3+ on twitch parameters, heart rate and coronary flow.     

Metabolism of extracellular phosphocreatine during changes in the ionic composition of the medium in the perfused rat heart]

Perfusion of isolated rat hearts with a phosphocreatine (10(-4) M) containing solution to which strophanthin or KCl had been added up to a concentration of 27 mM as well as Ca2+ depletion decreased phosphocreatine concentration in the perfusate with a simultaneous increase in creatine and phosphocreatine concentrations in the myocardium. Neither high extracellular concentrations of Na+ (200 mM), nor phosphocreatine increased creatine and phosphocreatine levels in the myocardium. The effect of high sodium perfusion media was completely reversed by strophanthin. Phosphocreatine decreased the lactate content in the perfusate. Strophanthin or potassium chloride enhanced the effect of phosphocreatine on the lactate release. Conversely, creatine augmented the lactate content in the perfusate. A high specificity of the phosphocreatine effect on the myocardium independently of the ionic composition of the perfusate was postulated. A mechanism of protective effects of phosphocreatine and high sodium perfusion media on "calcium paradox" is proposed.     

Evidence that isoproterenol-induced Ca2(+)-mobilization in rat parotid acinar cells is not mediated by activation of beta-adrenoceptors

The effects of isoproterenol (ISO), a beta-adrenoceptor agonist, on cytosolic free Ca2+ ([Ca2+]i) in rat parotid acinar cells were examined using the fluorescent Ca2(+)-indicator fura-2. At concentrations up to 1 mM, ISO caused a rapid increase in [Ca2+]i in a dose-dependent manner, while addition of 1 microM ISO, which evokes the maximum amylase secretion, had only a slight effect on [Ca2+]i. There was no such increase in [Ca2+]i with the addition (2 mM) of 8-bromo-cyclic AMP, a permeant cyclic AMP analogue. The alpha-adrenoceptor antagonist phentolamine blocked the ISO-induced [Ca2+]i increase better than the beta-adrenoceptor antagonist, propranol, and the muscarinic receptor antagonist, atropine. The IC50 value (the concentration which reduces the ISO-induced increase in [Ca2+]i by 50%) of phentolamine was estimated to be 7.6 nM, for propranolol 13.2 microM and for atropine 3.5 microM. The difference in potency between the three antagonists was similar to the difference in blocking the [Ca2+]i increase induced by phenylephrine, an alpha-adrenoceptor agonist. These results suggest that the Ca2(+)-mobilization in response to high concentrations of ISO results from an activation of alpha-adrenoceptors rather than beta-adrenoceptors.     

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.     

Amelioration of Cold Injury-Induced Cortical Brain Edema Formation by Selective Endothelin ETB Receptor Antagonists in Mice

Brain edema is a potentially fatal pathological condition that often occurs in stroke and head trauma. Following brain insults, endothelins (ETs) are increased and promote several pathophysiological responses. This study examined the effects of ET_B antagonists on brain edema formation and disruption of the blood-brain barrier in a mouse cold injury model (Five- to six-week-old male ddY mice). Cold injury increased the water content of the injured cerebrum, and promoted extravasation of both Evans blue and endogenous albumin. In the injury area, expression of prepro-ET-1 mRNA and ET-1 peptide increased. Intracerebroventricular (ICV) administration of BQ788 (ET_B antagonist), IRL-2500 (ET_B antagonist), or {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 (ET_A antagonist) prior to cold injury significantly attenuated the increase in brain water content. Bolus administration of BQ788, IRL-2500, or {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 also inhibited the cold injury-induced extravasation of Evans blue and albumin. Repeated administration of BQ788 and IRL-2500 beginning at 24 h after cold injury attenuated both the increase in brain water content and extravasation of markers. In contrast, {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 had no effect on edema formation when administrated after cold injury. Cold injury stimulated induction of glial fibrillary acidic protein-positive reactive astrocytes in the injured cerebrum. Induction of reactive astrocytes after cold injury was attenuated by ICV administration of BQ788 or IRL-2500. These results suggest that ET_B receptor antagonists may be an effective approach to ameliorate brain edema formation following brain insults.     

