Salt restriction reduces hyperfiltration, renal enlargement, and albuminuria in experimental diabetes.

The effects of dietary salt restriction on the renin-angiotensin system, glomerular filtration rate (GFR), renal size, and albuminuria were assessed in streptozotocin diabetic rats. Two series of experiments were performed: one short-term with severe salt restriction and the second long-term with moderate salt restriction. The first studied the effect of a very-low-salt diet for 4 weeks on GFR, renal size, and plasma angiotensin II concentration in diabetic and control rats. Diabetic and control male Sprague-Dawley rats received either a very-low-salt (0.005% NaCl) or a normal-salt (0.4% NaCl) diet. Diabetes was associated with a 49% increase in GFR, a 34% increase in kidney weight, and an 85% reduction in plasma angiotensin II when compared with control rats (P < 0.001). Sodium restriction in diabetic rats reduced GFR, restored plasma angiotensin II to control values, and retarded kidney growth when compared with diabetic rats receiving a normal sodium diet. GFR correlated negatively with plasma angiotensin II (r = -0.65, P < 0.001) and positively with kidney weight (r = 0.66, P < 0.001). In the second experiment, serial measurements of albuminuria and GFR were performed in control, diabetic, and salt-restricted (0.05% NaCl) control and diabetic rats over 24 weeks. Albuminuria showed a continuous rise in the diabetic rats when compared with control rats. Salt restriction attenuated the increase in albuminuria over the whole study period as well as reducing blood pressure and kidney weight in the diabetic rats. In conclusion, sodium restriction was associated with a lower GFR and kidney weight after 4 weeks and reduced levels of albuminuria, kidney weight, and blood pressure after 24 weeks in diabetic rats. Salt restriction may have an important role in the prevention and treatment of diabetic nephropathy.

Increased renal expression of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 in experimental diabetes.

It has been suggested that the cytokine vascular endothelial growth factor (VEGF) has an important role in the pathogenesis of diabetic retinopathy, but its role in nephropathy has not been clearly demonstrated. Assessment of VEGF, 125I-VEGF binding, and vascular endothelial growth factor receptor-2 (VEGFR-2) in the kidney was performed after 3 and 32 weeks of streptozotocin-induced diabetes. Gene expression of both VEGF and VEGFR-2 was assessed by Northern blot analysis and the localization of the ligand and receptor was examined by in situ hybridization. VEGF and VEGFR-2 protein were also evaluated by immunohistochemistry. Binding of the radioligand 125I-VEGF was evaluated by in vitro and in vivo autoradiography. Diabetes was associated with increased renal VEGF gene expression. VEGF mRNA and protein were localized to the visceral epithelial cells of the glomerulus and to distal tubules and collecting ducts in both diabetic and nondiabetic rats. Renal VEGFR-2 mRNA was increased after 3 weeks of diabetes but not in long-term diabetes. In situ hybridization and immunohistochemical studies revealed that glomerular endothelial cells were the major site of VEGFR-2 expression. In addition, VEGFR-2 gene expression was detected in cortical and renomedullary interstitial cells and on endothelial cells of peritubular capillaries. There was an increase in 125I-VEGF binding sites after 3 but not 32 weeks of diabetes. The major VEGF binding sites were in the glomeruli. 125I-VEGF binding was also observed in medullary rays and in the renal papillae. These studies indicate an early and persistent increase in renal VEGF gene expression in association with experimental diabetes. In addition, an early and transient increase in renal VEGF receptors was also observed in diabetic rats. These findings are consistent with a role for VEGF in mediating some of the changes observed in the diabetic kidney.

Evidence for atrial natriuretic peptide-(5-28) production by rat placental cytotrophoblasts.

