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.

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.

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.

Thiol redox modulation of doxorubicin mediated cytotoxicity in cultured AIDS-related Kaposi's sarcoma cells.

The chemotherapeutic, doxorubicin, is currently used empirically in the treatment of AIDS- related Kaposi's sarcoma (AIDS-KS). Although often employed in a chemotherapeutic cocktail (doxorubicin, bleomycin, vincristine) single-agent therapy has recently been attempted with liposome encapsulated doxorubicin. Although doxorubicin's mechanism of action against AIDS-KS is unknown, we hypothesized that doxorubicin's ability to undergo redox cycling is associated with its clinical efficacy. The current study was conducted to investigate the effects of doxorubicin on selected xenobiotic-associated biochemical responses of three cellular populations: KS lesional cells, nonlesional cells from the KS donors, and fibroblasts obtained from HIV- aged matched men. Our results show that during doxorubicin challenge, there are strong positive correlations between cellular glutathione (GSH) levels and viability (r = 0.94), NADPH levels and viability (r = 0.93), and GSH and NADPH levels (r = 0.93), and demonstrate that as a consequence of their abilities to maintain cellular thiol redox pools HIV- donor cells are significantly less susceptible to doxorubicin's cytotoxic effects relative to AIDS-KS cells. Additional studies further supported the contribution of reduced thiols in mediating doxorubicin tolerance. While pretreatment with the GSH precursor, N-acetylcysteine was cytoprotective for all cell groups during doxorubicin challenge, GSH depletion markedly enhanced doxorubicin's cytotoxic effects. Studies to investigate the effects of a hydroxyl scavenger and iron chelator during doxorubicin challenge showed moderate cytoprotection in the AIDS-KS cells but deleterious effects in the HIV control cells. Inactivation of the longer lived membrane generated ROI in the cytoprotective deficient AIDS-KS cells, as well as an impairment of endogenous defenses in the HIV- donor control cells, may account for these scavenger and chelator associated findings. In summary, our findings show that doxorubicin mediates, at least in part, its AIDS-KS cellular cytotoxic effects by a redox related mechanism, and provides a biochemical rationale for doxorubicin's clinical efficacy in AIDS-KS treatment.

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.

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.

Adenosine, inosine, and guanosine protect glial cells during glucose deprivation and mitochondrial inhibition: correlation between protection and ATP preservation.

The purpose of this study was to determine the mechanism by which adenosine, inosine, and guanosine delay cell death in glial cells (ROC-1) that are subjected to glucose deprivation and mitochondrial respiratory chain inhibition with amobarbital (GDMI). ROC-1 cells are hybrid cells formed by fusion of a rat oligodendrocyte and a rat C6 glioma cell. Under GDMI, ATP was depleted rapidly from ROC-1 cells, followed on a much larger time scale by a loss of cell viability. Restoration of ATP synthesis during this interlude between ATP depletion and cell death prevented further loss of viability. Moreover, the addition of adenosine, inosine, or guanosine immediately before the amobarbital retarded the decline in ATP and preserved cell viability. The protective effects on ATP and viability were dependent on nucleoside concentration between 50 and 1,500 microM. Furthermore, protection required nucleoside transport into the cell and the continued presence of nucleoside during GDMI. A significant positive correlation between ATP content at 16 min and cell viability at 350 min after the onset of GDMI was established (r = 0.98). Modest increases in cellular lactate levels were observed during GDMI (1.2 nmol/mg/min lactate produced); however, incubation with 1,500 microM inosine or guanosine increased lactate accumulation sixfold. The protective effects of inosine and guanosine on cell viability and ATP were >90% blocked after treatment with 50 microM BCX-34, a nucleoside phosphorylase inhibitor. Accordingly, lactate levels also were lower in BCX-34-treated cells incubated with inosine or guanosine. We conclude that under GDMI, the ribose moiety of inosine and guanosine is converted to phosphorylated glycolytic intermediates via the pentose phosphate pathway, and its subsequent catabolism in glycolysis provides the ATP necessary for maintaining plasmalemmal integrity.

Thiol redox modulation of tumor necrosis factor-alpha responsiveness in cultured AIDS-related Kaposi's sarcoma cells.