Functional characteristics of calcium-sensitive adenylyl cyclase of ciliate Tetrahymena pyriformis

Calcium-sensitive forms of adenylyl cyclase (AC) were revealed in most vertebrates and invertebrates and also in some unicellular organisms, in particular ciliates. We have shown for the first time that calcium cations influence the AC activity of ciliate Tetrahymena pyriformis. These cations at the concentrations of 0.2-20 microM stimulated the enzyme activity, and maximum of catalytic effect was observed at 2 microM Ca2+. Calcium cations at a concentrations of 100 microM or higher inhibited the AC activity. Calmodulin antagonists W-5 and W-7 at the concentrations of 20-100 microM inhibited the catalytic effect induced by 5 microM Ca2+ and blocked the effect at higher concentrations of Ca2+. Chloropromazine, another calmodulin antagonist, reduced Ca2+-stimulated AC activity only at the concentrations of 200-1000 microM. AC stimulating effects of serotonin, EGF and cAMP increased in the presence of 5 microM Ca2+. AC stimulating effects of EGF, cAMP and insulin decreased in the presence of 100 microM Ca2+, and AC stimulating effect of cAMP decreased also in the presence of calmodulin antagonists (1 mM). At the same time, stimulating effect of D-glucose in the presence of Ca2+ and calmodulin antagonists did not change essentially. The data obtained speak in favor of the presence of calcium-sensitive forms of AC in ciliate T. pyriformis which mediate enzyme stimulation by EGF, cAMP, insulin, and serotonin.     

The effect of adrenaline and high Ca2+ on the mechanical performance and oxygen consumption of the isolated perfused trout (Oncorhynchus mykiss) heart

In heart muscle from mammals, catecholamines frequently evoke an oxygen waste and reduce efficiency. It was examined if this also applies to fish in which heart muscle activity is often restricted by oxygen availability. In the isolated perfused heart from rainbow trout, adrenaline (0.5 microM) increased power output by approximately 97%, when afterload was adjusted to maximum both before and after adrenaline addition, and by approximately 68%, when afterload remained at the maximum obtained before adrenaline addition. Oxygen consumption was enhanced by a similar amount (approximately 70%) in both situations. Hence, efficiency, i.e. power output/oxygen consumption, increased significantly from 25 to 30% for the heart always exposed to maximal afterload, whereas it stayed unchanged at 24% for the heart exposed to control afterload only. Adrenaline increases the Ca2+ activity participating in activation, but doing this by elevating perfusate Ca2+ from 1.5 to 5 mM instead of applying adrenaline did not change the mechanical efficiency, although afterload was maximized. The results suggest that catecholamines enhance heart pump activity in the living trout without any oxygen waste. On the contrary, the oxygen budget may even improve when the peripheral resistance is increased by catecholamines.     

ETA receptor-mediated role of endothelin in the kidney of doca-salt hypertensive rats

Renal effects of FR139317, an endothelin ET_A receptor antagonist, were examined using anesthetized normotensive and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. The intravenous bolus injection of FR139317 (10 mg/kg) produced a slight decrease in mean blood pressure (MAP; −13%) in the control rats and this hypotension was accompanied by a moderate renal vasodilation (renal vascular resistance: RVR; −12%). In the DOCA-salt hypertensive rat, FR139317 had a more pronounced hypotensive effect (MAP; −26%) accompanied by a potent renal vasodilation (RVR; −33%). FR 139317 significantly increased renal blood flow only in the DOCAsalt rats. In contrast, FR139317 produced a significant decrease in urine flow and urinary sodium excretion only in control rats. Northern blot analysis revealed that the renal prepro endothelin-1 (ET-1) mRNA level was significantly increased in DOCA-salt hypertensive rats. Thus, it seems likely that endogenous ET-1 is responsible for the maintenance of DOCA-salt-induced hypertension. We also suggest that at least in part, ET-1 and £ta receptors are involved in renal hemodynamic abnormalities in DOCA-salt-induced hypertension. The augmentation of renal ET-1 production may possibly have a function in the development and maintenance of DOCA-salt-induced hypertension.     