Atrial natriuretic peptide (ANP), a 28-amino acid peptide, is produced and secreted by cardiac atriocytes to modulate cardiovascular and renal functions. We report here the production of ANP-(5-28) and its 15K mol wt (M(r)) presumptive precursors by cytotrophoblasts of rat placentae. Placental tissues were collected from Sprague-Dawley fetal rats on days 12, 16, 18, and 20 of gestation and acid extracted for immunoreactive (ir) ANP assay. The contents of placental irANP increased over the course of fetal growth, with the highest amount (mean +/- SE, 1083 +/- 125 pg/tissue; n = 7) found near term. Sephadex G-50 gel chromatographic profiles of the placental extract revealed a major peak of irANP coeluted with the 3K M(r) of synthetic rat (r) ANP-(1-28) and a minor peak in the position consistent with that of 15K M(r). HPLC analysis of the 3K M(r) species showed a single peak of immunoreactivity, which eluted with a retention time similar to that of rANP-(5-28). In placental sections, irANP and pro-ANP mRNA were localized by immunoperoxidase staining and colorimetric in situ hybridization in a subpopulation of placental cytotrophoblasts, but not in syncytiotrophoblasts or chorionic cells. Northern blot analysis showed a single band of pro-ANP mRNA in rat placental tissues similar in size to that found in the heart (approximately 0.85 kilobases), with the highest level of pro-ANP mRNA signal detected in the placentae of 16-day gestation fetuses. Our findings suggest that ANP is expressed and produced by a small population of rat placental cytotrophoblasts and that the 15K M(r) precursor peptide is extensively processed into the N-terminal-truncated form of ANP-(5-28) in the tissue.

Identification, distribution, and expression of angiotensin II receptors in the normal human prostate and benign prostatic hyperplasia.

The tissue distribution, cellular localization, and level of expression of angiotensin II (Ang II) receptors were examined in the normal human prostate and benign prostatic hyperplasia (BPH) by in vitro autoradiography, immunohistochemistry, and radioligand binding studies. In the normal human prostate, Ang II receptors were of the AT(1) subtype and localized predominantly to periurethral stromal smooth muscle. The AT(1) receptor antagonist losartan totally displaced specific [(125)I]-[Sar(1),Ile(8)]Ang II binding, in a concentration-dependent manner, whereas the AT(2) receptor antagonist PD123319 was without effect. There was no significant difference in receptor affinity, but AT(1) receptor density was markedly reduced in BPH compared with that in normal prostate. In rat prostate, Ang II (0.01-1 microM) produced a concentration-dependent increase in [(3)H]-noradrenaline release from sympathetic nerves. The findings of the present study suggest that angiotensin AT(1) receptors predominate in the human prostate. The high concentration of AT(1) receptors in the periurethral region suggests a role for Ang II in modulating cell growth, smooth muscle tone, and possibly micturition. Furthermore, down-regulation of AT(1) receptors in BPH may be due to receptor hyperstimulation by increased local levels of Ang II in BPH. Finally, Ang II may play a functional role in modulating sympathetic transmission in the prostate. These data support the novel concept that activation of the renin-angiotensin system may be involved in the pathophysiology of BPH.

Effect of dietary salt on hemodynamics of established renal hypertension in the rabbit. Implications for the autoregulation theory of hypertension.

Two groups of 10 rabbits were subjected to renal cellophane wrapping and sham operation. Their initial mean arterial pressures (MAP) were similar, 92 +/- 1.5 and 90 +/- 2.9 mm Hg. Six weeks later three experimental periods began, each of 2 weeks' duration, on low, normal, and high salt (1, 9, and 50 mmole Na/100 g food) diets. Each group had two subgroups: rabbits with both kidneys, and rabbits with only one kidney and previous nephrectomy. The hemodynamic findings were similar in each group. After sham operation, the range of dietary salt produced no significant circulatory changes. After wrapping, MAP was reduced on low compared with normal and high salt diets (122 vs 132 and 136 mm Hg; p = 0.01). This was entirely due to lowering of cardiac output (CO) on low salt; on normal and high salt CO was higher than in sham-operated rabbits. Total peripheral resistance (TPR) in the wrapped animals was unaffected by diet, i.e., 21.4, 20.5, and 21.2 units on low, normal, and high salt--about 35% above values of sham-operated rabbits. Volume-related CO changes therefore produce long-term changes in MAP without alteration in TPR, which is not in conformity with the autoregulation theory of hypertension. Evidence of impaired capacity of wrapped compared with sham-operated rabbits to handle salt included diet-related hematocrit changes, lower creatinine clearance, and some differences in renin responses to salt. Giving saralasin reduced TPR while the rabbits were on low salt; the fall was twice as great in wrapped compared with sham-operated rabbits.