Both clinical and experimental evidence indicates that AIDS-related Kaposi's sarcoma (AIDS-KS) has a multifactorial pathogenesis with factors such as HIV viral load, latent virus induction, and opportunistic infections contributing to disease progression. However, a consistent feature that unites these apparently diverse putative etiologic agents is sustained serum elevations of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha). While virtually every cell responds to TNF-alpha with gene activation, the extent of TNF-alpha-mediated cellular signaling is regulated by a delicate balance between signal activation and signal arresting events. Reactive oxygen intermediates (ROI), which are generated as a consequence of TNF-alpha membrane interaction, are part of this TNF-alpha-initiated cellular activation cascade. Previous studies in our laboratory have shown that AIDS-KS cells possess impaired oxygen intermediate scavenging capacities, thereby establishing conditions permissive for the intracellular retention of ROI. In this study, we used cellular capacity to upregulate the cytoprotective enzyme superoxide dismutase (SOD) to address the extent of cellular response to TNF-alpha. Concurrent with the SOD analyses, nucleotide profiles were obtained to assess cellular bioenergetic responses during TNF-alpha challenge. Proliferative growth levels of mitochondrial (Mn)SOD activities showed an activity spectrum ranging from lowest activity in AIDS-KS cells, to intermediate levels in matched, nonlesional cells from the AIDS-KS donors, to highest activities in HIV normal fibroblasts. In contrast, following TNF-alpha challenge, the AIDS-KS and KS donor nonlesional cells showed a 11.89- and 5.86-fold respective increase in MnSOD activity, while the normal fibroblasts demonstrated a 1.35-fold decrease. Subsequent thiol redox modulation studies showed that only the normal fibroblast cultures showed a potentiation of TNF-alpha-mediated MnSOD upregulation following GSH depletion. In addition, provision of the GSH precursor, N-acetylcysteine during TNF-alpha challenge only diminished MnSOD activity and mitochondrial compartmentalization in the AIDS-KS cells, a finding that likely reflects the lower levels of reduced thiols in this cellular population. Our data, which show that a perturbation in their cellular thiol redox status accentuates AIDS-KS cellular responsiveness to TNF-alpha, suggest a biochemical rationale for the recognized TNF-alpha AIDS-KS clinical correlation.

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.

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.

Verapamil accelerates the transition to heart failure in obese, hypertensive, female SHHF/Mcc-fa(cp) rats.

We sought to characterize the effects of the nonselective Ca2+ channel antagonist, verapamil, and the vascular-selective Ca2+ channel antagonist, felodipine, on obese, hypertensive, heart failure-prone, female SHHF/Mcc-fa(cp) rats. Rats were treated for < or = 2 months with verapamil (57 mg/kg/day) or felodipine (24 mg/kg/day). Blood pressures were determined at monthly intervals by the tail-cuff method. Heart weights and myosin isoforms were measured at the end of treatment. Direct cardiac effects of verapamil and felodipine were examined in electrically field stimulated, fura-2/AM-loaded cardiomyocytes. Both Ca2+ channel antagonists reduced systolic blood pressures. Verapamil, but not felodipine, increased heart weights and decreased expression of the myosin V1 isoform. In older animals, 75% of those treated with verapamil developed end-stage congestive heart failure. Age-matched control and felodipine-treated rats remained healthy. In isolated cardiomyocytes, 10(-9) M verapamil significantly reduced Ca2+ transient amplitudes but 10(-9) M felodipine did not. Both Ca2+ channel antagonists reduced blood pressures in obese, hypertensive, female SHHF rats. Verapamil, but not felodipine, produced heart failure in a large number of these animals. Differences between the in vivo effects of the two Ca2+ channel antagonists may be related to the differing effects on sarcolemmal Ca2+ influx.

Effects of calcium channel antagonists on Ca2+ transients in rat and canine cardiomyocytes.

