The relationship between plasma angiotensin and the reduction of blood pressure with the angiotensin converting enzyme inhibitor enalapril was studied in rats. Blood pressure was measured in conscious rats with indwelling arterial catheters. To measure angiotensin II, plasma was analyzed by physical separation of angiotensins using high performance liquid chromatography followed by radioimmunoassay. The effects of both single (acute) and long-term (chronic) dosages of enalapril were measured. After a single oral dose of enalapril (10 mg/kg), mean arterial pressure fell from 111 ± 3 to 86 ± 3 nun Hg (p<0.005). Despite the blood pressure reduction, plasma angiotensin II was unaffected (control, 9.9 ± 1.8 vs 9.7 ±1.1 pg/ml). After a higher single oral dose of enalapril (30 mg/kg), there was a reduction in both mean arterial pressure (81 ± 5 mm Hg, p<0.005) and plasma angiotensin II concentration (2.3 ± 0.6 pg/ml, p<0.01). The chronic effects of converting enzyme inhibition were evaluated in rats given enalapril in their drinking water (30 mg/kg/24 hr) for 1 week or 2 months. Mean arterial pressure remained equally low after chronic administration (for 1 week or 2 months), but plasma angiotensin II increased above normal (after 1 week, 28.9 ±8.7, p<0.01 vs control; after 2 months, 43.1 ± 16.2 pg/ml, p< 0.05 vs control). Although plasma angiotensin converting enzyme activity was undetectable at any time after enalapril administration, there was a partial return of the angiotensin I pressor response with chronic administration. The data are most compatible with actions of converting enzyme inhibitors independent of the blockade of plasma angiotensin II formation. (Hypertension 9 [Suppl III]: III-42- III-48, 1987) KEY WORDS • angiotensin II • converting enzyme inhibition • high performance liquid chromatography • radioimmunoassay • enalapril A NGIOTENSIN converting enzyme (ACE) in-/ \ hibitors were designed to lower blood pres-A. V . sure by blocking the formation of the potent vasopressor, angiotensin II (ANG II). 1 -2 Evidence from many studies seems to confirm this mechanism of ACE inhibitor action. The acute administration of ACE inhibitors lowers blood pressure, decreases levels of immunoreactive ANG II in plasma, 3 " 5 and prevents blood pressure rise in the two-kidney, one clip Goldblatt model. 6 Several recent observations suggest that the mechanism of action of ACE inhibitors is more complex than

Previous autoradiographic studies have delineated the renal medullas the predominant site of renal endothelin (ET) receptors. Accordingly, cultured rat renal medullary interstitial cells (RMICs) were studied as a target tissue for ET action. Scatchard analysis revealed presence of a single class of high-affinity receptor sites (Kd, 57 +/- 10 pM; receptor density, 749 +/- 124 fmol/mg protein). Relative potency order for displacing 125I-ET-1 was ET-1 greater than ET-2 greater than sarafotoxin greater than big endothelin (human) = big endothelin (porcine). ET-3, unrelated pressor substances, vasodilators, Ca2+ channel antagonists, atrial natriuretic factor, GTP, and GppNHp did not inhibit binding. Challenge of monolayers with ET-1 evoked a biphasic elevation in cytosolic free Ca2+ concentration [Ca2+]i). Initial transient rise in [Ca2+]i observed in absence of extracellular Ca2+ and accumulation of inositol trisphosphate (IP3) was consistent with activation of phosphatidylinositol-specific phospholipase C (PI-PLC). Half-maximal activation concentration of ET-1 for the process was 0.5 and 1 nM for [Ca2+]i and IP3, respectively. The late sustained phase in [Ca2+]i elevation was completely blocked by Ni2+, unperturbed by nimodipine, and accompanied by influx of Mn2+, indicating presence of receptor-operated Ca2+ channels. Ca2+ channel opening was detected at 10(-16) MET-1, whereas greater than 10(-12) M agonist was required to mobilize Ca2+ from intracellular stores and/or stimulate phosphoinositol hydrolysis, indicating that ET activation of PI-PLC and Ca2+ channel opening were independent events. ET-1 markedly stimulated prostaglandin E2 synthesis in a concentration-dependent manner that paralleled PI-PLC activation and mobilization of [Ca2+]i. In summary, cultured rat RMICs possess ET receptors that are linked to PI-PLC, Ca2+ channels, and perhaps phospholipase A2.