In vivo occupancy of angiotensin II subtype 1 receptors in rat renal medullary interstitial cells.

Angiotensin II receptor binding sites in type 1 interstitial cells in the inner stripe of the outer medulla are readily labeled in vitro by the radioligand but not in vivo after systemic radioligand administration. In anesthetized rats, we investigated if reduced vascular delivery due to angiotensin II-induced renal vasoconstriction or, alternatively, prior occupancy of these sites by endogenous angiotensins modulates angiotensin II subtype 1 receptor binding to renal medullary interstitial cells in vivo using electron microscopic autoradiography. Using 125I-angiotensin II, administered systemically, as a radioligand, binding in control rats occurred predominantly in the glomeruli and proximal tubules, while only low binding was observed in the inner stripe of the outer medulla. Pretreatment of rats with unlabeled [Sar1,Ile8]angiotensin II or with the angiotensin II subtype 1 receptor antagonist losartan before receiving the radioligand completely abolished binding to all sites. Renal vasodilatation induced by sodium nitroprusside or use of the radiolabeled antagonist analogue 125I-[Sar1,Ile8]angiotensin II did not alter binding to the inner stripe. In contrast, chronic salt loading or inhibition of angiotensin-converting enzyme by perindopril significantly increased binding not only to the cortical sites but also to the sites in the inner stripe of the outer medulla. Electron microscopic autoradiographs of the inner stripe detected binding in the interstitial cells only in rats treated with chronic salt loading or perindopril. These results suggest that endogenous angiotensins may modulate binding of circulating angiotensin II to the interstitial cells in vivo, and these angiotensin II receptor-bearing cells are more likely to be more responsive to interstitial angiotensin II than to the circulating hormone.

Role of angiotensin receptor subtypes in mesenteric vascular proliferation and hypertrophy.

The aim of this study was to explore the regulation of angiotensin receptors after chronic infusion with angiotensin II (Ang II) and to clarify the relative roles of the angiotensin type 1 (AT(1)) and type 2 (AT(2)) receptors in the mediation of Ang II-induced mesenteric vascular hypertrophy. In male Sprague-Dawley rats, Ang II infusion at a dose of 58.3 ng/min by subcutaneous osmotic minipumps for 14 days led to increased mesenteric weight and wall:lumen ratio of the vessels and proliferation of smooth muscle cells. These vascular changes were attenuated by either valsartan, an AT(1) receptor antagonist, at a dose of 30 mg. kg(-1). d(-1) by gavage, or PD123319, an AT(2) receptor antagonist, at a dose of 830 ng/min by intraperitoneally implanted osmotic minipumps. Ang II infusion was associated with hypertension, which was prevented by valsartan, but not PD123319. (125)I-Sar(1), Ile(8) Ang II binding to mesenteric vasculature was increased after Ang II infusion. Valsartan treatment was associated with reduced Ang II binding to both receptor subtypes, whereas PD123319 was associated with reduced Ang II binding to only the AT(2) receptor subtype. These findings suggest that the trophic and proliferative effects of Ang II on the mesenteric vasculature are mediated by both AT(1) and AT(2) receptors.

Atrial natriuretic peptide and plasma renin levels in assessment of mineralocorticoid replacement in Addison's disease.

