The role of arachidonic acid in rat heart cell metabolism.

Although it is known that arachidonic acid accumulates in the ischemic myocardium and that cardiac prostaglandin formation from the precursor arachidonic acid is altered during disease states, the role of arachidonic acid in the myocyte itself is not yet clear. Using isolated Ca-tolerant adult rat heart muscle cells, we were able to study cardiac metabolism of arachidonic acid without the effects induced by endothelial or other non-muscle tissue. Myocytes rapidly incorporate arachidonic acid as well as other fatty acids into their lipid pools, the predominant acceptor being the triacylglycerols at an extracellular fatty acid concentration of 20 microM. As exogenous arachidonic acid is decreased, the distribution pattern shifts to favor phospholipid esterification. Cardiocyte prostaglandin production from arachidonic acid added to the incubation medium was limited (less than 1% conversion of added arachidonic acid) and lipoxygenase pathway activity was not detected. Oxidation rates of arachidonic acid were 3-fold lower than for palmitic acid, indicating that it is of secondary importance in energy-yielding reactions. Our results suggest that arachidonic acid serves primarily as a structural component of myocardial membranes and that its release during ischemia would permit its use as a substrate for prostaglandin production by coronary vascular tissue.

Effects of obesity and hypertension on ventricular myocytes: comparison of cells from adult SHHF/Mcc-cp and JCR:LA-cp rats.

OBJECTIVE: The aim was to compare beta adrenergic receptors, cAMP production, and Ca2+ accumulation by the sarcoplasmic reticulum in ventricular cardiomyocytes from female SHHF/Mcc-cp and JCR:LA-cp rats. Whereas rats from both strains exhibit gross obesity when the animals are homozygous for the recessive "corpulent" gene, the SHHF rats, which are hypertensive, all develop heart failure during their second year of life. The normotensive JCR:LA-cp animals do not. METHODS: beta Adrenergic receptor number, ligand affinity, isoprenaline and forskolin stimulated cyclic AMP production, and ATP dependent, phosphate supported 45Ca2+ uptake by the sarcoplasmic reticulum were compared in ventricular cardiomyocytes isolated from 6 months old obese female SHHF/Mcc-cp and obese and lean female JCR:LA-cp rats. RESULTS: Bmax and Kd for (-)-[125iodo]-cyanopindolol (125ICYP) binding were each approximately 50% lower in SHHF/Mcc-cp v JCR:LA-cp myocytes. Cyclic AMP production in response to isoprenaline, isoprenaline plus isobutylmethylxanthine (IBMX), and forskolin plus IBMX was also significantly depressed in the SHHF/Mcc-cp cells. In addition, sarcoplasmic reticular 45Ca2+ uptake by SHHF/Mcc-cp cells was 35% lower than in lean or obese JCR:LA-cp myocytes. Isoprenaline stimulated cAMP production and sarcoplasmic reticular Ca2+ uptake by the lean JCR:LA-cp cells were comparable to that described previously for myocytes from normal Sprague-Dawley rats. By contrast, Bmax and Kd for 125ICYP binding by the JCR myocytes differed substantially from previously described results for normal Sprague-Dawley rats, whereas values for the SHHF cells did not. CONCLUSIONS: Declines in Ca sequestration by the sarcoplasmic reticulum of ventricular cardiomyocytes from obese, hypertensive SHHF rats are not related to their obesity. However, obesity may contribute to the decline in cAMP production. This may account, in part, for the exacerbation by obesity of cardiac dysfunction in essential hypertension.

Inosine and guanosine preserve neuronal and glial cell viability in mouse spinal cord cultures during chemical hypoxia.

