The concentration of atrial natriuretic peptide (ANP) is decreased in plasma but not in atria in hypophysectomised rats

To study the role of the pituitary gland in the release of Atrial Natriuretic Peptide (ANP) plasma and atrial concentrations were measured both in intact and in hypophysectomized rats. The plasma concentration of ANP (pg/ml) was significantly (p less than 0.01) decreased from 143 +/- 35 to 82 +/- 29 (mean +/- SD, n) while the tissue concentration (ng/wet tissue mg) remained unchanged, 192 +/- 46 and 194 +/- 39, respectively. The total atrial amount of ANP (ug) was, however, significantly (p less than 0.01) decreased from 29.7 +/- 7.8 to 17.0 +/- 3.3 after hypophysectomy. In intact animals, a volume load (1.1ml/100 body weight g 0.9% NaCl) resulted in 2-fold (p less than 0.001) increase in the plasma ANP levels whereas similar load had no effects on plasma ANP levels in hypophysectomized animals. In both groups, the right atrial pressure was increased from about 2 to about 6 mmHg. We conclude that in the absence of pituitary gland the right atrial pressure and the atrial ANP concentration do not change but plasma ANP levels and the response to volume stimulus are attenuated.     

Endotoxin stimulates endothelin-release in vivo and in vitro as determined by radioimmunoassay

A marked increase in immunoreactive endothelin was observed in rat serum collected within 10-15 min after infusion of endotoxin. Endothelin level was 117 +/- 11.5 pg/ml (mean +/- S.E., N = 4) in rats exposed to endotoxin as compared with undetectable levels (less than 2 pg/ml, N = 4) in controls. We have also observed a significant stimulation of endothelin-release by endotoxin from cultured bovine transformed thoractic aortic endothelial cells at concentrations of endotoxin ranging between 0.1 and 10.0 micrograms/ml. Serum was indispensable for the stimulating effect of endotoxin, although serum itself did not show any effect at the concentration used (1%). These results suggest that endothelin plays an important role in mediation of pathophysiological responses caused by endotoxin. The levels of endothelin were measured by radioimmunoassay with high sensitivity.     

Endothelin inhibits the atrial natriuretic factor stimulated cGMP production by activating the protein kinase C in rat aortic smooth muscle cells.

Preincubation of rat thoracic aortic smooth muscle cells with endothelin inhibits the atrial natriuretic factor (ANF)-induced cGMP accumulation in these cells in a concentration dependent manner. The maximal inhibition of 64% was afforded by 1 x 10(-6) M endothelin and the half maximal inhibition (IC50) was achieved with 1 x 10(-9) M endothelin. Endothelin (1 x 10(-6) M) also increased the plasma membrane bound protein kinase C (PKC) activity by 4 fold. Hormone-dependent increase in PKC activity was limited to plasma membranes only and some decrease in cytosolic PKC activity was observed. However, phorbol 12-myristate 13-acetate (PMA) (1 x 10(-6)M) provoked a total loss of cytosolic PKC activity and a net gain in membranous PKC activity indicative of the translocation of the enzyme. Pretreatment of these cells with H-7, a PKC inhibitor, released the endothelin and PMA-mediated attenuation of ANF-stimulated cGMP formation. These results suggest that PKC is involved in the regulation of ANF-induced cGMP accumulation and that the vasoconstrictor activity of endothelin might involve inhibition of the vasorelaxant activity of ANF through the inhibition of cGMP accumulation in smooth muscle cells (SMCs) of the rat aorta.     

内皮素在实验性主动脉狭窄—高盐摄入性高血压发病中的作用

内皮素是血管内皮释放的生物活性多肽,具有强烈的收缩血管和促进血管平滑肌细胞增生肥大的效应。本工作在大鼠腹主动脉狭窄和高盐摄入引起的高血压模型上,观察到高血压动物血浆内皮素水平成倍增加(9.70±0.68vs。假手术动物4.11±0.33pg/ml,P<0.01)。应用特异的内皮素抗血清治疗,显著缓解了动物血压的升高(18.97±1.32vs.27.33±0.90kPa,P<0.01)和心肌肥大损伤。实验结果提示:内皮素是高血压的重要发病因素之一;拮抗内皮素可能是高血压病防治的新途径。     

6-OHDA sympathectomy and exercise performance in the rat.