Assessment of mineralocorticoid replacement therapy in Addison's disease relies on clinical features and laboratory measurements, including plasma renin and potassium. Previous studies have questioned the value of measuring the plasma renin concentration (PRC), particularly in the setting of fludrocortisone overreplacement. The aim of this study was to evaluate the usefulness of plasma atrial natriuretic peptide (ANP) measurements as a marker of sodium and volume status in Addison's disease. Fourteen patients with Addison's disease receiving their usual glucocorticoid doses were placed on various doses of fludrocortisone (FC; 0 mg, 0.05 mg, 0.1 mg and 0.2 mg) in random order for four 2-week periods. At the end of each period, blood pressure and clinical symptoms were assessed, and blood was drawn for measurement of PRC and ANP levels. PRC was significantly elevated in patients receiving placebo (54.2 +/- 57.9 ng/mL x h) compared with PRC in those receiving baseline FC (24.7 +/- 42.4 ng/mL x h), 0.1 mg FC (15.2 +/- 25.9 ng/mL x h), and 0.2 mg FC (5.5 +/- 5.7 ng/mL x h). ANP levels were measured by either an extraction method (ANP(ext)) or directly from plasma (ANP(dir)). ANP(dir) was significantly elevated at 0.2 mg FC (87.1 +/- 20.1 pg/mL) compared with baseline (63.3 +/- 8.1 pg/mL), placebo (56.1 +/- 5.5 pg/mL), 0.05 mg FC (60.5 +/- 16.0 pg/mL), and 0.1 mg FC (65.4 +/- 13.7 pg/mL) values. ANP(ext) was elevated in patients receiving 0.2 mg FC (42.7 +/- 41.8 pg/mL) compared with that in patients receiving placebo (7.9 +/- 5.4 pg/mL), 0.05 mg FC (16.2 +/- 11.2 pg/mL), or 0.1 mg FC (19.7 +/- 11.1 pg/mL). Our data suggest that PRC is of value in determining mineralocorticoid underreplacement, whereas ANP is a more sensitive index of FC overreplacement. ANP levels may, therefore, be complementary to PRC in adjustment of mineralocorticoid doses in the upper dose range, where clinical symptoms and signs appear to be of little value.

Angiotensin II, sodium, and cardiovascular hypertrophy in spontaneously hypertensive rats.

Angiotensin II (Ang II) may cause cardiovascular hypertrophy as a consequence of increased blood pressure or possibly by direct trophic actions. To dissociate Ang II and blood pressure in young spontaneously hypertensive rats (SHR), we used sodium loading during angiotensin converting enzyme inhibitor treatment. Animals were treated between 6 and 10 weeks of age with perindopril to lower Ang II and blood pressure, or with perindopril and 1% saline drinking fluid or perindopril and aldosterone infusion to lower Ang II but maintain high blood pressure. Blood pressure, heart weight, and media/lumen ratio of mesenteric resistance arteries were studied while rats were on treatment at 10 weeks of age and 15 weeks after treatment at 25 weeks of age. Perindopril lowered blood pressure and inhibited the development of cardiovascular hypertrophy. Saline or aldosterone restored high blood pressure during perindopril treatment and resulted in increased heart weight/body weight and resistance artery media/lumen ratios in direct proportion to the elevation of blood pressure. Because increased structure occurred despite perindopril treatment, we conclude that direct trophic actions of Ang II are not essential for the development of cardiovascular hypertrophy in young SHR and that the antitrophic actions of angiotensin converting enzyme inhibitors depend more on changes in blood pressure than on Ang II. However, restoration of blood pressure and structure by sodium during perindopril treatment raises the possibility that the design of the cardiovascular system and blood pressure may depend indirectly on Ang II through effects on sodium metabolism.

Atrial natriuretic factor receptors and stimulation of cyclic GMP formation in normal and malignant osteoblasts.