Murine spinal cord primary mixed cultures were treated with the respiratory inhibitor, rotenone, to mimic hypoxic conditions. Under these conditions neurons rapidly underwent oncosis (necrosis) with a complete loss in viability occurring within 260 min; however, astrocytes, which accounted for most of the cell population, died more slowly with 50% viability occurring at 565 min. Inosine preserved both total cell and neuronal viability in a concentration-dependent manner. The time of inosine addition relative to hypoxic insult was critical with the most effective protection occurring when inosine was added just prior to or within 5 min after insult. Inosine was ineffective when added 30 min after hypoxic insult. The effect of guanosine was similar to that of inosine. Treatment of cultures with BCX-34, a purine nucleoside phosphorylase inhibitor, prevented protection by inosine or guanosine, suggesting involvement of a purine nucleoside phosphorylase in the nucleoside protective effect.

Effects of amiloride on calcium uptake by myocytes isolated from adult rat hearts.

Amiloride at high concentrations inhibits the uptake of Ca by rat heart myocytes containing elevated levels of intracellular Na and retards the development of Ca-dependent hypercontracture in these cells. In contrast, amiloride enhances the net uptake of Ca in Ca-tolerant myocytes containing normal levels of Na. The results suggest that amiloride may inhibit Na-Ca exchange across the sarcolemma of cardiac myocytes.

Vascular and contractile responses to extracellular ATP: studies in the isolated rat heart.

The dose-response characteristics for the effect of ATP upon cardiac function and vascular tone have been investigated in the isolated perfused rat heart. Vasodilation was observed with low ATP concentrations (0.01-0.1 mM) whereas severe vasoconstriction occurred with high concentrations (1.0-10.0 mM). At all doses studied, heart rate and pressure-rate product were reduced in a dose-dependent manner, with 10 mM ATP almost complete cardiac arrest was observed. Analysis of epicardial electrograms revealed that ATP induced arrhythmias, prolonged the P-R interval and induced partial blockade of S-A nodal activity and A-V conduction. Investigating possible mechanisms for the vascular and contractile effects of ATP, it was possible to exclude the calcium chelating properties of ATP and the effects of coincident ischemia arising as a consequence of ATP-induced vasoconstriction. Pharmacological studies revealed the ATP-induced vasoconstriction to be unresponsive to a range of coronary vasodilators and also allowed exclusion of prostaglandins, catecholamines and adrenergic receptors in the mediation of ATP effects. Investigations with acetylcholine revealed remarkably similar effects upon both contractile and vascular activity but studies with atropine suggested that the muscarinic receptor was not involved. Studies with theophylline allowed a dissociation of the vascular and contractile effects of ATP and indicated a possible involvement of the adenosine receptor in the cellular response to both high and low concentrations of ATP.

AMP deaminase in piglet cardiac myocytes: effect on nucleotide metabolism during ischemia.

The purpose of this study was to examine in situ regulation of AMP deaminase in newborn piglet cardiac myocytes and to determine its role in nucleotide metabolism during ischemia. When a rapid deenergization paradigm was used to assay AMP deaminase, enzyme activity depended on the hormonal and metabolic status of cells just before deenergization. Inosine 5'-monophosphate (IMP) formation was increased 150% in deenergized myocytes pretreated with phorbol 12-myristate 13-acetate (PMA; EC50 = 4.7 x 10(-8) M). This effect was 90% blocked with the protein kinase C (PKC) inhibitor staurosporine. In addition, the beta-adrenergic agonist isoproterenol stimulated AMP deaminase activity (EC50 = 1.5 x 10(-8) M), and IMP formation was directly correlated to intracellular cAMP levels (r2 = 0.9). Furthermore, adenosine increased IMP formation, whereas nonrespiring, glycolyzing piglet myocytes had reduced AMP deaminase activity. Pretreatment of perfused piglet hearts with adenosine, but not PMA, before exposure to global ischemia resulted in enhanced conversion of AMP to IMP during the ischemic period. Similar results were obtained in piglet myocytes preincubated with adenosine or PMA before exposure to simulated ischemia. These results may be relevant to the preconditioning phenomenon.

Response of isolated adult canine cardiac myocytes to prolonged hypoxia and reoxygenation.