6-hydroxydopamine (6-OHDA) was utilised for the study of the sympathetic nervous system in the resting rats and rats submitted to prolonged exercise. In order to reduce the acute physiological stress associated with an injection of 6-OHDA, beta-1 and alpha-1 adrenoceptors were blocked before the treatment leading to sympathectomy. Sympathectomised rats were divided in two groups: one sacrificed at rest, 24 hours after the treatment. The other group was sacrificed after a treadmill exercise to exhaustion. Running time to exhaustion was 36.0 +/- 4.5 min (mean +/- S.E.M.). This group ran significantly less than a control group brought to exhaustion in 73.7 +/- 10.0 min of exercise (P < 0.05). In order to make appropriate comparisons, another control group was run for 36 min. Some differences were observed between corresponding control and sympathectomized groups. At rest: 1) a lower plasma level of insulin, and 2) a higher plasma free fatty acid concentration were observed in sympathectomized rats. After 36 min of exercise: 1) a lower plasma concentration of norepinephrine, 2) no decrease of the plasma level of insulin, 3) no increase in the plasma glucagon concentration, and 4) a higher plasma glucose level were observed in sympathectomised rats when compared to control rats running for the same time. The lower plasma norepinephrine concentration in exercised sympathectomised rats suggests a lower sympathetic nervous activity in these animals than in control rats. The absence of a decrease of plasma insulin concentration and of an increase in glucagon can be attributed to this lower sympathetic activity in sympathectomised rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cardiovascular and hormonal response during a 4-week head-down tilt with and without exercise and LBNP countermeasures

To determine whether exercise and Lower Body Negative Pressure (LBNP) during 28 days of -6 degrees head-down tilt (HDT) would modify orthostatic tolerance and blood volume regulating hormones, twelve healthy men were assigned to either a no- countermeasure (No-CM, n=6), or a countermeasure (CM, n=6) group. LBNP sessions consisted of 15 minutes exposure to -30 mm Hg, on days 16, 18, 20 and 22-28 of HDT. Muscular exercise began on day 8 and consisted of combined graded dynamic and isometric resistance bilateral leg exercise on a specially designed supine ergometer, in two sessions of 15-20 min. each, every day, 6 days per week. A tilt test was performed before and at the end of HDT. Changes in resting plasma volume from control day (D-5) to HDT day 24 were -11.2% for No-CM and -2.2% for CM. After HDT three among the 6 subjects of the No-CM group presented presyncopal or syncopal symptoms, no tilt test was interrupted in CM group. Atrial Natriuretic Peptide (ANP) decreased at day 7 for the two groups and remained low during all the HDT period for No-CM group only. Plasma Renin Activity and Aldosterone increased at day 7 and remained elevated for the two groups. Norepinephrine and epinephrine were unchanged. Elevated diuresis and natriuresis were evident during the first day of HDT. However, renal excretory patterns were different between the two groups: indeed, a decrease of Na+, ANP and cGMP was observed only in No-CM at Day 13 during HDT. Our data showed that the subjects of the No-CM group experienced a greater increase in heart rate and a decrease in systolic blood pressure during tilt tests after HDT; nevertheless, after HDT, blood pressure was better maintained in CM group during the tilt test. The plasma volume decrease measured at the end of HDT was significantly lower in CM group, in contrast, these countermeasures were ineffective in preventing at least certain changes in blood volume regulating hormones.     

Plasma endothelin levels during myocardial ischemia and reperfusion

Endothelin, an endothelium-derived vasoconstrictive peptide, has a strong potency of coronary artery constriction. However, the role of endogeneous endothelin under pathophysiological conditions has not yet been known. In this study, we examined plasma endothelin concentration in dogs with myocardial ischemia and reperfusion. Anesthetized open-chest dogs underwent either 45 minutes occlusion of the left anterior descending coronary artery followed by 3 hours reperfusion, or 4-10 hours of continuous occlusion. Plasma concentration of endothelin from the central vein was measured by the highly sensitive enzyme-immunoassay. Plasma endothelin concentration increased 2.2-fold with the peak level at 60 minutes after release of the ligated artery, but occlusion per se caused no remarkable change. These data suggest that reperfusion of the occluded artery might be needed to increase the plasma concentration of endothelin in case of myocardial infarction.     

