Pulsed laser imaging of Ca(2+) influx in a neuroendocrine terminal.
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The surge of Ca(2+) that triggers vesicle fusion is shaped by the distribution of Ca(2+) channels and the physical relationship between those channels and the exocytotic apparatus. Although channels and the release apparatus are thought to be tightly associated at fast synapses, the arrangement at neuroendocrine cells is less clear. The distribution of Ca(2+) influx near release sites is difficult to determine because of spatial and temporal limitations on Ca(2+) imaging techniques. We now present spatially resolved images of Ca(2+) influx into rat neuroendocrine terminals on a millisecond time scale. Images of voltage-dependent Ca(2+) influx into neurohypophysial terminals were captured after excitation of Ca(2+)-sensitive dyes with pulses of laser light lasting a fraction of a microsecond. Submembranous Ca(2+) increases were detected during the first millisecond of an evoked Ca(2+) tail current. Steep gradients of Ca(2+) were evident, with concentrations near the membrane reaching above 1 microM during a 30 msec depolarization. Ca(2+) influx appeared evenly distributed, even when diffusion was restricted with an exogenous Ca(2+) chelator. During longer depolarizations, mean and peak Ca(2+) concentrations reached an asymptote in parallel, suggesting that Ca(2+) binding proteins near the membrane rapidly buffer Ca(2+) and do not become saturated during prolonged influx. These data support the hypothesis that exocytosis is activated in these terminals by the summation of influx through multiple, randomly spaced Ca(2+) channels. (+info)
Enhancement of cytochrome P-450 3A4 catalytic activities by cytochrome b(5) in bacterial membranes.
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Activities of testosterone, nifedipine, and midazolam oxidation by recombinant cytochrome P-450 (P-450) 3A4 coexpressed with human NADPH-P-450 reductase (NPR) in bacterial membranes (CYP3A4/NPR membranes) were determined in comparison with those of other recombinant systems and of human liver microsomes with high contents of CYP3A4. Growth conditions for Escherichia coli transformed with the bicistronic construct affected expression levels of CYP3A4 and NPR; an excess of NPR over P-450 in membrane preparations enhanced CYP3A4-dependent testosterone 6beta-hydroxylation activities of the CYP3A4/NPR membranes. Cytochrome b(5) (b(5)) and apolipoprotein b(5) further enhanced the testosterone 6beta-hydroxylation activities of CYP3A4/NPR membranes after addition to either bacterial membranes or purified enzymes. NPR was observed to enhance catalytic activity when added to the CYP3A4/NPR membranes, either in the form of bacterial membranes or as purified NPR (in combination with cholate and b(5)). Apparent maximal activities of testosterone 6beta-hydroxylation in CYP3A4/NPR membranes were obtained when the molar ratio of CYP3A4/NPR/b(5) was adjusted to 1:2:1 by mixing membranes containing each protein. Testosterone 6beta-hydroxylation, nifedipine oxidation, and midazolam 4- and 1'-hydroxylation activities in CYP3A4/NPR membranes plus b(5) systems were similar to those measured with microsomes of insect cells coexpressing CYP3A4 with NPR and/or of human liver microsomes, based on equivalent CYP3A4 contents. These results suggest that CYP3A4/NPR membrane systems containing b(5) are very useful models for prediction of the rates for liver microsomal CYP3A4-dependent drug oxidations. (+info)
Atrial L-type Ca2+ currents and human atrial fibrillation.
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Chronic atrial fibrillation (AF) is characterized by decreased atrial contractility, shortened action potential duration, and decreased accommodation of action potential duration to changes in activation rate. Studies on experimental animal models of AF implicate a reduction in L-type Ca2+ current (I(Ca)) density in these changes. To evaluate the effect of AF on human I(Ca), we compared I(Ca) in atrial myocytes isolated from 42 patients in normal sinus rhythm at the time of cardiac surgery with that of 11 chronic AF patients. I(Ca) was significantly reduced in the myocytes of patients with chronic AF (mean -3.35+/-0.5 pA/pF versus -9.13+/-1. 0 pA/pF in the controls), with no difference between groups in the voltage dependence of activation or steady-state inactivation. Although I(Ca) was lower in myocytes from the chronic AF patients, their response to maximal beta-adrenergic stimulation was not impaired. Postoperative AF frequently follows cardiac surgery. Half of the patients in the control group (19/38) of this study experienced postoperative AF. Whereas chronic AF is characterized by reduced atrial I(Ca), the patients with the greatest I(Ca) had an increased incidence of postoperative AF, independent of patient age or diagnosis. This observation is consistent with the concept that calcium overload may be an important factor in the initiation of AF. The reduction in functional I(Ca) density in myocytes from the atria of chronic AF patients may thus be an adaptive response to the arrhythmia-induced calcium overload. (+info)
Expression of Ca(2+)-mobilizing endothelin(A) receptors and their role in the control of Ca(2+) influx and growth hormone secretion in pituitary somatotrophs.
