Jorge Jaramillo

Tissue Specificity of Endothelin Binding Sites

A measurement was made of the binding of 125I-labeled endothelin (125I-ET) to crude membrane fractions prepared from rat aorta, atrium, ventricle, portal vein, trachea, lung parenchyma, vas deferens, ileum, bladder, and guinea-pig taenia coli and lung parenchyma. Scatchard analysis of 125I-ET binding in all tissues indicated binding to a single class of saturable sites. The affinity and density of 125I-ET binding sites varied between tissues. The Kd of 125I-ET binding was ≤0.5 nM for rat aorta, trachea, lung parenchyma, ventricle, bladder, and vas deferens, and guinea-pig taenia coli and lung parenchyma, 1.8 nM for rat portal vein and atrium, and 3.3 nM for ileum. The Bmax of 125I-ET binding had the following rank order of density in rat tissues: trachea > lung parenchyma = vas deferens ± aorta = portal vein = atrium > bladder > ventricle = ileum. The properties of 125I-ET endothelin binding were characterized in rat ventricular membranes. 125I-ET binding was time dependent, reaching a maximum within 45–60 min at 25°C. The calculated microassociation constant was 9.67 ± 105 s−1 M−1. Only 15–20% of 125I-ET dissociated from its binding site even when dissociation was studied as long as 3 h. Preincubation of ventricular membranes with ET prevented binding of 125I-ET. 125I-ET binding was destroyed by boiling of ventricular membranes and was temperature, pH, and cation (Ca2+, Mg2+, and Na+) dependent. F125I-ET binding was not affected by specific calcium channel ligands, N-methyl-D-aspartate (NMDA) receptor-gated ionophore antagonists, peripheral benzodiazepines, opiates, atrial natriuretic factor, angiotensin II, and sodium nitroprusside. There was no correlation between the density of ET binding sites and the contractile efficacy of ET in rat and guinea pig tissues. Exposure of rat atrium tissues to ET (10−7 M) in vitro resulted in a significant (25%) inhibition of ET binding to atrium membranes isolated from these tissues. These results indicate that specific high-affinity 125I-ET binding sites with varying properties exist in a number of tissues. ET binding does not display simple reversible bimolecular kinetics but instead demonstrates kinetics characteristic of irreversible binding.

Tissue selectivity and calcium dependence of contractile responses to endothelin

The tissue selectivity and calcium dependence of endothelin, a potent vasoconstrictor peptide obtained from endothelial cells, were investigated in a number of rat and guinea pig tissues. Endothelin produced a significant dose-dependent contraction of rat tissues with the following rank order of efficacy: aorta > portal vein > trachea >> vas deferens = lung parenchyma >> bladder. In the rat atrium and ventricle, endothelin possessed positive inotropic effects at low doses and arrhythmogenic effects at high doses. Endothelin also produced a significant contraction of guinea pig lung parenchyma and induced relaxations of guinea pig taenia coli precontracted with potassium chloride. The aortic ring contractile response to endothelin was characterized by both rapid and slow phases of contraction. The rapid phase of contraction was abolished by removal of extracellular calcium or preincubation of tissues with the dihydropyridine calcium channel antagonist, nifedipine. Removal of the endothelium from aortic rings resulted in an increase of approximately 10-fold in the contractile potency of endothelin. In comparison, the potency of the dihydropyridine calcium channel activator, (-)-S-BAY K 8644, was increased approximately 15-fold by the same treatment. These findings suggest that endothelin, while displaying some selectivity as a vascular smooth muscle spasmogen, is a general smooth and cardiac muscle spasmogen which utilizes both intra- and extracellular sources of calcium to support its contractions.

A novel renal hypertensive guinea pig model for comparing different inhibitors of the renin-angiotensin system

The present study describes a novel renal hypertensive guinea pig model for comparing different inhibitors of the renin-angiotensin system (RAS). Renal hypertension was induced by a two-step procedure consisting of ligation of the left caudal renal artery and right nephrectomy. Sham-operated animals were used as controls. Arterial blood pressure and heart rate were monitored in conscious animals. Left caudal renal artery ligation and subsequent right nephrectomy led to a significant increase (32% over sham-operated controls, p < .05) in mean arterial blood pressure (MABP), 3 to 4 weeks following surgery. Renal hypertensive animals had increased urine production (from 63 +/- 8 mL/kg per day to 143 +/- 29 mL/kg per day, p < .05) and an increased incidence of proteinuria (11/13 animals had urine protein levels higher than 20 mg/kg per day). Five of the 13 renal hypertensive animals also had hematuria. On autopsy, an 83% increase in the left kidney/body weight ratio and a 37% increase in the heart/body weight ratio were observed in the renal hypertensive animals, compared to the sham-operated controls. Changes in blood pressure and heart rate were assessed before and after an intravenous bolus injection of the drug to be tested. Captopril reduced MABP in both sham-operated and renal hypertensive animals with equal efficacy (up to a maximum of 42%). In contrast, BILA 2157 BS, one of our human renin inhibitors, produced a similar maximum MABP decrease but only in renal hypertensive animals. This selective antihypertensive effect was also observed with enalkiren, another renin inhibitor. These results indicate that the renal hypertensive guinea pig is an useful model for comparing and contrasting different RAS inhibitors.

Cutaneously applied acyclovir acts systemically in the treatment of herpetic infection in the hairless mouse

Using the SKH-1 hairless mouse (HM) we have addressed the issue as to whether topically applied acyclovir (ACV) may mediate some of its antiviral actions by a systemic effect. When topically applied in a formulation consisting of polyvinyl alcohol (25% w/v):DMSO:cremophor EL:linoleic acid (63:16:16:5, v/v/v/v), ACV penetrated hairless mouse skin in a concentration-dependent manner and dose-dependently reduced cutaneous herpes simplex virus 1 (HSV-1) KOS infection. Topically applied ACV also effectively reduced the mortality associated with disseminated HSV-2 HG-52 infection. At 1 h following topical application of 1.7% w/v ACV the plasma and skin concentrations of ACV were 5.5 nmoles/ml and 120 nmoles/g. At 1 h following an oral dose of ACV with antiviral efficacy comparable to topically applied ACV (1.7% w/v) the plasma and skin concentrations of ACV were 21.3 nmoles/ml and 51 nmoles/g. These findings imply that when applied topically to the HM, ACV can mediate a portion of its antiviral activity through a systemic mode of action.