Effect of Varying the Ionic Concentration of a Microdialysis Perfusate on Basal Striatal Dopamine Levels in Awake Rats

In vivo microdialysis was used to study the effects of Ca2+, Mg2+, and K+ ion concentrations on basal extracellular (EC) levels of striatal DA and metabolites in awake rats on the second day (48 h) after implantation of a microdialysis probe. Basal EC striatal dopamine (DA) levels were markedly (90%) and reversibly reduced by removal and subsequent replacement of Ca2+ ions from the microdialysis perfusate. This implies that the EC DA in this preparation is primarily of synaptic origin. The addition and subsequent removal of 1.7 mM MgCl2 to the Mg2(+)-free perfusate produced a reversible decrease (20%) in basal EC DA levels. This decrease may reflect a competitive interaction between Ca2+ and Mg2+ in the process of vesicular release. Basal EC DA levels were also reduced (27%) by decreasing the K+ concentration of the perfusate from 4 mM to 3 mM. However, after restoring the K+ concentration to 4 mM, EC DA levels were slow to return to pretreatment levels. Basal EC 3,4-dihydroxyphenylacetic acid and homovanillic acid levels exhibited a parallel but diminished response to each manipulation of the ionic concentration of the perfusate. This study demonstrates that small variations in the concentrations of Ca2+, Mg2+, and K+ in the perfusate employed in microdialysis preparations will affect basal EC striatal DA and metabolite levels.     

The effects of low concentrations of caffeine on spontaneous Ca release in isolated rat ventricular myocytes

We have investigated the effects on spontaneous SR Ca release of modulating the sarcoplasmic reticulum ryanodine receptor (RyR) with low (<0.5 mM) concentrations of caffeine. Experiments were performed on isolated rat ventricular myocytes. Intracellular Ca concentration was measured with Indo-1 or Fluo-3 in voltage-clamped cells. Spontaneous Ca release was produced by elevating external Ca to 5 mM. Caffeine application increased the frequency of spontaneous release. Both the magnitude of the spontaneous Ca transients and the integral of the resulting Na-Ca exchange current were decreased by caffeine. The combination of increased frequency of spontaneous release and decreased Ca efflux per event meant that the Ca efflux per unit time was unaffected by low concentrations of caffeine. The SR Ca content was reduced by caffeine. The extra Ca efflux calculated from the Na-Ca exchange current integrals occurring during the initial burst of spontaneous activity on application of caffeine accounted for this reduction of SR Ca content. In contrast to these maintained effects on spontaneous release, caffeine had only transient effects on stimulated Ca release produced by depolarizing pulses. We conclude that stimulation of the RyR results in spontaneous release at SR Ca contents lower than those at which release would normally occur. Therefore, the balance between normal and spontaneous Ca release can be shifted by modulation of the RyR. This will have important consequences for arrhythmogenesis due to spontaneous Ca release.     

Calcium fluxes in isolated acinar cells from rat parotid. Effect of adrenergic and cholinergic stimulation.

Rat parotid acinar cells dispersed by a combination of enzymatic treatments remain sensitive to adrenergic and cholinergic agonists. Previous studies have implicated Ca2+ in both adrenergic and cholinergic responses. This paper describes the effects of adrenergic and cholinergic stimulation upon 45Ca2+ fluxes in isolated parotid acinar cells. Suspensions of dispersed cells took up 45Ca2+ from the medium. The net rate of isotope influx was increased by the adrenergic agonists epinephrine, norepinephrine, isoproterenol, and phenylephrine, and by the cholinergic agonists acetylcholine and carbamylcholine. In 1 mM Ca2+, epinephrine was capable of increasing the 45Ca2+ influx in 40 min to three times that of resting cells. Isoproterenol, a beta-adrenergic agonist, was only half as effective as epinephrine in stimulating maximal calcium uptake although it was equally effective in stimulating maximal amylase release in the same cells. Experiments with the alpha-adrenergic antagonist phentolamine, the beta-adrenergic antagonist propranolol, and the cholinergic antagonist atropine confirmed that alpha- and beta-adrenergic and cholinergic stimulation each had a direct stimulatory effect on 45Ca2+ uptake. N6,O2'-Dibutyryl adenosine 3':5'-monophosphate also caused some stimulation of net calcium uptake. Direct measurement of Ca2+ efflux indicated that the increased calcium uptake in the presence of epinephrine was not the indirect result of a decrease in efflux. The rates of both basal and epinephrine-stimulated calcium uptake increased with increasing calcium concentration in the medium. Epinephrine had little effect on the rate of calcium uptake at 0.15 mM Ca2+. Although the energy poison NaCN had little effect on the basal rate of calcium uptake, the stimulable component of calcium uptake was inhibited by NaCN at all calcium concentrations tested (0.2 to 4.1 mM).     