Isolated adult canine ventricular myocytes incubated in the absence of glucose with the respiratory inhibitor rotenone retained 67% of ATP (control, 26.0 +/- 0.9 nmol/mg protein) during 3-h incubation, yet phosphocreatine fell to 23% of initial content. Lactate production proceeded at a constant rate of 5 nmol.mg-1.min-1 in rotenone-treated glucose-free myocytes. A 36% decline in rod-shaped cells and an increase in percent 22Na permeation from 37% in aerobic cells (approximately 13 mM intracellular sodium) to 68% in rotenone-treated glucose-free myocytes paralleled the loss of ATP. Total exchangeable calcium was maintained at control aerobic levels. Exposure of canine cells to 3-h hypoxia in the absence of glucose followed by 5-min reoxygenation resulted in a 73% decrease in ATP, a rise in calcium from 3.3 +/- 0.2 to 6.6 +/- 1.6 nmol/mg, and an increase in 22Na permeation to 111%. Under these conditions the number of rod-shaped myocytes declined by 77%, with corresponding increases in viable contracted and hypercontracted myocytes. The response of canine myocytes to severe hypoxia and reaeration contrasts greatly to earlier studies using adult rat cardiac myocytes [see Hohl et al. Am. J. Physiol. 242 (Heart Circ. Physiol. 11): H1022-H1030, 1982]. Species differences with respect to basal metabolism, rates of ATP production and degradation, and regulation of cation movements are most likely responsible for the observed differences.

FK506 alters sarcoplasmic reticulum calcium release in neonatal piglet cardiac myocytes.

Tacrolimus (FK506) is a potent immunosuppressive drug that, when complexed to a family of immunophilin proteins known as FK binding proteins, inhibits calcineurin in T lymphocytes. Although i.v. use of FK506 in pediatric transplant recipients has been linked to development of cardiomyopathies, its mechanism of cardiotoxicity has not been examined in a neonatal animal model. In our study the effects of FK506 were investigated in cardiac myocytes isolated from newborn piglets. The peak amplitude of electrically triggered fura-2 Ca2+ transients was increased in FK506-treated myocytes, but Ca2+ transient duration and baseline fura-2 Ca2+ ratios were not altered. 45Ca2+ uptake by digitonin-lysed piglet cells decreased at pCa < or = 6.0, indicating that sarcoplasmic reticulum efflux channels were leaky. The results suggest that elevated release of sarcoplasmic reticulum Ca2+ during systole contributes to cardiotoxic effects observed in children.

Isolation and regulation of piglet cardiac AMP deaminase.

The properties of piglet cardiac AMP deaminase were determined and its regulation by pH, phosphate, nucleotides and phosphorylation is described. AMP deaminase purified from the ventricles of newborn piglet hearts displayed hyperbolic kinetics with a Km of 2 mM for 5'-AMP. The enzyme had a pH optimum of 7.0 and was strongly inhibited by inorganic phosphate. ATP decreased the Km of the native enzyme 3-fold, but did not significantly block the inhibitory effects of phosphate. Kinetic parameters were not significantly altered in the presence of adenosine, cyclic AMP and NAD+, whereas, the Km was decreased by 50% in the presence of NADH. Piglet cardiac AMP deaminase was phosphorylated by protein kinase C, resulting in a 2-fold increase in Vmax with no change in Km. However, incubation with cAMP-dependent protein kinase did not affect enzyme kinetics. The 80-85 kD protein subunit of piglet cardiac AMP deaminase immunoreacted with antisera raised against human erythrocyte AMP deaminase, rabbit heart AMP deaminase and human recombinant AMP deaminase 3 (isoform E). These results are discussed in relation to in situ AMP deaminase activity in neonatal piglet heart myocytes.

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

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

Adenosine stimulation of AMP deaminase activity in adult rat cardiac myocytes.