Synthetic rat atrial natriuretic factor (Ile-ANF-26) stimulated cyclic GMP formation by up to several hundred-fold in osteoblast-rich cultures from newborn rat calvaria and in clonal osteogenic sarcoma cells (UMR 106-01) which are phenotypically osteoblast. ANF had no effect on the cyclic AMP response to parathyroid hormone in the same cells. Specific, high-affinity binding sites for ANF were identified in both cell types, with Kd and receptor numbers in normal osteoblasts of 1.2 +/- 0.1 X 10(-10) M and 42 +/- 4 X 10(3) per cell, and in UMR 106-01 cells of 1.4 +/- 0.1 X 10(-10) M and 22 +/- 4 X 10(3) per cell.

Interaction between atrial natriuretic peptide and the renin angiotensin aldosterone system. Endogenous antagonists.

The biologic actions of the cardiac peptide hormone atrial natriuretic peptide (ANP) of vasorelaxation, diuresis and natriuresis, suppression of aldosterone, vasopressin release, and thirst are the opposite of those of the renin angiotensin system. This close relationship is further strengthened by the complementary localization of their receptors in the brain, adrenal gland, vasculature, and kidney. In many physiologic situations including postural changes, volume expansion, water immersion, high altitude, and lower body negative pressure, the plasma levels of ANP and angiotensin II change inversely. In congestive heart failure, renin and aldosterone levels may initially be suppressed by high levels of ANP. Similarly the low renin levels associated with increasing age and with elderly hypertensive patients, may be the result of the elevation of plasma ANP that occurs with aging. ANP may thus be the endogenous antagonist of the renin angiotensin aldosterone system. These two opposing systems allow fine-tuning of volume and pressure by the body.

Hypotensive effect of ZD7155, an angiotensin II receptor antagonist, parallels receptor occupancy in two-kidney, one-clip Goldblatt hypertensive rats.

OBJECTIVE: To examine the effect of ZD7155, an angiotensin II receptor antagonist, on blood pressure, heart rate and occupancy of tissue angiotensin II receptor in two-kidney, one-clip Goldblatt hypertensive rats. METHODS: Goldblatt hypertension was produced in Sprague-Dawley rats. ZD7155 was administered orally at 3 mg/kg and blood pressure and heart rate were monitored for up to 48 h. In a second series of experiments, the rats were administered increasing doses of ZD7155 and monitored for 1 h. For each time and dose, rats were killed and their blood and tissues were collected. Tissue angiotensin II receptor binding was assessed by quantitative autoradiography in vitro. For a separate group of rats, the pressor response to 0.1 microg/kg angiotensin II was monitored before and after the administration of 3 mg/kg ZD7155. RESULTS: After oral administration of ZD7155, blood pressure was rapidly lowered and this lowering was sustained for up to 48 h. This effect was accompanied by sustained inhibition of angiotensin II receptor binding in the aorta, kidney and adrenal gland, together with an increase in plasma renin activity. Increasing doses of ZD7155 dose-dependently reduced blood pressure and inhibited angiotensin II receptor binding in the tissues. Degrees of inhibition varied among the different tissues and had different time courses. ZD7155 inhibited the pressor response to angiotensin II remarkably for 24 h. CONCLUSIONS: ZD7155 is a potent antihypertensive agent in two-kidney, one-clip Goldblatt hypertensive rats and this effect is accompanied by sustained inhibition of tissue angiotensin II binding.

The role of angiotensin II in the development of hypertension and in the maintenance of glomerular filtration rate during 48 hours of renal artery stenosis in conscious dogs.

The responses to 48 h of renal artery stenosis were compared in uninephrectomized, chronically-instrumented dogs with or without inhibition of angiotensin II (AII) formation by enalapril. Mean arterial pressure rose by an average of 29.9 mmHg (s.e.m. 3.5) in untreated dogs and by 14.5 mmHg (s.e.m. 2.8) in enalapril-treated dogs over the two days of stenosis. Renal artery stenosis reduced glomerular filtration rate (GFR) by 49% (s.e.m. 9) in untreated dogs and by 86% (s.e.m. 8) in enalapril-treated dogs. Compared to untreated dogs, enalapril-treated dogs also had lower renal artery pressure distal to the stenosis, drank less water and had larger rises in plasma K+ following renal artery stenosis. There were no differences in renal blood flow or urinary Na+ excretion in the two groups of dogs. Thus blockade of AII production did not prevent hypertension occurring in response to renal artery stenosis, but the rise in blood pressure was only about half that which occurred in normal dogs and GFR was much more severely reduced.

