Peter J.S. Chiu

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.

Renal Extraction of Gentamicin in Anesthetized Dogs

The tubular handling of gentamicin (G) and its intrarenal distribution were determined to elucidate the mechanism of G accumulation in the kidney. At a serum level of 11.1 ± 0.5 μg/ml (10 animals), as maintained by constant infusion for 5 h, serum Na+ and K+, arterial pressure, effective renal plasma flow and glomerular filtration rate remained undisturbed. The clearance values in milliliters per minute for G, inulin, and p-aminohippuric acid were 40.3 ± 1.8, 49.9 ± 2.8, and 132 ± 14, respectively. The ratio of clearance of G to clearance of inulin was 0.82 ± 0.04 (P < 0.005), suggesting net reabsorption of G by the renal tubules. The renal cortex/serum ratio for G was 11.9 ± 2.1, and the medulla/serum ratio was 2.7 ± 0.4, indicating greater uptake of G by the cortex. The extraction ratio of p-aminohippuric acid was 0.74 ± 0.03. In contrast, the extraction ratio of G was 0.20 ± 0.03, which was significantly lower than that of inulin (0.30 ± 0.04). It is concluded that the accumulation of G in the cortex was due to tubular reabsorption. Probably some of the reabsorbed G became trapped in the epithelial cells after crossing the luminal membrane, whereas some returned to the circulation.

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.

Effects of Hydration on Gentamicin Excretion and Renal Accumulation in Furosemide-Treated Rats

The effect of furosemide on gentamicin excretion and tissue accumulation was studied with clearance techniques in anesthetized rats, at two different infusion rates of saline or Ringer solution. Gentamicin (∼20 mg/kg) was administered by constant intravenous infusion over a period of 3 h. With the low fluid infusion rate, furosemide (25 mg/kg intravenously) caused severe reduction in glomerular filtration rate and diminished urinary output of gentamicin. Serum and renal tissue levels of the antibiotic were significantly elevated. High fluid infusion prevented the decline of the glomerular filtration rate, with near normalization of all measurements. A fluid deficit incurred by furosemide was noted at both the low and high infusion rates. Complete correction of this fluid deficit by continuous adjustment of the infusion rate fully restored normal renal handling of gentamicin. These results suggest that furosemide had no direct effect on renal excretion of gentamicin. In comparison, renal handling of gentamicin in rats did not respond to changes in the rate of fluid infusion in the absence of furosemide therapy. It appears that gentamicin excretion and gentamicin accumulation in the renal cortex in furosemide-treated rats, in contrast with those in untreated rats, are influenced significantly by the rate of fluid infusion. Fluid administration sufficient to maintain the glomerular filtration rate was found to be necessary for appropriate gentamicin elimination, with consequent reduction in serum and renal tissue levels of the drug.

RENAL UPTAKE AND NEPHROTOXICITY OF GENTAMICIN DURING URINARY ALKALINIZATION IN RATS

1. Effect of urine pH on accumulation of gentamicin in the renal cortex of rats was studied following constant intravenous infusion, and single or repeated i.v. injections with gentamicin.

Design and Optimization of Selective Protein Kinase C θ (PKCθ) Inhibitors for the Treatment of Autoimmune Diseases

Protein kinase C θ (PKCθ) has a central role in T cell activation and survival; however, the dependency of T cell responses to the inhibition of this enzyme appears to be dictated by the nature of the antigen and by the inflammatory environment. Studies in PKCθ-deficient mice have demonstrated that while antiviral responses are PKCθ-independent, T cell responses associated with autoimmune diseases are PKCθ-dependent. Thus, potent and selective inhibition of PKCθ is expected to block autoimmune T cell responses without compromising antiviral immunity. Herein, we describe the development of potent and selective PKCθ inhibitors, which show exceptional potency in cells and in vivo. By use of a structure based rational design approach, a 1000-fold improvement in potency and 76-fold improvement in selectivity over closely related PKC isoforms such as PKCδ were obtained from the initial HTS hit, together with a big improvement in lipophilic efficiency (LiPE).

The effects of atriopeptin II on calcium fluxes in rabbit aorta

Effects of atriopeptin II (AP II) at 10(-7) M on the 45Ca flux and contractile responses to vasoconstrictors including norepinephrine (NE), angiotensin II (angio II) and high K were studied in isolated rabbit aortic strips. Augmentation of 45Ca efflux from aorta in normal physiological saline (PSS) due to NE and angio II each at 3 X 10(-7) M was greatly inhibited by AP II, suggesting that stimulated increase in cytosolic Ca2+ was suppressed. In contrast, the 45Ca efflux responses to KCl (20 and 40 mM) were not affected by AP II. Furthermore, the marked inhibition of contractile responses to NE (-58%) and angio II (-57%) by AP II was accompanied by significant decreases in 45Ca influx, whereas AP II exhibited only a modest inhibition on KCl (40 mM)-induced contraction (-28%) without affecting the accompanying increase in 45Ca influx. The 45Ca efflux from aortae in Ca2+-free PSS due to NE was markedly diminished by AP II, suggesting impairment of intracellular Ca2+ release. With tissues in either a basal or post-stimulation state (tissue Ca2+ previously increased with KCl stimulation), AP II did not stimulate 45Ca efflux in Ca2+-free PSS, suggesting its lack of effect on Ca extrusion. It is concluded that AP II is preferentially antagonistic against vascular responses to NE and angio II vs. high K and that inhibition of Ca entry and release forms the primary basis of its potent vasorelaxant action against vasoconstrictors.