Cultured Chick Embryo Ventricular Cells Research Articles

Activation of furosemide-sensitive K+ fluxes in myocytes by ouabain and recovery from metabolic inhibition

Modulation of transsarcolemmal K+ flux mediated by the furosemide-sensitive K(+)-Cl- (or Na(+)-K(+)-Cl-) cotransport carrier was studied in cultured chick embryo ventricular cells. We defined at least three distinct K+ efflux pathways: 1) a Ba2(+)-sensitive efflux component, probably reflecting K+ movement through K+ channels; 2) a furosemide-sensitive component, reflecting K(+)-Cl- cotransport; and 3) a component insensitive to both Ba2+ and furosemide. With respect to K+ influx, there were 1) a ouabain-sensitive K+ uptake presumably mediated by Na(+)-K(+)-adenosinetriphosphatase and 2) a furosemide-sensitive K+ uptake. The effects of elevation of intracellular calcium concentration ([Ca2+]i) on Ba2+ and furosemide-sensitive K+ flux pathways were studied. Elevation of [Ca2+]i had minor effects on Ba2(+)-sensitive K+ flux. However, elevation of [Ca2+]i produced by exposure to ouabain for 60 min activated a furosemide-sensitive 42K+ efflux and a ouabain-resistant, furosemide-sensitive 42K+ influx. The activation of K+ influx, caused by an increase in [Ca2+]i, was completely inhibited by ATP depletion (produced by exposure to ouabain and metabolic inhibitors simultaneously) and was partially inhibited by the calmodulin inhibitor W7. Activation of the furosemide-sensitive K+ flux was also produced by washout of metabolic inhibitors, a condition in which ATP resynthesis occurs in the presence of an increased [Ca2+]i. Activation of furosemide-sensitive K+ fluxes by exposure to ouabain or washout of metabolic inhibitors caused a net K+ loss, which accounts in part for the cell shrinkage noted during recovery from metabolic inhibition in previous studies. These results suggest that [Ca2+]i and intracellular ATP concentration are important in the regulation of furosemide-sensitive K+ flux in these cells, perhaps via the involvement of a Ca2(+)-calmodulin-dependent protein kinase.

Hypothermia increases calcium content of hypoxic myocytes

To test the hypothesis that hypothermia prevents myocardial Ca2+ loading during reoxygenation, we examined the effects of 2 h of hypoxia with and without hypothermia on the Ca2+ content of cultured chick embryo ventricular cells. When compared with hypoxic cells at 37 degrees C, hypoxia at 11 degrees C (hypothermia) augmented the 45Ca content of cardiocytes after 30 min of normothermic reoxygenation from 3.85 +/- 0.2 to 4.7 +/- 0.1 nmol/mg protein (P less than 0.001). The Na+ content of hypoxic myocytes was also increased at the end of 2 h of hypoxia from 648 +/- 59 to 1,026 +/- 68 nmol/mg protein in cells exposed to hypoxia at 11 degrees C (P less than 0.001). Hypothermia ameliorated hypoxia-induced depression of cellular ATP content and did not result in significant membrane injury as determined by lactate dehydrogenase release. These data indicate that hypothermia augments rather than decreases the Ca2+ content of hypoxic myocytes during reoxygenation after hypoxia. Ca2+ loading appears to be secondary to an increase in Na+ content, creating a favorable gradient for Ca2+ influx through Na(+)-Ca2+ exchange or an unfavorable gradient for Ca2+ extrusion.

Myocyte Response to Stimulation of Receptors and Ion Channels

Ventricular myocytes dissociated from adult rat heart and cultured chick embryo ventricular cells were utilized to examine mechanisms by which neurotransmitters, hormones, and ontogeny modulate expression and function of beta-adrenergic receptors and L-type calcium channels. Either freshly dissociated cells or cultured cells were studied by an optical-video system to characterize contractility and, in some instances, by a microspectrofluorimeter to determine [Ca2+]i as reported by fura 2. Ligand binding studies in intact cells and membranes were conducted with receptor and ion channel antagonists and agonists. Exposure of intact cells to isoproterenol produced contractile de-sensitization, loss of high affinity receptors from the sarcolemma and closely coupled decline in hormone-sensitive adenylate cyclase activity. De-sensitization was by a microfilament-dependent process. Down-regulation depended upon microtubular function. During development of the chick heart, there was an increase in number of dihydropyridine binding sites, taken as a measure of number of L-type calcium channels, at a time when sensitivity to [Ca2+]o and to Bay k 8644 declined. Thyroid hormone was capable of up-regulating L-type calcium channels. Prolonged exposure to a beta-adrenergic agonist produced coordinate down-regulation of beta-receptors and calcium channels. Down-regulation was a cAMP-dependent process. Thus, the beta-adrenergic receptor and a distal component of the effector-response coupling system, the L-type calcium channel, can be regulated independently and in concert by physiologically and pathophysiologically important mechanisms.

