Subbarao Vemulapalli

Inhibition of endothelial derived relaxing factor (EDRF) aggravates ischemic acute renal failure in anesthetized rats

SummaryThe relative importance of endothelial derived relaxing factor (EDRF)/nitric oxide (NO) in maintaining kidney function in normal condition and in acute renal failure (ARF) were evaluated in inactin anesthetized rats. ARF was induced by unilateral occlusion of the left renal artery (40 min) followed by reperfusion, with the contralateral kidney serving as normal control. This protocol resulted in marked reductions in renal plasma flow (RPF), glomerular filtration rate (GFR) and increases in fractional sodium excretion (FENa) and urinary protein excretion in the post-ischemic kidney in comparison to the contralateral normal kidney. Administration of the nitric oxide (NO) synthase inhibitor NG — monomethyl-L-arginine (0.25 mg/kg per min, L-NMMA) exacerbated the ischemia-induced changes in renal functions as reflected by further reductions in urine flow (V), GFR, marked sodium wasting and renal edema. Pretreatment of the animals with NO precursor L-arginine (2.5 mg/kg per min, L-Arg) abolished the detrimental effects of L-NMMA in ARE In contrast, D-Arginine (2.5 mg/kg per min, DArg) failed to reverse the detrimental effects of L-NMMA. Infusion of L-Arg alone also resulted in improvements in RPF and GFR in the ischemic kidney. The results of the present study suggest that the function of the ischemic kidney is sustained by EDRF/NO and is thus more sensitive to NO synthase inhibition.

Modulation of circulating endothelin levels in hypertension and endotoxemia in rats

We have developed separate radioimmunoassays to measure circulating ET-1 and ET-3 levels in normotensive and different hypertensive rat models so that the role of endothelin in the regulation of vasomotor function can be studied. We also assessed the stimulatory effects of endotoxin on plasma and liver lymph ET-1 and ET-3 levels. The circulating ET-1 levels in normotensive rats, SHRs, and DOCA-salt hypertensive rats were 2.3 ± 0.5, 2.1 ± 0.4, and 2.1 ± 0.9 pg/ml, respectively. Similarly; the plasma ET-3 levels in normotensive and different hypertensive rats were similar, ranging from 19.7 ± 1.5 to 24.7 ± 2.2 pg/ml. The data indicate that steadystate circulating levels of endothelins are a poor correlate of the hypertensive state. Endotoxin (30 mg/kg i.v. over 15 min) reduced blood pressure significantly and augmented plasma ET-1 levels by sevenfold (29.1 ± 3.7 vs. 4.1 ± 0.6 pg/ml in the vehicle group; p < 0.05) and ET-3 levels by twofold (47.7 ± 7.0 vs. 22.7 ± 4.0 pg/ml in the vehicle group; p < 0.05).Human TNF-α (30 ng/kg/min x 30 min), a putative mediator of endotoxin shock, enhanced plasma ET-1 (18.3 ± 1.0 vs: 2.7 ± 0.4 pg/ml in the vehicle group; p < 0.05) by sevenfold and ET-3 levels by twofold (45.7 ± 2.0 vs. 27.1 ± 4.0 pg/ml in the vehicle group; p < 0.05) without affecting blood pressure. In con-trash; PAF (50 ng/kg/min x 30 min); another mediator liberated during endotoxin shock; exerted asimilar hypotensive response to endotoxin but did not alter either plasma ET-1 or ET-3 levels. In a separate study, endotoxin augmented plasma and liver lymph ET-1 levels by eight- and twofold, respectively. In comparison, endotoxin caused a twofold increase in both plasma and lymph ET-3 levels. Pretreatment with indomethacin (10 mg/kg p.o.)significantly attenuated the endotoxin-induced in-creases in plasma ET-1 and ET-3 levels without affecting the depressor responses to endotoxin. The endotoxininduced increase in ET-1 levels in the plasma and liver lymph is most likely derived from the endothelial cells. However, since ET-3 is not produced by endothelial cells, the source of endotoxin-induced increases in plasma and liver lymph ET-3 levels remains to beidentified. The data suggest that the endotoxin-stimulated re-lease of ET-1 and ET-3 can be dissociated from blood pressure changes.Eicosanoids may also be involved in the release of endothelins due to endotoxin.