Using an in situ assay for analyzing AMP deaminase activity in isolated adult rat ventricular myocytes, we have shown that IMP production is stimulated approximately twofold in cardiac cells incubated with 10 microM adenosine. This effect of adenosine was not blocked by the adenosine A1-receptor antagonist 8-cyclophenyl-1,3-dipropylaxanthine (0.01-1 microM) except at a concentration (100 microM) that may inhibit adenosine transport. Similarly, in situ AMP deaminase activity was not enhanced by treatment with the specific adenosine A1-receptor agonists N6-phenylisopropyl adenosine or cyclopentyladenosine, nor was it sensitive to prior treatment of cells with pertussis toxin. The nucleoside transport blockers S-4-nitrobenzyl-6-thioinosine, dipyridamole, and papaverine inhibited adenosine-induced increases in IMP production by 75-85%, suggesting an intracellular site of action. Modulation of enzyme activity via the transmethylation pathway could not be implicated since incubation of cardiac cells under conditions known to elevate intracellular S-adenosyl-L-homocysteine had no demonstrable effect on AMP deaminase. Furthermore, a direct allosteric effect of adenosine on the partially purified rat cardiac enzyme was not observed. The results indicate that intracellular adenosine modulates rat cardiac AMP deaminase by an unknown mechanism.

Metabolic compartmentalization in neonatal swine myocytes.

The transport of metabolites across the mitochondrial membrane is regulated by specific exchange and shuttle systems that are often dependent on the mitochondrial membrane potential. Thus, metabolite concentrations in the cytosol and mitochondrial compartments are largely determined by the energy state of the cardiac muscle cell. The purpose of this study was to investigate metabolic compartmentalization in ventricular myocytes isolated from newborn (< 24 h) swine hearts. Furthermore, the effect of respiratory inhibition on these distribution patterns was examined. Freshly isolated cells contained 33 nmol of ATP and 37 nmol of total adenine nucleotides (AN) per mg of myocyte protein. Rapid digitonin fractionation indicated that 95% of ATP and 86% of AN were cytosolic, whereas > 50% of the pyridine nucleotides were mitochondrial. With 11 mM added glucose, myocytes treated with the respiratory inhibitor, rotenone, maintained ATP at 88% of that of aerobic myocytes, but phosphocreatine declined by 50% over 30 min. Rotenone treatment caused the mitochondrial NAD/NADH ratio to decline from 1.2 to 0.06, whereas the cytosolic pyridine nucleotides remained > 90% oxidized. Total adenine and pyridine nucleotide content and their compartmentalization were unaffected by respiratory inhibition. Comparisons of metabolite content and respiratory activity between isolated piglet mitochondria and the mitochondrial compartment of piglet myocytes indicated that mitochondria account for approximately 30% of total myocyte protein. A similar value (29%) was obtained for the aqueous volume fraction of the in situ mitochondrial matrix using the 4000 Mr 14C-labeled polyethylene glycol-impermeable 3H2O spaces of intact and lysed myocytes. These results are comparable to literature values for myocardium from other species and age groups.

Polypharmacy, adverse drug-related events, and potential adverse drug interactions in elderly patients presenting to an emergency department.

STUDY OBJECTIVES: We sought to document the degree of polypharmacy, the frequency of adverse drug-related events (ADREs) leading to emergency department presentation that were recognized by emergency physicians, and the frequency of potential adverse drug interactions (PADIs) in medication regimens of elderly patients in the ED. METHODS: We conducted a retrospective chart review on 300 randomly selected ED visits made by patients 65 years of age and older between January 1 and December 31, 1998. ADREs were defined according to a standardized algorithm. PADIs were identified by using the drug interaction database PharmVigilance. RESULTS: After excluding 17 patient visits with inadequate documentation, 283 were left for review. Of these, 257 (90.8%) patients were taking 1 or more medications (prescribed or over the counter). The number of medications consumed ranged from 0 to 17 and averaged 4.2 (SD+/-3.1) drugs per patient. ADREs accounted for 10.6% of all ED visits in our patient group. The most frequently implicated classes of medications were nonsteroidal anti-inflammatory drugs, antibiotics, anticoagulants, diuretics, hypoglycemics, beta-blockers, calcium-channel blockers, and chemotherapeutic agents. Thirty-one percent of all patients in our group had at least 1 PADI in their medication list. Among patients who presented because of an ADRE, 50% had at least 1 PADI in their medication list that was unrelated to the ADRE with which they presented. CONCLUSION: ADREs are an important cause of ED presentation in the elderly. PADIs are found in a significant proportion of medication lists. Emergency physicians must be vigilant in monitoring elderly patients for medication-related problems.