Coregulation of calcium channels and beta-adrenergic receptors in cultured chick embryo ventricular cells.

To examine mechanisms whereby the abundance of functional Ca channels may be regulated in excitable tissue, Ca channel number was estimated by binding of the dihydropyridine (DHP) antagonist 3H (+)PN200-110 to monolayers of intact myocytes from chick embryo ventricle. Beta adrenergic receptor properties were studied in cultured myocytes using [3H]CGP12177, an antagonist ligand. Physiological correlates for alterations in DHP binding site number included 45Ca uptake and contractile response to (+)BAYk 8644, a specific L-type Ca channel activator. All binding and physiological determinations were performed in similar intact cell preparations under identical conditions. 4-h exposure to 1 microM isoproterenol reduced cell surface beta-adrenergic receptor number from 44 +/- 3 to 17 +/- 2 fmol/mg (P less than 0.05); DHP binding sites declined in number from 113 +/- 25 to 73 +/- 30 fmol/mg (P less than 0.03). When protein kinase A was activated by a non-receptor-dependent mechanism, DHP binding declined similarly to 68% of control. Exposure to diltiazem, a Ca channel antagonist, for 18-24 h had no effect on number of DHP binding sites. After 4-h isoproterenol exposure, 45Ca uptake stimulated by BAYk 8644 declined from 3.3 +/- 0.2 nmol/mg to 2.9 +/- 0.3 nmol/mg (P less than 0.01) and BAYk 8644-stimulated increase in amplitude of contraction declined from 168 +/- 7 to 134 +/- 11% (P = 0.02). Thus, elevation of [cAMP] in myocytes is associated with a time-dependent decline in Ca channel abundance as estimated by DHP binding and a decline in physiological responses that are in part dependent on abundance of Ca channels. Binding of a directly acting Ca channel antagonist for 18-24 h does not modulate the number of DHP binding sites.

Interaction between isoproterenol and dihydropyridines in heart cells

The negative inotropic potencies (IC 50) of the calcium channel blockers nicardipine (12 ± 2 nM) and nifedipine (9 ± 2 nM) measured in cultured chick embryo ventricular cells were not significantly different. Exposure of the cells to isoproterenol potentiated the negative inotropic effect of low concentrations of nicardipine (10 −9-10 −8 M) and decreased the effect of higher concentrations (10 −7 M). In contrast, isoproterenol shifted the entire dose-response curve for nifedipine to the right. These results provide evidence that activation of β-adrenoceptors modulates the negative inotropic effect of nifedipine and nicardipine differently.

Atrial natriuretic peptide decreases contractility of cultured chick ventricular cells

To determine whether atrial natriuretic peptide (ANP) has an inotropic effet, the contractility of spontaneously beating cultured chick embryo ventricular cells was studied in response to rat-ANP (1–23) superfused at concentrations ranging from 10 −10 M to 2.5 × 10 −7 M. r-ANP reversibly decreased contractility with a threshold concentration of 10 −8 M; at the highest concentration, r-ANP decreased contractility to a moderate extent (−30 ± 4 %) r-ANP increased dose-dependently intracellular cGMP levels. Stimulation of contractility with [Ca2+], the calcium-channel agonist BAY K 8644 or isoproterenol attenuated to various degrees the inhibitory effect of r-ANP. By contrast, the inhibitory effect of r-ANP on contractility was unchanged or even enhanced after stimulation of contractility by angiotensin II. There was no difference in r-ANP-induced increase in cGMP whether cells were pre-incubated with angiotensin II or not. These results indicate that r-ANP was able to decrease contractility of cultured cardiac myocytes and suggest a preferential antagonism of the inotropic effect of angiontensin II.