Effects of Endogenous Glutamate on Extracellular Concentrations of GABA, Dopamine, and Dopamine Metabolites in the Prefrontal Cortex of the Freely Moving Rat: Involvement of NMDA and AMPA/KA Receptors

Using microdialysis, interactions between endogenous glutamate, dopamine, and GABA were investigated in the medial prefrontal cortex of the freely moving rat. Interactions between glutamate and other neurotransmitters in the prefrontal cortex had already been studied using pharmacological agonists or antagonists of glutamate receptors. This research investigated whether glutamate itself, through the increase of its endogenous extracellular concentration, is able to modulate the extracellular concentrations of GABA and dopamine in the prefrontal cortex. Intracortical infusions of the selective glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) were used to increase the endogenous extracellular glutamate. PDC (0.5, 2, 8, 16 and 32 mM) produced a dose-related increase in dialysate glutamate in a range of 1–36 M. At the dose of 16 mM, PDC increased dialysate glutamate from 1.25 to 28 M. PDC also increased extracellular GABA and taurine, but not dopamine; and decreased extracellular concentrations of the dopamine metabolites DOPAC and HVA. NMDA and AMPA/KA receptor antagonists were used to investigate whether the increases of extracellular glutamate were responsible for the changes in the release of GABA, and dopamine metabolites. The NMDA antagonist had no effect on the increase of extracellular GABA, but blocked the decreases of extracellular DOPAC and HVA, produced by PDC. In contrast, the AMPA/KA antagonist blocked the increases of extracellular GABA without affecting the decreases of extracellular DOPAC and HVA produced by PDC. These results suggest that endogenous glutamate acts preferentially through NMDA receptors to decrease dopamine metabolism, and through AMPA/KA receptors to increase GABAergic activity in the medial prefrontal cortex of the awake rat.     

The mechanism of the effect of K+ on the steroidogenesis of rat zona glomerulosa cells of the adrenal cortex: role of cyclic AMP

The effects of various concentrations of extracellular K+ (3.6-13 mM) on the steroid (corticosterone and aldosterone) and cyclic AMP outputs of capsular cells (95% zona glomerulosa) of the rat adrenal cortex were studied at different concentrations of extracellular Ca2+. Small amounts of EGTA (50 microM) were added to reduce the free Ca2+ concentrations effectively to zero at the lowest possible total Ca2+ concentration. At a total extracellular concentration of 2.5 mM Ca2+, in 27 experiments the mean values of the steroid and cAMP outputs showed a maximum at 8.4 mM K+. The increase in steroid and cAMP outputs at 5.9, 8.4 and 13 mM K+ compared with that at 3.6 mM were highly significant (p less than 0.01). The overall correlation of either corticosterone or aldosterone with cAMP outputs was also highly significant and was even better from 3.6 to 8.4 mM K+. Lowering the effective free concentration of Ca2+ to zero decreased the steroid and cAMP outputs significantly at all K+ concentrations, and no output was then significantly higher than at 3.6 mM. With the pooled data on outputs at all total Ca2+ (2.5, 0.5, 0.25, 0.10, 0.05 and 0.0 mM) and K+ (3.6, 5.9, 8.4 and 13 mM) concentrations, the correlation of either steroid with cAMP outputs was highly significant (but again optimally from 3.6 to 8.4 mM K+). Nifedipine (10(-6) to 10(-4) M) was added to the incubations with the aim of specifically inhibiting Ca2+ influx at total extracellular Ca2+ concentrations of 2.5, 1.25 and 0.25 mM and with the usual K+ concentrations. The cAMP outputs were reduced at all K+ concentrations above 3.6 mM K+. The effect was highly significant at 10(-4) M nifedipine and a total Ca2+ of 1.25 mM, which with the incubation conditions used, corresponds to the free Ca2+ concentrations in vivo. These results indicate that cAMP plays a significant role in the stimulation of steroid output by K+ particularly between 3.6 and 8.4 mM K+. In this range of K+ concentrations the stimulation of cAMP seems to be controlled by increases in Ca2+ influx. The correlation of steroid and cAMP output at the higher K+ concentrations (between 8.4 and 13 mM K) and at the various total Ca2+ concentrations is less significant. Also, with all concentrations of added nifedipine there is an 'anomalous' increase in steroid output at 13 mM K+ and at total Ca2+ concentrations of 2.5 and 1.25 mM. However, at the same K+ concentrations and at 0.25 mM Ca2+, nifedipine decreases steroid outputs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reversible elimination of myofibrillar Ca2+ sensitivity by diamide and other sulfhydryl reagents: comparison with reversible contracture produced in intact cells