内皮素在休克中的发病学意义

内皮素是新近发现的血管内皮细胞产生的一种生物活性多肽,本工作用特异性放免法测定发现晚期败血症休克病人血浆内皮素水平显著增加;给健康大鼠持续滴注内皮素即能复制出典型休克模型(Endothelin-shock);给失血性休克大鼠滴注小剂量内皮素则恶化休克促进其不可逆发展。实验结果提示内皮素可能是机体的内源性致损伤因子,是参与休克发生发展的重要体液因素之一。     

Endothelin inhibits renin release from isolated rat glomeruli

The effect of endothelin on renin release from isolated rat glomeruli was examined. Endothelin inhibited basal renin release in a dose-dependent manner with an IC50 of 1.0 x 10(-9) M. Endothelin also inhibited renin release stimulated by isoproterenol (10(-5) M). Nifedipine (10(-5) M), a calcium channel blocker, induced an increase in renin release. Endothelin did not affect this nifedipine-induced renin release. These results suggest that endothelin inhibits renin release via a calcium entry mechanism and increases intracellular calcium.     

Protective effect of a calcium antagonist against renal vasoconstriction induced by endothelin in the normotensive rat

We studied the effect of nifedipine, a dihydropyridine calcium antagonist, on the hemodynamic changes induced by endothelin, in awake normotensive rats. Endothelin (0.07-1.40 nmol/kg, e.v.) caused an initial hypotensive effect, followed by long lasting hypertension. Renal blood flow was reduced immediately and still remained below basal levels, at 30 minutes after endothelin injection. Nifedipine (1 mg/kg, i.p.) significantly prevented the effect of endothelin on mean blood pressure and induced a right-ward shift in the dose response curve of renal hemodynamic changes induced by endothelin. We conclude that treatment with calcium antagonist could be very useful in all those conditions in which systemic and regional vasocostriction is provoked by endothelin.     

Plasma concentrations of endothelin in patients with abnormal vascular reactivity. Effects of ergometric exercise and acute saline loading

We measured circulating concentrations of endothelin, a recently discovered vasoconstrictor peptide produced by vascular endothelial cells, in healthy subjects and in patients with abnormal vascular reactivity. Endothelin concentrations were determined by radio-immunoassay after extraction of plasma using Sep-Pak C-18 cartridges in healthy subjects (n = 20), in patients with diabetes mellitus type I (n = 10), in patients with mild to moderate essential hypertension (n = 12) and in non-dialyzed patients with stable chronic renal failure (n = 12). Plasma concentrations were similar in healthy controls, in diabetics and in hypertensive patients averaging 5.0 +/- 0.6 pg/ml, 4.7 +/- 0.2 pg/ml and 6.5 +/- 1.0 pg/ml, respectively. In contrast, plasma concentrations of endothelin were markedly elevated in patients with chronic renal failure averaging 16.6 +/- 2.9 pg/ml (p less than 0.005). No correlations were observed between serum creatinine concentrations ranging from 124 to 850 mumol/l or blood pressure and plasma concentrations of endothelin. Bicycle ergometric exercise in six healthy subjects and an acute modest i.v. saline load of 1,000 ml of 0.45% NaCl administered within 60 min in six patients with mild essential hypertension did not affect plasma concentrations of endothelin. Thus, it is unlikely that vascular synthesis of endothelin is related to acute physiological changes in systemic hemodynamics or to the circulatory and renal responses to acute extracellular fluid volume (ECFV) expansion. A potential role of endothelin, however, in the control of regional blood flow cannot be excluded. Elevated plasma concentrations of endothelin observed in patients with chronic renal failure require further investigations.     