Endothelin has potent direct inotropic and chronotropic effects in cultured heart cells

To determine whether endothelin, a highly potent vasoconstrictor peptide, may affect cardiac myocyte contractility directly, we studied the effects of synthetic porcine endothelin-1 in cultured chick embryo ventricular cells. Endothelin-1 had a potent chronotropic effect (EC50 0.17 nmol/l) in spontaneously beating cells. The increase in the beating rate was accompanied by a frequency-dependent decrease in the amplitude of contraction. In electrically driven cells (1 Hz), endothelin-1 increased the amplitude of contraction dose-dependently, with an EC50 of 0.3 nmol/l, smaller than that of the calcium channel blocker BAY K 8644 (EC50 3.3 nmol/l), and with an efficacy close to that of BAY K 8644 and isoproterenol. Nicardipine (100 nmol/l) shifted to the right, by two orders of magnitude, the dose-response curve of endothelin-1 for inotropism. These results indicate that endothelin-1 is one of the most potent inotropic agents in cultured cardiac myocytes, and suggest that this effect involves, at least in part, a calcium ion influx through voltage-sensitive calcium channels.

Beta-adrenergic receptor regulation during hypoxia in intact cultured heart cells

Regulation of cardiac beta-adrenergic receptors during hypoxia and ischemia is an area of active investigation, with some investigators reporting an increase in sarcolemmal beta-receptor number after ischemia. Previous studies have been limited by the necessity of examining beta-adrenergic receptor properties in membrane preparations from hypoxic or ischemic cardiac tissue and drawing conclusions about receptor localization in intact tissue from the behavior of a fraction of total receptors in membrane populations. As an approach to examining beta-receptor properties under well-defined pathophysiological conditions in intact heart cells, we studied cell-surface beta-receptors and adenylate cyclase activity in intact cultured chick embryo ventricular cells under conditions of controlled hypoxia and reoxygenation. During 2 h of hypoxia (PO2 less than 1.5 Torr) there was a progressive decline in cell surface beta-receptors from 26 +/- 2 to 10 +/- 6 fmol/mg (P less than 0.003) with no change in antagonist or agonist affinity. Receptor number recovered fully during 2 h of reoxygenation. Basal adenosine 3',5'-cyclic monophosphate (cAMP) production was unchanged, but response to isoproterenol in the absence or presence of a phosphodiesterase inhibitor decreased to about half of the response for normoxic cells but fully recovered during reoxygenation in a pattern similar to that for receptor number. Although [ATP] declined significantly during hypoxia (from 35 to 25 nmol/mg), the decline in [GTP] was marginal (4.3 to 3.9 nmol/mg), making it unlikely that substrate for guanine nucleotide regulatory protein was limiting for beta-adrenergic signal transduction.(ABSTRACT TRUNCATED AT 250 WORDS)

A dihydropyridine calcium channel blocker with phosphodiesterase inhibitory activity: Effects on cultured vascular smooth muscle and cultured heart cells

To define the cellular mechanism of action of a dihydropyridine Ca channel antagonist in an experimental model system devoid of neural influences and reflex effects, we studied the actions of RS93522 on cultured vascular smooth muscle cells and on cultured chick embryo ventricular cells. 45Ca uptake by monolayer cultures of vascular smooth muscle cells was inhibited in a concentration-dependent manner. The IC 50 for this effect was 10 n m, similar to that for nifedipine (7 n m) in the same system. 10 −6 m RS93522 inhibited 45Ca uptake more fully than 10 −6 m nifedipine ( P < 0.05). Using an optical-video system, the effect of RS93522 on amplitude of contraction of spontaneously beating cultured ventricular cells was studied. Amplitude of contraction was inhibited with IC 50 = 7.9 × 10 −8 m. 45Ca uptake in myocytes was depressed by 15% at 5 min. RS93522 had the additional property of inhibiting phosphodiesterase activity in myocardial homogenates with IC 50 = 1.6 × 10 −5 m; the potency and efficacy of phosphodiesterase inhibition was similar to that for milrinone in the same system. As expected of Ca channel antagonists, it has a negative inotropic effect on cultured myocardial cells. The compound also has phosphodiesterase inhibitory activity that possibly may potentiate vasodilatation and ameliorate, in part, negative inotropic effects. Thus, RS93522 has two distinct pharmacodynamic effects in myocytes and is a potent calcium channel blocker.

Effects of contrast media on calcium transients and motion in cultured ventricular cells.