Effects of calcium on the permeability of isolated adult rat heart cells to sodium.

The permeability of rat heart myocytes to Na increases when extracellular Ca (Ca0) is decreased. This increased permeability is reflected in elevated Na/K ratios in nonenergized myocytes and in increased ouabain-sensitive lactate production in anaerobic myocytes supplemented with glucose. Myocytes treated with ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid (1 mM) maintain low Na/K ratios, but expend considerable glycolytic ATP in ouabain-sensitive cation cycling. The data suggest that Ca0 bound to the sarcolemma can restrict transmembrane movement of Na via pathways that are not yet defined. The lack of significant net accumulation of Ca argues against the explanation that Ca0 maintains low internal Na levels as a result of Na-Ca exchange. Both the increased uptake of Na and increased utilization of ATP in the absence of Ca0 may be relevant to the phenomenon of "Ca-paradox" in situ.

The effect of anoxia on lipid metabolism in isolated adult rat cardiac myocytes.

In ischemic myocardium abnormal lipid metabolism results in accumulation of compounds that are deleterious to membrane structural integrity and membrane dependent functions. In this study isolated adult rat ventricular myocytes were used to investigate anoxia-induced alterations in cellular lipid composition and metabolism. Myocyte phospholipid content declined 19% on average during 60 min anoxia and intracellular arachidonic acid increased 3-fold, without affecting myocyte ATP content. Anaerobic incubation in the absence of glucose depleted cellular ATP to 2 nmol/mg protein, elicited a 23% decrease in phospholipids, and reduced triacylglycerol content by 51%. Intracellular levels of C16-C22 fatty acids were significantly elevated, especially palmitic and arachidonic acids. Myocytes presented with 0.08 mM [1-14C]-palmitic or arachidonic acid acylated 85% (25-26 nmol/mg) of the fatty acid taken up into triacylglycerols. Anoxia decreased this esterification by 46-60%. Formation of [14C]-CO2 was also depressed 70-90% by anaerobiosis. The results demonstrate that anoxia stimulates degradation of complex lipids, with a concomitant increase in non-esterified fatty acids, especially arachidonic acid.

Phorbol esters and cyclic AMP activate AMP deaminase in adult rat cardiac myocytes.

Using rapid deenergization as a probe for adenylate deaminase activity in intact adult rat cardiac myocytes, we have previously established that IMP formation is enhanced by alpha-adrenergic agonists. In the present study, the effect of adrenergic agents on adenylate deaminase was further characterized. Phenylephrine (PE)3 increased IMP production in a dose-dependent fashion with an EC50 of 8 x 10(-7) M. The response to PE was reversed within 10 min by the alpha 1-antagonist, prazosin. Likewise, adenylate deaminase was also activated in ventricular myocytes challenged with phorbol 12-myristate 13-acetate (PMA, EC50 = 5 nM); cardiac cells presented with 100 nM PMA increased IMP production from 4.4 +/- 0.5 (control) to 15.7 +/- 0.9 nmol/mg protein when subsequently deenergized. The effects of PMA and PE were attenuated 85 +/- 5% and 96 +/- 4%, respectively, by pretreatment of cells with 150 nM staurosporine, an inhibitor of protein kinase C. Furthermore, incubation of cardiac cells with 1 microM PMA for 24 h blunted the response to both PMA and phenylephrine 85-90%. Elevating cyclic AMP (cAMP) content to greater than 15 pmol/mg by treatment with forskolin or isoproterenol plus isobutylmethylxanthine also resulted in enhanced adenylate deaminase activity, but this stimulatory effect was not abolished by 24 h incubation with 5 microM PMA. Forskolin and PMA-induced increases in IMP production appeared to be additive. However, 0.5 microM isoproterenol inhibited the cellular response to phenylephrine by about 30% but did not affect PMA-stimulated adenylate deaminase activity. We conclude that both cAMP and protein kinase C stimulate adenylate deaminase, perhaps through selective activation of different isoforms. However, cAMP also exerts partial inhibition on alpha-adrenoreceptor-mediated increases in IMP production.