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The expression and coupling of endothelin (ET) receptors were studied in rat pituitary somatotrophs. These cells exhibited periods of spontaneous action potential firing that generated high-amplitude fluctuations in cytosolic calcium concentration ([Ca(2+)](i)). The message and the specific binding sites for ET(A), but not ET(B), receptors were found in mixed pituitary cells and in highly purified somatotrophs. The activation of these receptors by ET-1 led to an increase in inositol 1,4,5-trisphosphate production and the associated rise in [Ca(2+)](i) and growth hormone (GH) secretion. The Ca(2+)-mobilizing action of ET-1 lasted for 2-3 min and was followed by an inhibition of action potential-driven Ca(2+) influx and GH secretion to below the basal levels. As in somatostatin-treated cells, the ET-1-induced inhibition of spontaneous electrical activity and Ca(2+) influx was accompanied by the inhibition of adenylyl cyclase and by the stimulation of inward rectifier potassium current. In contrast to somatostatin, ET-1 did not inhibit voltage-gated Ca(2+) channels. During prolonged agonist stimulation a gradual recovery of Ca(2+) influx and GH secretion occurred. In somatotrophs treated with pertussis toxin overnight, the ET-1-induced Ca(2+)-mobilizing phase was preserved, but it was followed immediately by facilitated Ca(2+) influx and GH secretion. Both somatostatin- and ET-1-induced inhibitions of adenylyl cyclase activity were abolished in pertussis toxin-treated cells. These results indicate that the transient cross-coupling of Ca(2+)-mobilizing ET(A) receptors to the G(i)/G(o) pathway in somatotrophs provides an effective mechanism to change the rhythm of [Ca(2+)](i) signaling and GH secretion during continuous agonist stimulation. (+info)
Continuous infusion cyclosporine and nifedipine to day +100 with short methotrexate and steroids as GVHD prophylaxis in unrelated donor transplants.
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Unrelated donor marrow transplantation is associated with an increased incidence of graft-versus-host disease (GVHD) compared with sibling donor transplants. Forty-one patients undergoing unrelated donor transplants were treated with a GVHD prophylaxis regimen that consisted of continuous infusion cyclosporine from day -1 to 100 days post transplant along with nifedipine, glucocorticoids and short-course methotrexate. The regimen was well-tolerated in this cohort with mostly high risk disease. Fifty-one percent of patients developed acute GVHD, which was grade III-IV in 22% of patients. Six of 22 patients at risk for chronic GVHD developed extensive chronic GVHD, five of whom were adults. In patients <18 years of age, there was a >40% chance of 2 year disease-free survival. Use of continuous infusion cyclosporine with nifedipine as an immunosuppressant and protectant against cyclosporine-induced toxicities in unrelated donor transplants is well-tolerated, and results in acute GVHD incidence favorable to that reported with bolus cyclosporine. (+info)
Nifedipine inhibits pulmonary hypertension but does not prevent decreased lung eNOS in hypoxic newborn pigs.