To investigate the mechanisms of the negative inotropic effects of contrast media, we superfused spontaneously contracting cultured chick embryo ventricular cells with Renografin-76 and iohexol (12% solutions), and hypertonic sucrose during simultaneous measurement of [Ca2+]i transients (indo-1) and motion (video-motion detector system). Exposure to contrast agents caused a significant reduction of contractility, with Renografin-76 having a much greater effect on amplitude of motion than iohexol. Renografin-76 significantly depressed [Ca2+]i transient amplitude, whereas iohexol had no effect. Addition of Ca2+ to correct for calcium binding by Renografin-76 completely reversed its depression of [Ca2+]i transients but only partially reversed the negative inotropic effects. Hypertonic sucrose caused a significant decrease in contraction amplitude, with no significant effects on [Ca2+]i transient amplitude. We conclude that the marked negative inotropic effect of Renografin-76 is caused by both calcium binding and hypertonicity. The less marked depression of contractility produced by iohexol likely is a result of hypertonicity and not caused by alteration of [Ca2+]i.

Central and peripheral cardiovascular effects of the enantiomers of the calcium antagonist PN 200-110

The negative inotropic effects and the central and peripheral hypotensive effects of (+) and (−) PN 200-110 were investigated in cultured chick heart cells and in spontaneously hypertensive rats, respectively. There was a large difference in negative inotropic potency between the two enantiomers in cultured chick embryo ventricular cells: the (+) enantiomer was 140 fold more potent (IC 50 = 1.1 ±0.2 nM) than the (−) enantiomer (IC 50 = 160 ± 20 nM). (+) PN 200-110 was 10 fold more potent than (−) PN 200-110 in lowering blood pressure after intravenous injection and only three fold more potent after intra-cerebroventricular injection (i.c.v.) into pentobarbital-anaesthetized spontaneously hypertensive rats. I.c.v. administered (+) PN 200-110 (1 μg/kg) partially antagonized the hypertensive response to i.c.v. administered BAY K 8644 (30 μg/kg), a calcium channel agonist, while the same dose of the (−) enantiomer did not change the i.c.v. BAY-induced increase in blood pressure. These results suggest that the dihydropyridine calcium channel antagonist, PN 200-110, may act centrally and stereoselectively at the level of the dihydropyridine receptor sites involved in the control of blood pressure in spontaneously hypertensive rats.

Cellular mechanisms underlying calcium-proton interactions in cultured chick ventricular cells.

1. Cytosolic free Ca2+ concentration ([Ca2+]i) in cardiac muscle cells is influenced by many factors including intracellular pH. Intracellular alkalinization has been shown to reduce, whereas acidification has been shown to augment [Ca2+]i. We examined the cellular mechanisms underlying Ca2+-H+ interactions using cultured chick embryo ventricular cells. 2. Cells were loaded with fura-2 or BCECF (2,7-biscarboxyethyl-5(6)-carboxy-fluorescein) and changes in time-averaged [Ca2+]i or pHi were monitored continuously using a dual-wavelength spectrofluorometer. 3. Exposure of cells to 20 mM-NH4Cl (intracellular alkalinization) produced a rapid decrease in [Ca2+]i; subsequent wash-out of NH4Cl (intracellular acidification) resulted in an increase in [Ca2+]i to levels above control. Intracellular acidification produced by elevated CO2 content also resulted in an increase in [Ca2+]i. The Na+-H+ exchange inhibitor ethylisopropylamiloride (10 microM) inhibited completely the rise but not the fall in [Ca2+]i in response to manipulation of pHi with NH4Cl. 4. In the presence of caffeine (20 mM), NH4Cl produced a decrease in [Ca2+]i similar to that observed in the absence of caffeine, but subsequent removal of NH4Cl resulted in an increase in [Ca2+]i that was distinctly smaller than that observed in the absence of caffeine. Ryanodine (10 microM) had no significant influence on NH4Cl-induced changes in [Ca2+]i. 5. Following treatment with the mitochondrial inhibitors sodium cyanide (5 mM), CCCP (carbonyl cyanide m-chlorophenyl hydrazone, 10 microM) or rotenone (10 microM), the NH4Cl-induced decrease in [Ca2+]i was markedly diminished, but wash-out of NH4Cl resulted in increases in [Ca2+]i similar to those observed in control cells. 6. Inhibition of glycolysis with 20 mM-2-deoxyglucose did not significantly alter the changes in [Ca2+]i induced by NH4Cl addition or its wash-out, but 2-deoxyglucose plus cyanide abolished the decrease in [Ca2+]i produced by intracellular alkalinization and nearly completely blocked the increase in [Ca2+]i produced by acidification. 7. In all experiments, the increase in [Ca2+]i during wash-out of NH4Cl was inhibited by ethylisopropylamiloride.(ABSTRACT TRUNCATED AT 400 WORDS)

Simultaneous measurement of calcium transients and motion in cultured heart cells.