Effects of simulated ischemia and reperfusion on the sarcoplasmic reticulum of digitonin-lysed cardiomyocytes.

ATP-dependent, inorganic phosphate-supported 45Ca2+ uptake by digitonin-lysed adult rat ventricular cardiomyocytes was used to evaluate the effects of simulated ischemia and reperfusion on the physically intact sarcoplasmic reticulum. Mitochondrial reactions were inhibited with rotenone and oligomycin. 45Ca2+ accumulation in the presence of the calcium efflux inhibitors, procaine (10 mM) and ruthenium red (30 microM), was used to characterize unidirectional uptake kinetics. A decrease in pH from 7.2 to 6.6 increased the [Ca2+] K0.5 from 0.5 to 2.0 microM and reduced the apparent Vmax by 28%. In the absence of procaine and ruthenium red, at a free [Mg2+] of 0.5 mM, maximum net uptake occurred at pCa 6.2 when pH was 7.2 and at pCa 6.0 when pH was 6.6. At lower pCa, net Ca2+ accumulation declined. Increasing free [Mg2+] from 0.5 to 1 mM at pH 6.6 or to 2.5 mM at pH 7.2 increased net 45Ca2+ accumulation in the absence of procaine and ruthenium and shifted maximum uptake to pCa 5.6 and 6.0, respectively. Increases in cytosolic free [Mg2+] thought to occur during myocardial ischemia are therefore capable of inhibiting calcium efflux from the sarcoplasmic reticulum. Reducing [ATP] from 10 to 1 mM reduced maximum net 45Ca2+ uptake by 30% both in the presence and absence of efflux inhibitors. Preincubation of intact myocytes under conditions designed to simulate ischemia and reperfusion decreased 45Ca2+ uptake greater than or equal to 50%. The data indicate that myocardial ischemia and reperfusion can alter both Ca2+ accumulation and calcium release by the sarcoplasmic reticulum.

Mechanical alternans and the force-frequency relationship in failing rat hearts.

We examined contractile performance in perfused ventricles from normal rats and from SHHF/Mccfacp rats with end-stage heart failure. Changes in pacing frequency from 3 to 5 Hz evoked a complex response in normal rat myocardium. The first beat after a switch to 5 Hz was extremely weak, but each successive beat was stronger until force exceeded the 3 Hz steady state value by approximately 30%. Force then gradually declined to a new steady state where developed pressure was depressed but rate-pressure product was slightly greater than that at 3 Hz. By contrast, in failing SHHF/Mcc-facp hearts, an increase in pacing frequency from 3 to 5 Hz did not increase force development. Instead, the isovolumic left ventricles exhibited mechanical alternans. This alternation between weak and strong beats was abolished by 1 mM caffeine but restored by its washout. Inhibition of SR Ca2+ accumulation by 50-500 nM thapsigargin in normal ventricles did not evoke alternans when pacing frequencies were increased. The results indicate that mechanical alternans in failing rat hearts is due to altered reactions of the sarcoplasmic reticulum, but a decreased rate of Ca2+ accumulation is not the primary cause.