Atrial natriuretic factor-potentiating and antihypertensive activity of SCH 34826. An orally active neutral metalloendopeptidase inhibitor.

The effects of SCH 34826, an orally active neutral metalloendopeptidase inhibitor, on responses to atrial natriuretic factor-(103–125) or -(99–126) and on blood pressure were evaluated in rats. SCH 34826 (10,30, and 90 mg/kg s.c. and 90 mg/kg p.o.) potentiated the antihypertensive action of atrial natriuretic factor (30 /tg/kg i.v.) in conscious spontaneously hypertensive rats. SCH 34826 (90 mg/kg) also potentiated the diuretic and natriuretic responses to atrial natriuretic factor (30 /tg/kg i.v.) as well as the plasma levels achieved after pcptide injection. SCH 34826 significantly reduced blood pressure in the conscious deoxycorticosterone acetate-salt hypertensive rat, at doses of 90 mg/kg s.c. (−35±12 mm Hg), 10 mg/kg p.o. (−30±7 mm Hg), and 90 mg/kg p.o. (−45±6 mm Hg). SCH 34826 was devoid of acute antihypertensive activity in the spontaneously hypertensive rat but reduced blood pressure by day 3 of a 5-day treatment schedule. SCH 34826 (90 mg/kg s.c.) enhanced urine volume output in the deoxycorticosterone acetate-salt rat (2.78±0.6 vs. l27±03 ml/100 g/3 hr in vehicle-control rats, p < 0.05). SCH 34826 (90 mg/kg s.c) increased plasma levels of atrial natriuretic factor at 1 hour (753±89 vs. 451 ±79 pg/ml in vehicle-treated rats, p< 0.05) but not 3 hours after dosing. The renal excretion of atrial natriuretic factor (3,092 ±1,089 vs. 21 ±6 pg/100 g/3 hr in vehicle-treated rats, p< 0.05) and cyclic guanosine monophosphate (2,131 ±509 vs. 879±168 pg/100 g/3 hr in vehicle-treated rats, p < 0.05) was markedly elevated by SCH 34826 in deoxycorticosterone acetate-salt rats. These studies suggest that neutral endopeptidase inhibition may represent a new approach to treatment of some forms of hypertension.

Antiplatelet and antiproliferative effects of SCH 51866, a novel type 1 and type 5 phosphodiesterase inhibitor.

SCH 51866 is a potent and selective PDE1 and PDE5 inhibitor. The antiplatelet, antiproliferative, and hemodynamic effects of SCH 51866 were compared with those of E4021, a highly selective PDE5 inhibitor. SCH 51866 inhibited PDE1 and PDE5 isozymes with a 50% inhibitory concentration (IC50) of 70 and 60 nM, respectively. SCH 51866 and E4021 inhibited washed human platelet aggregation induced by collagen with an IC50 of 10 and 4 microM, respectively, and attenuated (p < 0.05) the adhesion of 111indium-labeled platelets to the nylon filament-injured rat aorta. The doses of SCH 51866 and E4021 that inhibited platelet adhesion caused significant increases in platelet cyclic guanosine monophosphate (cGMP; p < 0.05). SCH 51866 (1-10 mg/kg, p.o. twice daily) but not E4021 (3-30 mg/kg, p.o twice daily) inhibited neointima formation in the carotid arteries of spontaneously hypertensive rats (SHRs) subjected to balloon angioplasty. Moreover, SCH 51866 (0.3-10 mg/kg, p.o.) elicited dose-dependent reduction in blood pressure in SHRs, whereas E4021 (3-30 mg/kg, p.o.) did not affect blood pressure in SHRs. In conclusion, the data suggest that inhibition of PDE1 and PDE5 isozymes by SCH 51866 exerts antiplatelet and vascular protective effects. In comparison, inhibition of PDE5 alone by E4021 exhibited antiplatelet effects without affecting neointima formation.