The fluorescent Ca2+ probe indo-1 is a new intracellular Ca2+ concentration [( Ca2+]i) indicator that may be suitable for measurement of [Ca2+]i transients in intact heart cells. We exposed spontaneously contracting cultured chick embryo ventricular cells (37 degrees C) to the membrane-permeable indo-1-acetoxymethyl ester (indo-1 AM). Indo-1 loading was associated with a decrease in the amplitude of contraction measured with a video motion detector, but contractility returned to control levels during a subsequent 30-min wash. Analysis of emission spectra of dye obtained by digitonin permeabilization of cells loaded in indo-1 AM showed that the active intracellular dye was not pure indo-1 but probably includes partially deesterified molecules. With the use of an inverted X40 objective epifluorescence system, washed cells containing indo-1 were excited at 360 nm, and fluorescence intensity was measured at 410 nm (increases with increasing [Ca2+]) and 480 nm (decreases with increasing [Ca2+]). Calibration of the [Ca2+]i signals, reflected by the ratio of 410 to 480 nm fluorescence, was achieved by use of ethylen-glycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA)-Ca2+ buffered solutions containing the nonfluorescent Ca2+ ionophore Bromo-A23187. Average end-diastolic and peak-systolic [Ca2+]i were 328 +/- 32 and 813 +/- 72 nM (means +/- SE, n = 8). The onset of the [Ca2+]i transient preceded motion by 27 +/- 5 ms (means +/- SE, n = 4), but generally resembled the motion signals in contour. These findings indicate that indo-1 may be used to detect [Ca2+]i transients in isolated ventricular cells without causing significant alterations in mechanical performance.

Role of changes in [Ca2+]i in energy deprivation contracture.

Mechanisms of energy deprivation contracture were investigated in cultured chick embryo ventricular cells. In the presence of zero-extracellular-Na+, (choline chloride substitution)-nominal-zero-Ca2+ [( Ca2+] approximately 5 microM), exposure of ventricular cells to 1 mM cyanide (CN) and 20 mM 2-deoxyglucose (2-DG)-zero-glucose solution resulted in the development of a contracture (video motion detector) in 5.9 +/- 0.5 minutes. Early after contracture development, the resupply of extracellular Na+, in the continued presence of CN + 2-DG, resulted in a rapid partial relaxation (t1/2 = 1.9 +/- 0.3 seconds), associated with an increase in 45Ca efflux, presumably due to transsarcolemmal Ca2+ extrusion due to Na+-Ca2+ exchange. Resupply of glucose and removal of CN + 2-DG, in the continued absence of Na+, resulted in an initially slower (t1/2 = 11.6 +/- 2.5 seconds), but more complete relaxation of contracture, which was not associated with increased Ca2+ efflux. Pretreatment with 20 mM caffeine delayed the onset of contracture (9.2 +/- 1.1 minutes) and resulted in a contracture that could not be relaxed by resupply of external Na+ only. Studies using the fluorescent Ca2+ probe indo 1 demonstrated that in zero-Na+-zero-Ca2+ solutions, contracture due to CN + 2-DG was associated with an initial rise in [Ca2+]i but that this did not account for all of contracture force development. In cells exposed to CN + 2-DG in the presence of normal extracellular Na+ and Ca2+ concentrations, a small rise in [Ca2+]i was associated with initial contracture development, consistently preceding the development of a larger accelerated contracture presumably due to ATP depletion.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of simultaneous electrophysiologic and mechanical effects of verapamil and nifedipine in cultured chick embryo ventricular cells

In order to examine possible differences in mechanisms of action of nifedipine and verapamil, simultaneous recordings of membrane potential and cell motion were made during exposure of cultured chick embryo ventricular cells to these drugs. Both verapamil and nifedipine produced a rapid negative inotropic effect and excitation-contraction coupling delay presumably related to the ability of both agents to inhibit Ca2+ influx via the slow Ca channel, and thus decrease the [Ca2+]i transient. However, for an equivalent negative inotropic effect, verapamil produced relatively more membrane diastolic depolarization and slowing of repolarization than did nifedipine. Verapamil also produced a more marked decrease in +dV/dtmax. In ion flux studies, it was demonstrated that verapamil produced a significant inhibition of 24Na influx, relative to control, whereas nifedipine did not. Verapamil also produced a slight decrease in 42K efflux, of borderline significance. These findings support previous results which indicate there are differences in the mechanisms of action of these Ca2+-channel blocking drugs.