First-generation Ca2+ channel antagonists depress myocardial contractility, but many of the newer Ca2+ channel blockers have a high degree of "vascular selectivity". This study compares the effects of the Ca2+ antagonists felodipine, amlodipine, mibefradil, verapamil and nifedipine, and the Ca2+ channel agonist. (S)(-)-Bay K-8644 on Ca2+ transient amplitudes in fura-2/AM-loaded rat and canine ventricular cardiomyocytes. At 10(-11) and 10(-10) M, felodipine increased [Ca2+]i transient amplitudes by 10-25% in field-stimulated fura-2-loaded cells from both species while at 10(-6) M it depressed [Ca2+]i transients by 80%. Mibefradil increased [Ca2+]i transient amplitudes by 16% at 10(-11) and 10(-10) M and decreased the transients by 25% at 10(-6) M. The calcium channel agonist, (S)(-)-Bay K-8644 increased [Ca2+]i transient amplitudes at 10(-10)-10(-6) M (maximally 37% at 10(-7) M) but depressed [Ca2+]i transients by 10% at 10(-5) M. Nifedipine was inhibitory at all concentrations tested (10(-11)-10(-6) M) in canine myocytes, but in rat cells. 10(-10) M nifedipine increased [Ca2+]i transient amplitudes by 37%. All concentrations of verapamil and amlodipine (10(-11)-10(-6) M) depressed [Ca2+]i transients in both rat and canine myocytes. We conclude that: (1) felodipine and mibefradil may be positive rather than negative inotropes at low concentrations, which are therapeutically relevant: and (2) low concentrations of nifedipine may have a positive inotropic effect in the rat but not the dog heart.

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.

Mechanism of GroEL action: productive release of polypeptide from a sequestered position under GroES.

The chaperonin GroEL is a large, double-ring structure that, together with ATP and the cochaperonin GroES, assists protein folding in vivo. GroES forms an asymmetric complex with GroEL in which a single GroES ring binds one end of the GroEL cylinder. Cross-linking studies reveal that polypeptide binding occurs exclusively to the GroEL ring not occupied by GroES (trans). During the folding reaction, however, released GroES can rebind to the GroEL ring containing polypeptide (cis). The polypeptide is held tightly in a proteolytically protected environment in cis complexes, in the presence of ADP. Single turnover experiments with ornithine transcarbamylase reveal that polypeptide is productively released from the cis but not the trans complex. These observations suggest a two-step mechanism for GroEL-mediated folding. First, GroES displaces the polypeptide from its initial binding sites, sequestering it in the GroEL central cavity. Second, ATP hydrolysis induces release of GroES and productive release of polypeptide.

Cultured AIDS-related Kaposi's sarcoma (AIDS-KS) cells demonstrate impaired bioenergetic adaptation to oxidant challenge: implication for oxidant stress in AIDS-KS pathogenesis.

Despite its recognition as the most prevalent HIV associated cancer, speculation still abounds regarding the pathogenesis of AIDS-related Kaposi's sarcoma (AIDS-KS). However, it has been established that both cytokines, e.g. IL-6, and HIV-associated products, e.g., Tat, are integral in AIDS-KS cellular proliferation. Further, both experimental and clinical evidence is accumulating to link reactive oxygen intermediates (ROI) with both cytokine induction (primarily via nuclear factor-kappa B[NF-kappa B] dependent routes) as well as the subsequent cytokine, tumor necrosis factor alpha (TNF alpha) stimulation of HIV replication. Features of AIDS-KS patients, such as retention of phagocytes, presence of sustained immunostimulation, and a frequent history of KS lesions arising at traumatized sites, make oxidant stress a viable clinical factor in AIDS-KS development. Time course nucleotide profile analyses show that AIDS-KS cells have an inherent, statistically significant, biochemical deficit, even prior to oxidant stress, due to 1) a more glycolytic bioenergetic profile, resulting in lower levels of high energy phosphates (impairing capacity for glutathione [GSH] synthesis and DNA repair); 2) lower levels of NADPH (compromising the activities of GSSG reductase and peroxidase function of catalase); and 3) reduced levels of GSH (impeding both GSH peroxidase and GSH-S-transferases). Following exposure to physiologically relevant levels of H2O2, only the human microvascular endothelial cells (a putative AIDS-KS progenitor cell) responded with bioenergetic adaptations that reflected co-ordination of energy generating and cytoprotective pathways, e.g., retention of the cellular energy charge, increased NAD+, and an accentuation of the ATP, NADPH, and total adenine nucleotide differences relative to AIDS-KS cells. Also, some of the AIDS-KS strains retained intracellular GSSG subsequent to oxidant challenge, inviting the formation of deleterious protein mixed disulfides. While the results of our study address some AIDS-KS issues, they also raise an etiological question, i.e., Does the inability to tolerate oxidant stress arise in conjunction with AIDS-KS neoplastic development, or is it pre-existing in the population at risk? Regardless, use of antioxidant therapy (low risk/ potentially high benefit) in both the "at risk" population as well as in those individuals with active disease may prove a useful preventative and/or treatment modality.