Vascular expression of angiotensin type 2 receptor in the adult rat: influence of angiotensin II infusion.

OBJECTIVE: The aim of this study was to investigate the relative role of the angiotensin type 1 (AT1) and type 2 (AT2) receptors in mediating angiotensin II-induced regulation of AT2 receptor in mesenteric artery. DESIGN: Sprague-Dawley rats were infused with either angiotensin II or vehicle for 14 days at a dose of 58.3 ng/min. Ang II-infused rats were allocated to receive either an AT1 antagonist, valsartan at a dose of 30 mg/kg per day or the AT2 receptor antagonist PD123319 at a dose of 830 ng/min. METHODS: Gene and protein expression of the AT2 receptor in the mesenteric vasculature was assessed by quantitative reverse transcriptase polymerase chain reaction, immunohistochemistry and by in vitro autoradiography with a specific radioligand, 1251-CGP 42112B. RESULTS: The AT2 receptor mRNA and protein were detected in the mesenteric artery from adult rats. Both nuclear emulsion and immunohistochemical staining showed expression of the AT2 receptor in the adventitial and medial layers. Compared to control rats, angiotensin II infusion was associated with a significant increase in the AT2 receptor expression. Valsartan treatment significantly reduced AT2 receptor gene expression, with no significant effect of PD123319 on this parameter. CONCLUSIONS: This study confirms that the presence of the AT2 receptor in mesenteric arteries in adult rats, shows an up-regulation of the AT2 receptor following angiotensin II infusion and suggests a role for the AT1 receptor in this regulation. In view of the recently demonstrated effects of the AT2 receptor, these findings may be relevant to the role of the AT2 receptor in the pathophysiology of vascular remodeling.

In-vitro and in-vivo inhibition of rat neutral endopeptidase and angiotensin converting enzyme with the vasopeptidase inhibitor gemopatrilat.

OBJECTIVES: Vasopeptidase inhibitors are single molecules that simultaneously inhibit neutral endopeptidase (NEP) and angiotensin converting enzyme (ACE). The aim of this study was to characterize in-vitro and in-vivo inhibition of NEP and ACE in the rat with the vasopeptidase inhibitor gemopatrilat. DESIGN AND METHODS: In-vitro NEP and ACE inhibition was studied by radioinhibitory binding assay using rat renal membranes and the specific NEP inhibitor radioligand 125I-RB104 and the specific ACE inhibitor radioligand 125I-MK351A, respectively (n = 3 per curve). In-vivo NEP and ACE inhibition was studied using in-vitro autoradiography in rats that received oral gemopatrilat (1, 3, 10 mg/kg; n = 4 per dose) and were killed 1 h later, or received oral gemopatrilat (3, 10 mg/kg) and were killed at time points 1, 2, 4, 8, 18, 24 and 48 h (n = 4 per time point). RESULTS: Gemopatrilat caused a concentration-dependent displacement of specific radioligands from renal membrane NEP (IC50 305 +/- 5.4 nmol/I) and ACE (IC50 3.6 +/- 0.02 nmol/). In the dose-response study gemopatrilat (1, 3 and 10 mg/kg) caused significant inhibition of plasma ACE (P< 0.01), and renal ACE and NEP (3, 10 mg/kg, P < 0.01). In the time course experiment, gemopatrilat (10 mg/kg) increased plasma renin activity for 8 h (P< 0.01) and inhibited plasma ACE (P< 0.05), renal NEP (P< 0.01) and renal ACE (P< 0.05) for 48 h. CONCLUSIONS: Gemopatrilat is a potent in-vitro vasopeptidase inhibitor that also causes prolonged inhibition of circulating and renal ACE and renal NEP after a single oral dose. The data suggest that gemopatrilat may be a useful addition to existing vasopeptidase inhibitors in the treatment of cardiovascular disease.