Effect of atrial natriuretic peptide on arterial pressure and renal function in cirrhotic rats with ascites

Glomerular filtration rate, urine volume, sodium excretion and mean arterial pressure were measured in 10 rats with Cl4C induced cirrhosis presenting sodium retention and ascites, and in 10 control rats before and during the iv administration of the 28 aminoacid rat alpha-Atrial Natriuretic Peptide (alpha-ANP) (a bolus of 1 microgram followed by a constant infusion of 33 ng/min). alpha-ANP induced a similar increase in glomerular filtration rate and filtered sodium load in both groups of rats. In contrast, the increase in urine volume and sodium excretion produced by alpha-ANP was significantly lower in cirrhotic rats (from 13.8 +/- 1.9 to 37.9 +/- 9.1 microliters/min., and from 0.5 +/- 0.1 to 3.3 +/- 1.0 microEq/min) than in control animals (from 14.6 +/- 1.3 to 102.5 +/- 17.7 microliters/min., p less than 0.005; and from 1.0 +/- 0.3 to 14.1 +/- 3.2 microEq/min., p less than 0.001). The results indicate that in rats with experimental cirrhosis and ascites there are blunted diuretic and natriuretic responses to alpha-ANP, probably as a consequence of the exaggerated tubular sodium reabsorption present in these animals.     

Endothelin-induced sudden death and the possible involvement of platelet activating factor (PAF)

Endothelin (5 nmol/kg, i.v.) caused a transient hypotension followed by a lasting hypertension in rats. However, an abrupt fall in the blood pressure was observed in most rats 6 to 30 min after the injection of endothelin and sudden death followed with lethality noted over 60 min. An abnormal electrocardiogram (ECG) (ventricular arrhythmias) was observed in rats injected with endothelin. Endothelin (i.v.) also caused sudden death in mice. Pretreatment (5 or 60 min) with specific PAF antagonists, CV-6209 (0.1-3 mg/kg, i.v.) and WEB 2086 (30 mg/kg, p.o.), and a calcium channel blocker, diltiazem (60 mg/kg, p.o.) prevented death and attenuated the ECG changes induced by endothelin, but CV-6209 did not prevent the blood pressure changes induced by endothelin. CV-6209 (0.5-3 mg/kg, i.v.), WEB 2086, diltiazem and dexamethasone (5 mg/kg, i.v.) protected mice against the death induced by endothelin. On the other hand, aspirin (cyclooxygenase inhibitor, 100 mg/kg, p.o.) did not protect mice from the death. Thus, endothelin is a highly toxic peptide with cardiotoxic effects, and PAF may be involved in the pathogenesis of the sudden death.     

The second messenger system(s) mediating the secretion of atrial natriuretic peptide (ANP) from the isolated rat heart during rapid cardiac pacing

This study demonstrates that rapid cardiac pacing elevates Atrial Natriuretic Peptide (ANP) levels, independently from atrial stretch. The second messenger system mediating this response was examined. The phosphoinositide system, generally regarded to be important in mediating ANP release, was shown to play only a modulating role during rapid cardiac pacing. The main mediator would appear to be calcium, and a non-calmodulin dependent, calcium mediated system controlling ANP release during rapid cardiac pacing is suggested.     

Role of ET-1 receptor binding and [Ca(2+)](i) in contraction of coronary arteries from DOCA-salt hypertensive rats

Hypertension is associated with an increase in coronary artery disease, but little is known about the regulation of coronary vascular tone by endothelin-1 (ET-1) in hypertension. The present study evaluated the mechanisms mediating altered contraction to ET-1 in coronary small arteries from deoxycorticosterone acetate (DOCA)-salt hypertensive rats. DOCA-salt rats exhibited an increase in systolic blood pressure and plasma ET-1 levels compared with placebo rats. Contraction to ET-1 (1 x 10(-11) to 3 x 10(-8) M), measured in isolated coronary small arteries maintained at a constant intraluminal pressure of 40 mmHg, was largely reduced in vessels from DOCA-salt rats compared with placebo rats. To determine the role of endothelin receptor binding in the impaired contraction to ET-1, (125)I-labeled ET-1 receptor binding was measured in membranes isolated from coronary small arteries. Maximum binding (fmol/mg protein) and binding affinity were similar in coronary membranes from DOCA-salt rats compared with placebo rats. Changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) were measured in freshly dissociated coronary small artery smooth muscle cells loaded with fura 2. ET-1 (10(-9) M) produced a 30 +/- 9% increase in [Ca(2+)](i) in smooth muscle cells from placebo rats, but had no effect on cells from DOCA-salt rats (2 +/- 2%). In summary, the ET-1-induced coronary artery contraction and increase in [Ca(2+)](i) are impaired in DOCA-salt hypertensive rats, whereas endothelin receptor binding is not altered. These results suggest endothelin receptor uncoupling from signaling mechanisms and indicate that impaired [Ca(2+)](i) signaling contributes to the decrease in ET-1-induced contraction of coronary small arteries in DOCA-salt hypertensive rats.     