Response of failing canine and human heart cells to beta 2-adrenergic stimulation.

BACKGROUND: Failing human hearts lose beta 1- but not beta 2-adrenergic receptors. In canine hearts with tachypacing failure, the ratio of beta 2- to beta 1-adrenergic receptors is increased. The present study was designed to determine whether heart failure increases sensitivity to beta 2-adrenergic stimulation in isolated canine ventricular cardiomyocytes and to verify that myocytes from failing human ventricles contain functional beta 2-adrenergic receptors. METHODS AND RESULTS: Myocytes from healthy dogs, dogs with tachypacing failure, and human transplant recipients were loaded with fura 2-AM and subjected to electric field stimulation in the presence of zinterol, a highly selective beta 2-adrenergic agonist. Zinterol significantly increased [Ca2+]i transient amplitudes in all three groups. The failing canine myocytes were significantly more responsive than normal to beta 2-adrenergic stimulation. We also measured isotonic twitches, indo-1 fluorescence transients, and L-type Ca2+ currents in healthy canine myocytes. Zinterol (10(-5) mol/L) elicited large increases in the amplitudes of simultaneously recorded twitches and [Ca2+]i transients. Zinterol also increased L-type Ca2+ currents in the normal canine myocytes; this augmentation was abolished by 10(-7) mol/L ICI 118,551. cAMP production by suspensions of healthy and failing canine myocytes was not increased by zinterol (10(-9) to 10(-5) mol/L), nor did 10(-5) mol/L zinterol elicit phospholamban phosphorylation. CONCLUSIONS: Failing human ventricular cardiomyocytes contain functional beta 2-adrenergic receptors. Canine myocytes also contain functional beta 2-adrenergic receptors. The canine ventricular response to beta 2-agonists is increased in tachypacing failure. Positive inotropic responses to beta 2-stimulation are not mediated by increases in cAMP or cAMP-dependent phosphorylation of phospholamban.

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.

Evidence against norepinephrine-stimulated efflux of mitochondrial Mg2+ from intact cardiac myocytes.

We investigated the hypotheses that norepinephrine stimulates Mg2+ efflux from intact isolated adult rat ventricular cardiomyocytes and that adenosine 3',5'-cyclic monophosphate stimulates Mg2+ efflux from permeabilized myocytes and isolated mitochondria. Norepinephrine stimulation of Mg2+ release from cardiac myocytes was observed only when cells at approximately 20 mg protein/ml in Mg(2+)-containing buffer were diluted 50- to 60-fold into an Mg(2+)-free medium. Under these conditions, > 30% of total cellular lactic acid dehydrogenase activity was also released, indicating that a significant portion of the cells had died. In other protocols, where Mg2+ efflux from myocytes was not observed, extracellular Mg2+ removal and administration of 10 microM norepinephrine increased 45Ca2+ accumulation by cells in suspension. In single myocytes, Mg2+ removal and norepinephrine administration increased intracellular free [Ca2+] as measured by fura-2 fluorescence microscopy, and this was accompanied by vigorous spontaneous contractile activity followed by Ca2+ overload hypercontracture. With permeabilized myocytes and isolated mitochondria from a variety of sources, adenosine 3',5'-cyclic monophosphate did not stimulate Mg2+ efflux. These results suggest that recent evidence for direct hormonal regulation of myocardial Mg2+ homeostasis may need to be reevaluated.

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.

Cardiac nucleotide levels and mitochondrial respiration in copper-deficient rats.

1. Male weanling Long-Evans rats were randomly assigned to either copper-adequate (6.0 mg Cu/kg feed) or copper-deficient (< 1.0 mg Cu/kg feed) diets for a 5 week period. 2. Cardiac ATP levels and energy charges did not differ between the two groups. Levels of NAD and phosphocreatine as well as the sum of pyridine nucleotides were greater in copper-deficient rats compared with copper-adequate rats using HPLC analysis. 3. Mitochondrial respiratory control ratios and oxygen consumption rates from copper-deficient rat hearts were depressed, although ADP:O ratios were similar to copper-adequate rat hearts.