Cardiac contractile activity is usually controlled by intracellular Ca2+, but it can also be modified by oxidizing agents. Incubation of guinea pig heart myofibrils with diamide (3 mM, 1 h) increased basal (no Ca2+) ATPase activity by 580% and abolished Ca2+ dependence. The effect was proportional to diamide concentration (0.01-1 mM) and duration of preincubation (up to 2 h). Dithiothreitol (5 mM, 1 h) reversed most of the basal ATPase activation and restored Ca2+ sensitivity. Other sulfhydryl reagents produced a similar effect but also produced inhibition of total ATPase. In intact cell preparations, diamide produced a slow tonic contraction, consistent with myofibril activation. In the perfused rat heart, 1 mM diamide slowly increased diastolic ventricular pressure; this increase was partially reversed by dithioerythritol. In isolated rat heart myocytes, 1 mM diamide produced a slow tonic contraction, increased contractility in response to stimulation. Cardiocytes superfused for 1 h with buffer containing EGTA to deplete Ca2+ did not contract in response to stimulation but showed a slow tonic contraction with diamide. This contraction could be slowly and only partially reversed by dithioerythritol. Response to stimulation was restored by addition of Ca2+. The results show that diamide can produce contraction in viable cells. This contraction does not require extracellular Ca2+ and is unlikely to involve intracellular Ca2+. The direct activation of myofibrillar ATPase may contribute to the increased myocardial stiffness seen in ischemia and to ischemic contracture.     

Glucose and octanoate utilization by isolated adult rat heart cells

Rat heart muscle cells continue to beat in the isolated state apparently independent of any innervation. The oxidation of ¹⁴C-glucose to ¹⁴CO₂ was linear for at least 60 minutes of incubation. The rate of glucose oxidation rose rapidly up to a medium glucose concentration of 2.5 mM and then plateaued. Lactate production reached a maximum at 5 mM glucose. Glucose uptake was linearly related to the concentration up to 40 mM. The addition of octanoate reduced, but did not eliminate, glucose oxidation. Octanoate utilization increased with increasing concentration and reached a maximum at 2 mM. The oxidation of octanoate was linearly related to the time of incubation for at least 90 minutes. The presence of glucose, at a concentration of 1.25 mM or higher, increase the oxidation of octanoate by the heart cells. The metabolic parameters measured with the isolated heart cells gave values comparable to those obtained with the perfused rat heart. Decreasing or increasing the concentration of sodium, potassium or magnesium did not effect the oxidation of either glucose or octanoate with the exception that when sodium was increased above 200 mM, a significant increase in glucose oxidation was observed. In contrast, the addition of calcium to a calcium free medium increased glucose oxidation, reaching a maximum at 0.2 mM calcium. The oxidation of octanoate reached a maximum at 0.2 mM and then decreased significantly with increasing calcium concentration. The metabolic activity appears to be independent of the concentration of sodium, potassium or magnesium. In contrast, the isolated heart cell is very sensitive to a change in calcium concentration.