Lack of a direct regulatory effect of atrial natriuretic factor on prostaglandins and renin release by isolated rat glomeruli

We have tested the direct regulatory effect of synthetic Atrial Natriuretic Peptide (ANP, 8-33aa) on prostaglandins and renin release by isolated rat glomeruli. Variable incubation times and doses of ANP did not modify the rate of PGE₂, PGF_2a and TXB₂ production. Similar results were obtained for renin release. These data do not support a role for ANP in the regulation of prostaglandins and renin release by rat glomeruli.     

The effect of endothelin-1 on Na+/H+ metabolism in vascular smooth muscle cells]

The effects of endothelin on intracellular pH (pHi) were examined in cultured rat vascular smooth muscle cells (VSMC) using the fluorescent probe BCECF. Endothelin induced biphasic changes in pHi: initial decrease followed by a subsequent increase above the basal level due to activation of the Na+/H+ exchange. The elevation of pHi was slow and sustained, but depended on the dose of endothelin: IC50 was about 3 x 10(-8) M. Na+/H+ exchange inhibition by EIPA (10(-7) M) or by equimolar replacement of external Na+ by choline abolished the pHi increase by enhancing the first phase of cytoplasm acidification. Effects of endothelin were compared with the action of protein kinase C (PK-C) activator phorbol 12-13 myristate ester (PMA). PMA induced a monophasic slow and sustained increase in pHi. The treatments of VSMC with H-7 and staurosporine (PK-C) inhibitors prevented the pHi response to endothelin and PMA. These results suggest that protein kinase C may play an important role in mediating the effects of endothelin on Na+/H+ exchange in VSMC.     

Evidence for endothelin-1 release from resistance vessels of rats in response to hypoxia

To elucidate further the contribution of endothelin into endothelium-dependent vasoconstriction evoked by hypoxia, we observed endothelin release during hypoxia. Endothelin was detectable in perfusate from mesenteric artery. Immunoreactive endothelin was confirmed as endothelin-1 by a reverse phase-HPLC. Endothelin release increased 4.1 +/- 1.3 to 12.4 +/- 2.0 pg/30 min without changing perfusion pressure. Thirty minutes of hypoxia stimulated endothelin release by 71 +/- 11% (P less than 0.05) and was associated with an elevation of perfusion pressure. These results suggest that endothelin contributes to endothelium-dependent vasoconstriction by hypoxia in mesenteric artery and may play an important role in the local peripheral vascular tone.     

Endothelin secretion is regulated by cyclic AMP and phosphatase 2A in endothelial cells

Endothelin is a 21 amino acid peptide secreted by endothelial cells and is the most potent vasoconstrictor known. The present study examines regulatory mechanisms of endothelin secretion, focusing on the role of protein phosphorylation. Endothelin secretion was measured by radioimmunoassay in primary cultures of human umbilical vein endothelial cells. While treatment that raised cAMP levels reduced the basal endothelin secretion rate, agents that elevated cGMP had no effect. Downregulation or inhibition of protein kinase C resulted in decreased endothelin secretion, suggesting that protein kinase C regulates endothelin secretion in the opposite direction to cAMP dependent protein kinases Okadaic acid, at concentrations that selectively inhibit protein phosphatases 2A, reduced the endothelin secretion and the effects of okadaic acid and db-cAMP were additive. Endothelin production was stimulated by fetal calf serum and by the protein kinase inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H7), but was inhibited by the calmodulin antagonist trifluoperazine. The present findings that regulators of cAMP-dependent protein kinases, protein kinase C, calmodulin, and protein phosphatase 2A all affect endothelin secretion suggest that endothelin secretion is controlled by phosphorylation/dephosphorylation of as yet unidentified regulatory proteins within the cell. © 1994 Wiley-Liss, Inc.