Calcium handling by sarcoplasmic reticulum of neonatal swine cardiac myocytes.

In recent years, because of similarities to human infants, neonatal piglets have increasingly become the model of choice for studying neonatal heart function. However, the cardiac sarcoplasmic reticulum (SR) has not been thoroughly characterized in this species. Accordingly, Ca2+ pump kinetics, efflux channel characteristics, Ca2+ transients, and contractile movements were examined in isolated newborn piglet cardiac ventricular myocytes. Maximum uptake rate (Vmax) and concentration required to produce a half-maximal effect (K0.5) for oxalate-supported, ATP-dependent 45Ca2+ uptake by the SR of digitonin-lysed myocytes were 285 +/- 17 nmol 45Ca2+.min-1.mg-1 and 0.69 +/- 0.07 microM, respectively. In the absence of phospholamban phosphorylation, Vmax was reduced to 195 +/- 26 nmol 45Ca2+.min-1.mg-1 (P < 0.05 vs. control) and K0.5 increased to 1.28 +/- 0.13 microM (P < 0.05 vs. control). [3H]ryanodine binding studies yielded a maximum binding capacity of 181 +/- 12 fmol/mg and a dissociation constant of 1.7 +/- 0.2 nM. Raising extracellular Ca2+ (0.5-5 mM) increased peak amplitude and decreased the duration of electrically stimulated fura 2 Ca2+ transients and recordings of cell length changes. Both ryanodine and 2,5-di-tert-butylhydroquinone, an inhibitor of SR calcium adenosinetriphosphatase, completely abolished Ca2+ transients in piglet myocytes. These studies indicate that the SR has a significant role in excitation-contraction coupling in neonatal piglet myocytes.

Beta2-adrenergic receptor antagonists protect against ventricular fibrillation: in vivo and in vitro evidence for enhanced sensitivity to beta2-adrenergic stimulation in animals susceptible to sudden death.

BACKGROUND: The ventricular myocardium contains functional beta2-adrenergic receptors that when activated increase intracellular Ca2+ transients. Because elevated Ca2+ has been implicated in the induction of ventricular fibrillation (VF), it is possible that the activation of these receptors may also provoke malignant arrhythmias. METHODS AND RESULTS: To test this hypothesis, a 2-minute occlusion of the left circumflex coronary artery was made during the last minute of exercise in 28 dogs with healed anterior myocardial infarctions: 17 had VF (susceptible) and 11 did not (resistant). On a subsequent day, this test was repeated after administration of the beta2-adrenergic receptor antagonist ICI 118,551 (0.2 mg/kg). This drug did not alter the hemodynamic response to the coronary occlusion, yet it prevented VF in 10 of 11 animals tested (P<.001). However, heart rate was reduced in 6 animals. Therefore, the ICI 118,551 exercise-plus-ischemia test was repeated with heart rate held constant by ventricular pacing (n=3). ICI 118,551 still prevented VF when heart rate was maintained. Next, the effects of increasing doses of the beta2-adrenergic receptor agonist zinterol on Ca2+ transient amplitudes were examined in ventricular myocytes. Zinterol elicited significantly greater increases in Ca2+ transient amplitudes at all doses tested (10(-9) to 10(-6) mol/L) in myocytes prepared from susceptible versus resistant animals. The cardiomyocyte response to isoproterenol (10(-7) mol/L) in the presence or absence of the selective beta1- (CGP-20712A, 300 nmol/L) or beta2- (ICI 118,551, 100 nmol/L) adrenergic receptor antagonist was also examined. Isoproterenol elicited larger Ca2+ transient increases in the susceptible myocytes, which were eliminated by ICI but not by CGP. CONCLUSIONS: When considered together, these data demonstrate that canine myocytes contain functional beta2-adrenergic receptors that are activated to a greater extent in the susceptible animals. The resulting cytosolic Ca2+ transient increases may lead to afterpotentials that ultimately trigger VF in these animals.