Russell B. Melchert

Inhibition of L-Type Ca2+ Channel Current in Rat Ventricular Myocytes by Terfenadine

To elucidate possible mechanisms underlying the cardiotoxicity of terfenadine, the effect of this antihistamine on L-type Ca2+ channel current (ICa,L) was studied in adult rat ventricular myocytes using the whole-cell patch-clamp technique. Myocytes were held at -70 mV and internally dialyzed and externally perfused with Na(+)- and K(+)-free solutions; exposure to terfenadine (10(-9) to 5 x 10(-6) mol/L) resulted in a concentration-dependent inhibition of peak ICa,L with a half-maximum inhibition concentration (IC50) of 142 nmol/L. The terfenadine-induced inhibition of ICa,L was not mediated via effects on histamine H1 receptors, because 1 mumol/L triprolidine, a more selective and potent H1 antagonist, had no effect on ICa,L. In this study, we found that terfenadine (1) increased both the fast and slow time constants of ICa,L inactivation, (2) shifted the steady state inactivation of ICa,L to more negative potentials, and (3) elicited a tonic block and a use-dependent block of ICa,L. The terfenadine-induced tonic and use-dependent block and the steady state inhibition of ICa,L were voltage dependent. Both tonic and use-dependent blocks of ICa,L by terfenadine at -40 mV were greater than that at -70 mV, and blocks were partially released by applying a long hyperpolarizing prepulse to -90 mV. These results suggest that terfenadine binds to L-type Ca2+ channels in inactivated and rested states and inhibits ICa,L predominantly by interacting with the inactivated state with an apparent dissociation constant of 60 nmol/L. Open-state block could be observed only at high concentrations of terfenadine. The high-affinity interaction of terfenadine with the inactivated state of L-type Ca2+ channels may play an important role in its cardiotoxicity under pathophysiological conditions, such as ischemia.

Gender differences in blood pressure and heart rate in spontaneously hypertensive and Wistar-Kyoto rats

1. In general, premenopausal women are known to have lower blood pressure than men and animal models have shown a similar sexual dimorphism. However, many studies in animals have been performed using anaesthetized or restrained models. Current experiments were conducted to investigate the relationships among resting heart rate, blood pressure and gender in conscious, unrestrained normotensive Wistar‐Kyoto (WKY) rats and spontaneously hypertensive rats (SHR).

Linoleic acid metabolites act to increase contractility in isolated rat heart

Previous in vivo studies in dogs suggest that the 9,10-monoepoxide of linoleic acid (9,10-cis-epoxyoctadecenoic acid [9,10-EOA]) has toxic cardiovascular effects that result in death at higher doses. More recent work with rabbit renal proximal tubule cells suggests that the 12,13-metabolites of linoleic acid are more toxic than the 9,10-isomers. Thus, in the current study, we tested the hypothesis that 12,13-EOA and 12,13-dihydroxyoctadecadienoic acid (12,13-DHOA) have direct adverse effects on the heart. Langendorff-perfused rat hearts were exposed to 30 μM linoleic acid, 30 μM 12,13-EOA, or 30 μM 12,13-DHOA for 60 min followed by a 30-min recovery period. As indicated by peak left intraventricular pressure and/or +dP/dtmax, all three of the agents elicited moderate increases in contractile function that peaked within 10–20 min. The effects of linoleic acid and 12,13-EOA returned to control values during the remainder of the 60-min exposure, whereas the positive inotropic response to 12,13-DHOA was maintained until washout. Sustained arrhythmias and negative inotropic actions were not observed with any of the three compounds. Subsequently, the monoepoxides were infused into conscious rats (35 mg/kg/h) while blood pressure, heart rate, and EKG were monitored for 24 h using biotelemetry techniques. The only effect observed was a slight decline in blood pressure. Thus, current data suggest that linoleic acid and its oxidative metabolites do not have direct cardiotoxic effects during acute exposure.

Lovastatin inhibits phenylephrine-induced ERK activation and growth of cardiac myocytes

The 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) exert numerous cellular effects through the inhibition of cholesterol synthesis. The objectives of these experiments were to determine the following: (1) whether lovastatin (LOV) inhibits phenylephrine (PE)-induced growth of neonatal rat cardiac myocytes without inducing cytotoxicity and (2) whether growth-inhibiting effects of LOV are associated with reduced PE activation of extracellular signal regulated kinases 1 and 2 (ERK 1/2). After 48 h of exposure, LOV alone (0.1–10 μM) inhibited 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction without significant changes in propidium iodide staining, and 100 μM mevalonic acid prevented the effect of LOV on MTT reduction. PE (50 or 100-μM for 48 h) induced significant increases in protein-to-DNA ratios. PE (100 μM for 5 min) significantly increased the phosphorylated forms of ERK 1 and ERK 2 and activity of ERK. After 24 h pretreatment or 48 h cotreatment, LOV (10 μM) significantly inhibited PE-induced growth. In addition, LOV pretreatment significantly inhibited the stimulatory effect of PE on ERK 2 phosphorylation and ERK activity. These results demonstrate that LOV, at concentrations that do not alter membrane integrity, inhibits PE-induced growth of cardiac myocytes, potentially through reduced activation of ERK 1/2.