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Therapies to prevent the onset or progression of pulmonary hypertension in newborns have received little study compared with those in adult models. We wanted to determine whether nifedipine treatment prevents the increased pulmonary vascular resistance, blunted pulmonary vascular responses to acetylcholine, and reduced lung endothelial nitric oxide synthase (eNOS) amounts that we have found in a newborn model of chronic hypoxia-induced pulmonary hypertension. Studies were performed with 1- to 3-day-old piglets raised in room air (control) or 10% O2 (hypoxia) for 10-12 days. Some piglets from each group were given nifedipine (3-5 mg/kg sublingually three times a day). Pulmonary arterial pressure, pulmonary wedge pressure, and cardiac output were measured in anesthetized animals. Pulmonary vascular responses to acetylcholine and eNOS amounts were assessed in excised lungs. The calculated value of the pulmonary vascular resistance for nifedipine-treated hypoxic piglets (0.09 +/- 0.01 cmH(2)O. ml(-1). min. kg) was almost one-half of the value for untreated hypoxic piglets (0.16 +/- 0.01 cmH(2)O. ml(-1). min. kg) and did not differ from the value for untreated control piglets (0.05 +/- 0.01 cmH(2)O. ml(-1). min. kg). Pulmonary arterial pressure responses to acetylcholine and whole lung homogenate eNOS amounts were less for both nifedipine-treated and untreated hypoxic piglets than for untreated control piglets. Nifedipine treatment attenuated pulmonary hypertension in chronically hypoxic newborn piglets despite the persistence of blunted responses to acetylcholine and reduced lung eNOS amounts. (+info)
Nifedipine and arotinolol in combination for accelerated-malignant hypertension: results of one year follow-up.
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The effects of a combined therapy with a calcium channel antagonist and alphabeta-blocker in patients with accelerated-malignant hypertension on blood pressure and renal function were examined. Thirteen patients presented with the clinical features of malignant hypertension (diastolic blood pressure >130 mmHg, retinal damage and progressive renal failure) at our hospital, over the 3 yr period from 1995 to 1997. These patients were treated with both a calcium antagonist, 60-80 mg/d dose of long acting nifedipine, and an alphabeta-blocker, 20 mg/d dose of arotinolol, for over 12 mo. At admission, the average blood pressure of the patients was 233+/-8/144+/-3 mmHg. The level of serum creatinine in these patients was 6.2+/-1.0 mg/dl. Intermittent hemodialysis therapy was introduced in 7 patients. Three days after treatment, blood pressure decreased to 162+/-4/102+/-4 mmHg. A month later, blood pressure decreased to 148+/-3/89+/-2 mmHg and serum creatinine levels were 3.6+/-0.4 mg/dl. Renal function in these patients improved, and they completely recovered from renal dysfunction, allowing withdrawal of haemodialysis therapy. One year later, the blood pressure in all of these patients was well controlled and no further renal deterioration was observed, except in one patient. Despite the reduction in blood pressure, one patient was on hemodialysis three times a week after 8 mo of treatment. From these finding, it is concluded that combination therapy with a calcium antagonist and alphabeta-blocker is effective in both the reduction of highly elevated blood pressure and protection of the kidneys, resulting in amelioration of accelerated-malignant hypertension. (+info)
Pharmacokinetics and pharmacodynamics of nifedipine in untreated and atorvastatin-treated hyperlipidemic rats.
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Nifedipine, a hypertensive calcium channel blocker, is commonly administered to subjects with coronary heart disease who often exhibit hyperlipidemia. In general, the pharmacokinetic consequences of hyperlipidemia include increased total drug concentrations and decreased unbound fraction in plasma. However, the pharmacodynamic consequences of hyperlipidemia are conflicting; unaltered, increased, or decreased pharmacological effects are reported. In this study, the effect of experimental hyperlipidemia on pharmacokinetic and pharmacodynamic consequences of nifedipine was studied. After establishing a dose (0.05-0.3 mg.kg(-1))-effect relationship, single 0.1 mg.kg(-1) i.v. doses of nifedipine were administered to control and poloxamer 407-induced hyperlipidemic (with and without cholesterol-lowering agent atorvastatin) rats. Mean arterial pressure, total as well as unbound nifedipine plasma concentrations, and total cholesterol were monitored. Hyperlipidemia significantly decreased systemic clearance of nifedipine by 40% and increased T(1/2) and area under the plasma concentration-time curve by 85 and 65%, respectively. Compared with the hyperlipidemic group, atorvastatin-treated rats had significantly lower total plasma cholesterol (0-70%), increased systemic clearance (39%), and decreased T(1/2) (27%) and area under the plasma concentration-time curve (24%). Hyperlipidemia prolonged pharmacological T(1/2) of nifedipine by 300%. Atorvastatin treatment significantly reduced this prolongation to 46%. There was a significant correlation between mean blood pressure and the total but not unbound nifedipine plasma concentrations. Hyperlipidemia potentiates the hypotensive effect of nifedipine by increasing its total plasma concentrations despite decreased unbound drug concentration. (+info)