Reduction of endothelin levels by the dihydropyridine calcium antagonist nisoldipine and a 'natural factor' in cultured human endothelial cells.

OBJECTIVE: Endothelin is thought to be related to cardiovascular disease. The purpose of this study was to determine whether endothelin levels could be reduced by a calcium antagonist and a 'natural factor'. DESIGN: Since calcium ionophores can induce endothelin-1 messenger RNA synthesis in cultured endothelial cells, the calcium antagonist nisoldipine was used in this study to determine whether it could reduce endothelin levels. It has been reported that coculture of endothelial cells and smooth muscle cells from different species and different parts of the body can reduce endothelin levels. This study was also designed to determine whether coculture of the two cell types from the same species and the same section of an artery could reduce endothelin levels. METHODS: Cultured endothelial cells from human umbilical artery (HUAEC) and umbilical vein (HUVEC) were treated with increasing concentrations of nisoldipine. HUAEC were cocultured with human umbilical artery smooth muscle cells (HUASMC). Endothelin levels were measured by a radioimmunoassay. RESULTS: Incubation of the HUAEC with nisoldipine for either 7 or 24 h resulted in a dose-dependent (10(-8)-10(-5) mol/l) reduction in endothelin levels in the conditioned media. Endothelin levels in cell lysates were not detectable in either the absence or the presence of nisoldipine. This suggests that the reduction of endothelin levels in the media could be due to inhibition of endothelin synthesis. Under the same conditions, incubation of HUVEC with the same concentrations of nisoldipine produced a similar concentration-dependent reduction in endothelin levels. Endothelin levels were undetectable in the conditioned media from HUASMC. Coculture of HUAEC with HUASMC significantly reduced endothelin levels (P < 0.01) compared with HUAEC cultured alone. CONCLUSIONS: Endothelin levels can be reduced by the calcium antagonist nisoldipine and a 'natural factor' associated with smooth muscle cells.

Localization and characterization of endothelin receptor binding sites in the rat brain visualized by in vitro autoradiography.

Endothelin binding sites in rat brain were mapped by quantitative in vitro autoradiography employing [125I]endothelin-1 as radioligand. [125I]Endothelin-1 bound with high affinity and specificity to rat cerebellar sections and was potently displaced by unlabelled endothelins (endothelin-1 greater than endothelin-2 = endothelin-3) and sarafotoxin 6B. The highest densities of endothelin binding sites were found in the cerebellum (especially Purkinje cell layer), choroid plexus and median eminence. High densities were found in the supraoptic and paraventricular hypothalamic nuclei, anterior hypothalamic area, ventromedial hypothalamic nucleus, mammillary nuclei and glomerular layer of olfactory bulb. Moderate densities were found in many thalamic nuclei, the pretectal region, interpeduncular nucleus, suprachiasmatic nucleus, raphe nuclei, tegmental nuclei, olfactory ventricle, red nucleus, subthalamic nucleus, central gray, reticular nuclei, vestibular nuclei, oculomotor and trochlear nuclei, hypoglossal nucleus, motor trigeminal nucleus, nucleus of the trapezoid body and lateral cerebellar nucleus. Low but detectable densities of endothelin binding sites were found in medial geniculate nucleus, fields of Ammon's horn, caudate-putamen, globus pallidus, entopeduncular nucleus, substantia nigra, anterior commissure, internal capsule, anterior pituitary, median preoptic nucleus, septohypothalamic nucleus, superior colliculus and area postrema. These patterns were completely abolished by 1 microM unlabelled endothelin-1, -2 and -3 and sarafotoxin S6B. Brain endothelin binding sites show high affinity for endothelin-1, -2 and -3 and sarafotoxin 6B with highest affinity for endothelin-1. Endothelin binding sites show a non-vascular pattern of distribution in the brain, suggesting that the peptide may have widespread functions as a modulator of neuronal function.