The distribution of endothelin-like immunoreactivity was examined in normal rat kidneys using the immunoperoxidase technique. A specific polyclonal antibody to endothelin 1, which recognized endothelin 1 and its precursor molecule, big endothelin, was raised in rabbits. Immunoperoxidase staining revealed specific endothelin-like immunoreactivity in the renal cortex, medulla, and papilla. Immunostaining density was greatest in the renal papilla where staining was predominantly localized to the vasa rectae of the distal nephron segments. Cytoplasmic immunostaining was noted focally in collecting duct cells in the renal papilla. In the renal medulla, intense immunostaining was identified in the vasa rectae. Cortical immunostaining was localized to the endothelial surfaces of arcuate arteries, veins, arterioles and peritubular capillaries. Glomerular immunostaining followed a capillary loop distribution and appeared to be predominantly localized to endothelial cells with smaller amounts of reaction product overlying the mesangium. The most proximal portion of the proximal tubule brush border and papillary collecting duct epithelium demonstrated focal endothelin-like immunostaining. We conclude that endothelin-like immunoreactivity is widely distributed in renal tissue compatible with important autacrine and paracrine actions in the kidneys.

This investigation was performed to study the potential role of endothelin in the modulation of fetoplacental vascular resistance in the human placenta. Full-term placentas from uncomplicated pregnancies were studied within 30 min of delivery. The umbilical artery and vein to a single placental cotyledon were cannulated and the artery perfused with RPMI media (0.82 ml/min). Endothelin 1 caused a sustained dose-dependent increase in perfusion pressure. Infused endothelin 1 (50 nM) stimulated thromboxane release 2.3-fold compared with basal values. Thromboxane release persisted for 15 min after discontinuation of endothelin. Properties of human placental endothelin 1 receptors were defined in binding studies performed on a crude membrane fraction of placental cotyledons. Binding was saturable, reached steady state by 3 h at 25 degrees C, and was linear with protein concentration. Scatchard analysis of binding data indicated a single class of high-affinity binding sites with a Kd of 36.1 +/- 9.7 pM and a density of 185.4 +/- 9.6 fmol/mg protein (n = 5). The potency order for competitive inhibition of the binding of 125I-labeled endothelin 1 was endothelin 1 greater than endothelin 2 = endothelin 3 = sarafotoxin S6b greater than big endothelin (human) = big endothelin (porcine). Phenylephrine, bradykinin, norepinephrine, atrial natriuretic factor, diltiazem, U46619, and angiotensin II did not displace 125I-endothelin 1 from its receptors. These experiments demonstrate that endothelin 1 is a potent pressor substance in the human fetoplacental cotyledon. Pressor effects of endothelin may be mediated by a combination of direct effects and stimulation of vasoconstrictor prostanoids.

This investigation was performed to study the renin-angiotensin system in the human fetoplacental circulation. Full-term placentas from uncomplicated pregnancies were studied within 30 min of delivery. The umbilical artery and vein to a single placental cotyledon were cannulated and the artery perfused with RPMI media (0.764 ml/min). Angiotensin II caused a dose-dependent increase in perfusion pressure that was blunted by the administration of the competitive angiotensin II receptor antagonist saralasin. The properties of human placental angiotensin II receptors were further defined in binding studies performed on a crude membrane fraction of placental cotyledons. In experiments performed at 22 degrees C, saturable binding reached steady state at 30 min and was linear with protein concentration. Scatchard analysis of binding data indicated a single class of high-affinity binding sites. The potency order to competitive binding of analogues and antagonists of angiotensin II was [Ile5]angiotensin II = [Sar1, Ala8]-angiotensin II greater than [Val5]angiotensin II greater than angiotensin III greater than angiotensin II-(3-8) hexapeptide. Further evidence for the physiological significance of angiotensin II binding sites was provided by measurements of the circulating components of the renin-angiotensin system in umbilical venous blood (n = 7). Plasma renin activity, angiotensin I, angiotensin-converting enzyme activity, angiotensin II, and aldosterone were each present in elevated amounts. These experiments provide evidence for an active renin-angiotensin system in the human fetal circulation that may modulate placental perfusion and function under physiological conditions.