Sildenafil relaxes rabbit clitoral corpus cavernosum.

The role of phosphodiesterase type 5 inhibition in the modulation of female sexual dysfunction was investigated by assessing its effects on in vitro relaxation of rabbit clitoris. Stimulation of the non-adrenergic, non-cholinergic neurons of the clitorus elicited a frequency-dependent relaxation response. Inhibition of NO synthase with L-NAME (100 microM) or inhibition of soluble guanylate cyclase with ODQ (1.0 microM) almost completely abolished the electrical field stimulation-induced relaxation of clitorus suggesting that NO-cGMP pathway mediates the relaxation response to electrical field stimulation. Similarly, tetrodotoxin, a neuronal sodium channel blocker abolished the electrical field stimulation-induced clitoral relaxation implying a neuronal release of NO contributes to the electrical field stimulation elicited relaxation. Pretreatment of the clitoral corpus cavernosum strips with sildenafil (100 nM) enhanced the electrical field stimulation-induced relaxations both in magnitude and duration. The results suggest that sildenafil enhances electrical field stimulation elicited clitoral relaxation by a NO-cGMP dependent pathway. These data also imply that sildenafil may be useful to treat female sexual dysfunction.

Attenuation of Ischemic Acute Renal Failure by Phosphoramidon in Rats

The protective effects of phosphoramidon, a dual inhibitor of endothelin-converting enzyme and neutral endopeptidase (E.C. 24.11), on renal function in ischemic acute renal failure were investigated in anesthetized rats. Intravenous infusion of phosphoramidon (0.03 and 0.1 mg/kg per min) significantly suppressed tubular sodium wasting (measured by fractional excretion of sodium) and proteinuria in the postischemic kidney without modifying functional parameters in the contralateral normal kidney. Phosphoramidon (0.1 mg/kg/min) was associated with increased glomerular filtration in the ischemic kidney. In comparison, SCH 42354, a highly selective inhibitor of neutral endopeptidase at 0.3 mg/kg/min, did not inhibit endothelin-converting enzyme or afford renal protection. The data suggest that the protective action of phosphoramidon against ischemic acute renal failure is most likely mediated by inhibition of endothelin formation.

Phosphoramidon does not inhibit endogenous endothelin-1 release stimulated by hemorrhage, cytokines and hypoxia in rats

The role of phosphoramidon-sensitive endothelin converting enzyme in the release of endogenous endothelin-1 was investigated in anesthetized rats. Intravenous infusion of phosphoramidon 0.3 mg/kg/min did not suppress the release of endothelin-1 stimulated by hemorrhage or cytokines. Elevation of endothelin-1 in rats subjected to hypoxia was not modified by phosphoramidon (0.1 or 0.3 mg/kg/min for 2 h). A high dose of phosphoramidon (10 mg/kg i.v. +0.1 mg/kg/min) significantly potentiated the hypoxia-induced increases in plasma endothelin-1 levels. Increases in endothelin-1 release caused by bilateral nephrectomy were further enhanced by hypoxia. It is concluded that the release of endogenous endothelin-1 release stimulated by hemorrhage, cytokines and hypoxia is resistant to inhibition by phosphoramidon, and at high doses, phosphoramidon potentiates hemorrhage- and hypoxia-induced increases in endothelin-1 levels, most likely by preventing its degradation.

Cardiovascular and renal action of platelet-activating factor in anesthetized dogs.