Altered Ca fluxes and contractile state during pH changes in cultured heart cells.

We studied mechanisms underlying changes in myocardial contractile state produced by intracellular (pHi) or extracellular (pHo) changes in pH using cultured chick embryo ventricular cells. A change in pHo of HEPES-buffered medium from 7.4 to 6.0 or to 8.8 changed the amplitude of cell motion by -85 or +60%, and 45Ca uptake at 10 s by -29 or +22%, respectively. The pHo-induced change in Ca uptake was not sensitive to nifedipine (10 microM), but was Na gradient dependent. Changes in pHi produced by NH4Cl or preincubation in media at pH values ranging from 6.0 to 8.8 failed to alter significantly 45Ca uptake or efflux. However, larger changes in pHi were associated with altered Ca uptake. Changes in pHo from 7.4 to 6.0 or to 8.8 were associated with initial changes in 45Ca efflux by +17 or -18%, respectively, and these effects were not Na dependent. Exposure of cells to 20 mM NH4Cl produced intracellular alkalinization and a positive inotropic effect, whereas subsequent removal of NH4Cl caused intracellular acidification and a negative inotropic effect. There was, however, a lack of close temporal relationship between pHi and contractile state. These results indicate that pHo-induced changes in contractile state in cultured heart cells are closely correlated with altered transsarcolemmal Ca movements and presumably are due (at least in part) to these Ca flux changes. In contrast, pHi-induced changes in contractile state appear principally to involve altered Ca handling within the cell and/or altered Ca sensitivity of myofibrils.

Effects of ryanodine and caffeine on contractility, membrane voltage, and calcium exchange in cultured heart cells.

To investigate the mechanisms of action of ryanodine and caffeine, changes in mechanical and electrical activity caused by these agents were correlated with alterations in 45Ca fluxes and cell Ca contents in chick embryo ventricular cell monolayer cultures. Ryanodine (10(-10)-10(-5) M) irreversibly decreased contraction amplitude by 10-70% relative to control in a concentration-dependent manner with minimal effects on electrical activity. Ryanodine caused a slight decrease in rapid 45Ca uptake, but no change in total exchangeable calcium content or rapid 45Ca efflux. Caffeine (1-20 mM) caused a transient (less than 10 seconds) 5-12% increase in contraction amplitude followed by a sustained 9-76% decrease in contraction amplitude and a 10 mV decrease in diastolic membrane voltage. Caffeine caused a decrease in rapid 45Ca uptake, a decrease in total exchangeable calcium content, and an increase in rapid 45Ca efflux. These results suggest that caffeine produces a decrease in sarcoplasmic reticulum (SR) Ca2+ uptake, and/or an increase in SR Ca2+ release that eventually depletes the SR of Ca2+, presumably accounting for the negative inotropic effect. The ryanodine effects on contraction are more difficult to account for solely in terms of alterations of transsarcolemmal Ca2+ fluxes and Ca2+ contents. Our data indicate an important role for the SR in excitation-contraction coupling in cultured chick embryo ventricular cells and suggest that SR Ca2+ is part of the rapidly exchanging Ca2+ compartment noted in 45Ca flux studies.