Streptozotocin-induced diabetes reduces the density of [125I]-endothelin-binding sites in rat cardiac membranes.

The effect of acute, streptozotocin-induced diabetes on the affinity (KD), density (Bmax) and selectivity of specific, high affinity binding sites for [125I]-endothelin [( 125I]-ET) in rat cardiac membrane fragments was determined. Three days after a single i.v. bolus dose of streptozotocin (60 mg kg-1), the density of [125I]ET binding sites was reduced (P less than 0.01) without changes in affinity or selectivity.

Atrial natriuretic peptide is involved in the ACTH response to stress and glucocorticoid negative feedback in the rat.

The role of atrial natriuretic peptide (ANP) in the ACTH response to stress and the glucocorticoid negative feedback control of ACTH release was investigated in adult male homozygous Brattleboro and adrenalectomized Wistar rats respectively, using the technique of immunoneutralization. The relatively low ACTH response to stress and the lack of arginine vasopressin make the homozygous Brattleboro rat a more rigorous and simpler preparation in which to test the hypothesis that ANP is involved in the ACTH response to stress. In both sets of experiments, blood sampling and injection of sheep anti-ANP or control serum were carried out in conscious animals through intra-atrial cannulae implanted 2 days previously under halothane anaesthesia. A 30-s exposure to ether resulted in a brisk twofold increase in the plasma ACTH concentrations in homozygous Brattleboro rats infused with anti-ANP, but not control serum. The injection of either dexamethasone, a potent glucocorticoid receptor agonist, or corticosterone resulted in a rapid and marked reduction in the plasma concentrations of ACTH in Wistar rats which had been adrenalectomized, under halothane anaesthesia, at least 21 days before experimentation. The inhibitory action of dexamethasone, but not corticosterone, was significantly reduced in animals infused with anti-ANP serum. These results show that the inhibition of ANP release into hypophysial portal blood is probably important for triggering the ACTH response to stress and that ANP may play a role in corticosteroid negative feedback control of ACTH release mediated by type II (glucocorticoid) receptors.

Atrial natriuretic peptide is a physiological inhibitor of ACTH release: evidence from immunoneutralization in vivo.

The brain is thought to exert a predominantly stimulatory action on ACTH secretion mediated mainly by corticotrophin-releasing factor-41 (CRF-41) and arginine vasopressin (AVP). Several data, however, also point to the existence of an ACTH-inhibiting factor. Atrial natriuretic peptide (ANP), at concentrations found in hypophysial portal blood, inhibits ACTH release in vitro. The aim of the present studies was to use ANP immunoneutralization to determine whether ANP does in fact inhibit ACTH release in vivo. Intracerebroventricular infusion (1 microliters/min for 30 min) of sheep anti-ANP serum into male rats anaesthetized with sodium pentobarbitone had no significant effect on jugular venous plasma concentrations of ACTH or LH but did decrease significantly the plasma concentrations of prolactin. Intravenous infusion of 0.8 ml sheep anti-ANP serum but not control (non-immune) sheep serum, through an indwelling intra-atrial cannula in conscious male rats resulted in a marked and significant increase in plasma ACTH and corticosterone concentrations. The ACTH and corticosterone response to a 30-s ether stress was not significantly potentiated in the same conscious rats infused with anti-ANP serum. Intra-atrial infusion of anti-ANP did not significantly affect plasma prolactin, LH, glucose or sodium concentrations or plasma osmolality. These results show for the first time that ANP is a potent inhibitor of ACTH secretion in the conscious male rat and that, therefore, ANP is a hypothalamic neurohormone which is likely to play an important inhibitory role in the neural control of ACTH release.