Platelet-activating factor (PAF) has hypotensive effects similar to those of antihypertensive polar renomedullary lipid (APRL), a potent endogenous hypotensive lipid. In this study the cardiovascular and renal effects of PAF were characterized in anesthetized dogs. Intravenous infusion of PAF at 0.1 micrograms/kg/min for 1 hour caused marked reduction in arterial blood pressure and cardiac output and was accompanied by minimal changes in heart rate. Concomitantly, renal blood flow, glomerular filtration rate, urine flow, and fractional excretion of Na+ and K+ fell significantly. Plasma renin activity was greatly stimulated (11.9 +/- 1.66 vs 3.26 +/- 0.45 ng/angiotensin I/ml/hr for the placebo group). There were no significant alterations in any of these parameters following PAF at a lower dose (0.03 micrograms/kg/min for 1 hour). In a separate study, PAF at 0.1 micrograms/kg/min for 20 minutes produced a decrease in left ventricular myocardial contractile force, concomitant with bradycardia and hypotension, which indicated the presence of a negative inotropic activity. It is concluded that systemic administration of PAF has a deleterious effect on kidney function due to arterial hypotension and diminished cardiac output.

Design and synthesis of xanthine analogues as potent and selective PDE5 inhibitors.

We have discovered potent and selective xanthine PDE5 inhibitors. Compound 25 (PDE5 IC(50)=0.6 nM, PDE6/PDE5=101) demonstrated similar functional efficacy and PK profile to Sildenafil (PDE5 IC(50)=3.5 nM, PDE6/PDE5=7).

Disparate effects of phosphoramidon on blood pressure in SHR and DOCA-salt hypertensive rats

Phosphoramidon inhibits both endothelin converting enzyme (ECE) and neutral metalloendopeptidase (NEP). The contribution of ECE and NEP inhibition to the antihypertensive effects of phosphoramidon was investigated in SHR and DOCA-salt hypertensive rats. SCH 32615, an active acid of the potent and selective NEP inhibitor prodrug SCH 34826 was used as a reference compound. Intravenous infusion of SCH 32615 (1.0 mg/kg/min x 2 hr) or phosphoramidon (0.3 and 1.0 mg/kg/min x 2 hr) did not reduce blood pressure (BP) in conscious SHR. The combination of SCH 32615 (100 mg/kg + 1.0 mg/kg/min) and phosphoramidon (0.3 mg/kg/min) also did not alter BP in SHR. In comparison, the BP of conscious DOCA-salt rats was significantly reduced by phosphoramidon (0.01, 0.1 and 1.0 mg/kg/min x 2 hr) (-28 +/- 6, -51 +/- 5 and -85 +/- 6 mmHg, respectively). SCH 32615 (100 mg/kg, i.v.) over 5 min followed by a sustained infusion of 1.0 mg/kg/min for 2 hr also reduced BP by 49 +/- 7 mmHg (P < .05) in DOCA-salt rats. However, phosphoramidon (0.1 mg/kg/min x 2 hr) failed to cause a further reduction in BP in DOCA-salt rats concurrently receiving SCH 32615. In contrast, a higher dose of phosphoramidon (0.3 mg/kg/min) in combination with SCH 32615 caused a greater reduction in BP in DOCA-salt rats than SCH 32615 alone. In anesthetized normotensive rats, phosphoramidon (0.01-1.0 mg/kg/min x 30 min) dose-dependently inhibited the BP responses to big endothelin-1 (BET-1) without blocking the pressor responses to ET-1. SCH 32615 failed to attenuate the pressor responses to either BET-1 or ET-1. The results indicate that SCH 32615 lacks in vivo ECE inhibitory activity. It is concluded that the antihypertensive action of SCH 32615 and low doses of phosphoramidon can be attributed to the inhibition of NEP which may presumably cause an accumulation of ANF. In comparison, at higher doses phosphoramidon causes a further reduction of BP in DOCA-salt hypertensive rats by inhibition of endothelin bioconversion.