Calcium flux measurements during hypoxia in cultured heart cells

We tested the hypothesis that cultured chick embryo ventricular cells grown in monolayer could be used to study calcium fluxes across the myocardial sarcolemmal membrane during simulated ischemia. A specially adapted anaerobic chamber allowed the exposure of heart cells to profound hypoxia (PO2 less than 1.5 Torr) for prolonged periods of time. Ca2+ flux studies were conducted in this chamber following hypoxia and substrate deprivation to simulate important components of myocardial ischemia. To prove we were measuring cellular rather than interstitial cation contents we conducted 51Cr-EDTA interstitial space marker washout studies and showed that our procedures were sufficient to remove at least 99.7% of the extracellular fluid. The addition of lanthanum (1 mM) to the wash solution reduced nonspecific 45Ca2+ binding to the polystyrene culture plates to less than 0.03% of applied counts, but did not alter 45Ca2+ uptake under normoxic conditions. Following 2 h of hypoxia and substrate deprivation the Ca2+ content of the rapidly exchangeable Ca2+ pool of cultured monocytes increased 282% compared to control values during normoxia. We conclude that cultured chick embryo ventricular cells grown in monolayer are suitable for investigation of cation fluxes during simulated ischemia. Under the conditions studied, lanthanum displaceable Ca2+ did not make a major contribution to Ca2+ influx. The system permitted clear resolution of alterations in Ca2+ flux kinetics under conditions of profound hypoxia.

Enkephalins increase cyclic adenosine monophosphate content, calcium uptake, and contractile state in cultured chick embryo heart cells.

Peripheral vascular effects of opioid peptides are well known, but direct myocardial effects have not been established. We studied the inotropic response of spontaneously beating cultured chick embryo ventricular cells to the enkephalin analogue [D-Ala2]-enkephalin. Amplitude of cell motion increased in a concentration-dependent manner with 0.53 microM [D-Ala2]-enkephalin producing half-maximal response. The mechanism of this positive inotropic effect was investigated by examining alterations in 45Ca influx, cyclic AMP accumulation and adenylate cyclase activity in response to [D-Ala2]-enkephalin. At maximally inotropic concentrations, the 45Ca influx rate increased 39%, adenylate cyclase was stimulated by 30%, and cyclic AMP content rose more than twofold. Thus, in contrast to neural tissue, receptors for enkephalin in cultured heart cells are coupled to adenylate cyclase in a stimulatory manner. Occupancy of these receptors produces an increase in cyclic AMP levels and exerts a positive inotropic effect via a verapamil-sensitive enhancement of Ca influx.

Effects of thyroid hormone on calcium handling in cultured chick ventricular cells.

Mechanisms underlying thyroid hormone-induced changes in myocardial contractile state were investigated by studying the effects of triiodothyronine (T3) on Ca2+ fluxes across the sarcolemmal membrane and Ca2+ handling by the sarcoplasmic reticulum, using spontaneously contracting monolayers of cultured chick embryo ventricular cells. Cells were grown in serum-free medium containing either no T3 or 10(-8) M-T3 for 48 h. At [Ca2+]o levels of 0.6 and 1.2 mM, the velocity of cell contraction was significantly greater in cells grown in 10(-8) M-T3 than in its absence. At higher [Ca2+]o, no differences in the velocity of contraction were noted. 45Ca2+ exchange kinetic studies showed a biexponential pattern with a rapid and a slow component of uptake in cells grown both with and without 10(-8) M-T3. The rate of the rapid phase of uptake and total Ca2+ content were higher in cells grown in T3, with the increment in content ascribable to the rapidly exchangeable Ca2+ pool. Verapamil partially inhibited the T3-induced increase in the rapidly exchangeable pool. 45Ca2+ uptake in response to a step change to Na+-free medium in the presence of 1 microM-verapamil was significantly greater in cells grown in 10(-8) M-T3 than in T3-free medium. Cells grown in T3 showed 20% greater beating rate than cells grown in its absence. A similar increase in beating rate achieved by lowering [K+]o from 4.0 to 3.0 mM or by electrical stimulation failed to affect the rate of 45Ca2+ uptake or the size of the rapidly exchangeable pool; pacing-induced increases in rate resulted in reduction rather than augmentation of contractile state. Ca2+ efflux rate was greater in cells grown in 10(-8) M-T3 than in T3-free medium, whereas cells loaded with various levels of Ca2+ acutely by incubation at selected [Ca2+]o levels had similar efflux rates. Replacement of Na+ by choline in the efflux medium resulted in elevated Ca2+ efflux rates in cells grown both with and without T3; however, it remained greater in cells grown in 10(-8) M-T3 than in its absence. Caffeine (20 mM) in the efflux medium increased Ca2+ efflux to a greater degree in cells grown in T3 than without it. Caffeine also produced a greater tonic contraction in T3-treated cells than in cells grown in absence of T3 in Na+- and Ca2+-free medium.(ABSTRACT TRUNCATED AT 400 WORDS)