Cervical cancer isolate PT3, super-permissive for adeno-associated virus replication, over-expresses DNA polymerase δ, PCNA, RFC and RPA

BackgroundAdeno-associated virus (AAV) type 2 is an important virus due to its use as a safe and effective human gene therapy vector and its negative association with certain malignancies. AAV, a dependo-parvovirus, autonomously replicates in stratified squamous epithelium. Such tissue occurs in the nasopharynx and anogenitals, from which AAV has been clinically isolated. Related autonomous parvoviruses also demonstrate cell tropism and preferentially replicate in oncogenically transformed cells. Combining these two attributes of parvovirus tropism, squamous and malignant, we assayed if AAV might replicate in squamous cervical carcinoma cell isolates.ResultsThree primary isolates (PT1-3) and two established cervical cancer cell lines were compared to normal keratinocytes (NK) for their ability to replicate AAV. One isolate, PT3, allowed for high levels of AAV DNA replication and virion production compared to others. In research by others, four cellular components are known required for in vitro AAV DNA replication: replication protein A (RPA), replication factor C (RFC), proliferating cell nuclear antigen (PCNA), and DNA polymerase delta (POLD1). Thus, we examined PT3 cells for expression of these components by DNA microarray and real-time quantitative PCR. All four components were over-expressed in PT3 over two representative low-permissive cell isolates (NK and PT1). However, this super-permissiveness did not result in PT3 cell death by AAV infection.ConclusionThese data, for the first time, provide evidence that these four cellular components are likely important for AAV in vivo DNA replication as well as in vitro. These data also suggest that PT3 will be a useful reagent for investigating the AAV-permissive transcriptome and AAV anti-cancer effect.

Acute dilatory and negative inotropic effects of homocysteine are inhibited by an adenosine blocker

1 Previous studies have shown that homocysteine elicits acute negative inotropic and coronary vasodilatory effects in rat hearts. In addition, this earlier work suggested that the inotropic action is mediated via an endothelium‐derived agent that is neither nitric oxide (NO) nor a cyclooxygenase product, while the coronary actions were found to be antagonized by the NOS inhibitor l‐NNA. Current experiments, which utilized coronary‐perfused rat hearts, were designed to determine if muscarinic or adenosine receptors are involved in these acute actions of homocysteine. 2 Left ventricular developed pressure was used as a measure of systolic function in electrically paced, Langendorff‐perfused heart with coronary pressure being used to monitor coronary vascular tone. Acute effects of homocysteine (10–300 mmol/L) were examined in the presence and absence of 1 ¥ 10‐6 mol atropine or 7 ¥ 10‐5 mol 8‐(p‐sulfophenyl) theophylline (SPT), a non‐selective adenosine receptor antagonist. 3 Atropine had no effect on either the inotropic or vascular actions of homocysteine. In contrast, SPT partially antagonized both actions of the amino acid with the antagonism of the vasodilation being much greater than its inhibition of the negative inotropic effect. Experiments with adenosine demonstrated that the selected dose of SPT elicited marked rightward shifts in the dose–response curves for both the inotropic and vascular actions. 4 Current results suggest that adenosine plays a role in both the negative inotropic and vasodilatory actions of homocysteine. However, the relatively minor antagonistic action of SPT on the inotropic effect of homocysteine suggests that additional endothelium‐derived mediators underlie its effects on contractility.

Interaction of xenobiotics with myocardial signal transduction pathways

Although the understanding of how toxicants alter cardiac ion-channel function has matured rapidly over the past 20–30 yr, little is known about how xenobiotics may alter the signaling pathways of cardiac myocyte growth and death. Signaling molecules and pathways responsible for the growth of cardiac myocytes include the mitogen-activated protein kinases (MAPKs), janus kinase-signal transducer and activator of transcription (JAK-STATs), nuclear receptor signaling, calcineurin, and the mobilization of free calcium. Signaling molecules and pathways responsible for programmed cardiac myocyte death include the death receptors, mitochondrial proteins, p53 tumor suppressor protein, ceramide signaling, and caspases. Overlap or “crosstalk” between the various growth and death pathways in the myocardium is evident, and these pathways likely exist in a delicate balance where, for example, slight reductions in growth signaling may favor pathways leading to cardiac myocyte apoptosis. Several classical cardiotoxicants are now known to alter signaling pathways in cardiac myocytes; however, the significance of these effects is not entirely clear. Furthermore, xenobiotics that alter the interstitium or extracellular matrix, or both, may significantly alter signaling pathways in cardiac myocytes. The goal of this review is to summarize current findings regarding the interaction of xenobiotics with myocardial signal transduction pathways in the hope of stimulating new insights and highlighting important areas for future research.

Effect of Interleukin 6 on the Hepatic Metabolism of Itraconazole and Its Metabolite Hydroxyitraconazole Using Primary Human Hepatocytes

A potential cytokine-drug interaction between interleukin 6 (IL-6) and itraconazole (ITZ) was studied using human hepatocytes in primary culture. Cultures from 5 adult males (mean age 42 ± 15 years) who had not received any medicines known to interact with CYP3A4 were studied. Cultures were exposed to ITZ 500 ng/ml, and the effects of 120 µg/ml cimetidine, 50 ng/ml human IL-6, or IL-6 plus IL-6 receptor antagonist were analyzed for 2, 4, 8, and 12 h. Intracellular ITZ and hydroxyitraconazole concentrations were measured using HPLC and normalized to total cellular protein. Mean intracellular concentrations between groups were compared using one-way Anova (f test; p < 0.10) and corresponding Bonferroni versus control test for multiple comparisons (p < 0.02). Mean intracellular ITZ concentrations between the groups were similar at all time points. Human hepatocytes in primary culture can metabolize ITZ. However, IL-6 did not inhibit hydroxyitraconazole formation, but it may inhibit its subsequent metabolism.