Vasoconstrictor action of angiotensin I-convertase and the synthetic substrate (Pro11,D-Ala12)-angiotensin I.

A chymase (also referred to as angiotensin I-convertase) specific for the conversion of angiotensin (Ang) I to Ang II has been identified in human heart. This serine protease is also present in dog and marmoset vasculature. We examined the vasoconstrictor effects of Ang II putatively generated from an angiotensin-converting enzyme (ACE)-resistant convertase synthetic substrate (SUB) in vivo and in vitro. In marmosets, SUB (7 to 700 micrograms/kg i.v.) or Ang I (0.1 to 30 micrograms/kg) caused similar dose-dependent increases in mean arterial pressure (10 to 100 mm Hg) and decreases in heart rate. Pressor effects of SUB were slightly attenuated at low (but not high) doses by captopril (CAP, 1 mg/kg i.v.) and blocked by losartan (5 mg/kg i.v.); in contrast Ang I pressor effects were substantially blocked by both. In isolated canine superior mesenteric artery, Ang I-induced contraction was eliminated by losartan and reduced but not eliminated by 10 mumol/L CAP. When combined with the serine protease inhibitor chymostatin, CAP eliminated Ang I-induced contraction, but chymostatin alone had no effect. SUB-induced contraction was not blocked by CAP but was equally blocked by chymostatin (25 mumol/L) alone or by the combination of CAP (10 mumol/L) and chymostatin (25 mumol/L); losartan (10 mumol/L) eliminated SUB-induced responses. Previous studies have suggested that Ang I-convertase is important for production of Ang II in the heart. Our results are consistent with a potential role for Ang I-convertase in the production of Ang II in the vasculature, resulting in Ang II-mediated vasoconstriction.

Synergistic effect on reduction in blood pressure with coadministration of the renin inhibitor, CP-80,794, and the angiotensin converting enzyme inhibitor, captopril.

The effects of coadministration of a renin inhibitor, CP-80,794, and an angiotensin converting enzyme inhibitor, captopril, on blood pressure of sodium-depleted guinea pigs was studied. Dose-response curves for CP-80,794 (0.3-3.0 mg/kg i.v.) and captopril (0.03-1.0 mg/kg i.v.) were obtained either alone or in the presence of a submaximal dose of the other inhibitor. The hypotensive response calculated for each compound individually was subtracted from the combined dose response. The results showed that statistically significant synergy with captopril and CP-80,794 occurred when the area rather than the peak drop or duration of change in blood pressure was measured. The degree of the synergy indicated that to achieve the same reduction in blood pressure, the dose of each drug, below the high end of its response range, could be decreased approximately sixfold when administered in combination. It was determined that the plasma pharmacokinetics of CP-80,794 were not altered during coadministration, as plasma concentrations of CP-80,794 were similar in the presence and absence of 0.1 mg/kg i.v. of captopril. These results indicate that by inhibiting sequential enzymes in the renin-angiotensin system, synergistic effects can be produced. The relative safety of each inhibitor could be improved by large reductions in dose when used concurrently.

Regulation of plasma atrial natriuretic peptide by the central nervous system.

The anterior ventral region of the third ventricle (AV3V) is a major central site in the regulation of cardiovascular and renal function. To examine the role of the central nervous system in the regulation of atrial natriuretic peptide (ANP) secretion, the effect of lesions in this region on basal, volume-stimulated, and osmotic-stimulated plasma ANP was determined. Basal levels of plasma ANP were not different in sham- and AV3V-lesioned rats. Volume expansion with a continuous infusion of saline or with a bolus administration of saline increased plasma ANP in sham-lesioned rats. In AV3V-lesioned rats, continuous infusion of saline had no effect on plasma ANP, and a bolus administration of saline decreased plasma ANP. Osmotic stimulation with hypertonic saline increased plasma ANP in sham-lesioned rats but had no effect on plasma ANP in AV3V-lesioned rats. These results suggest that the central nervous system is involved in the regulation of ANP secretion and that altered ANP regulation may contribute to the cardiovascular and renal deficiencies in AV3V-lesioned rats.

Erythrocyte sodium concentration and 86Rb uptake in weanling Dahl rats.

Alterations in Na, K ATPase pump activity as well as erythrocyte (RBC) intracellular sodium concentration (Nai) have been demonstrated in humans and rats with established hypertension. The contribution of hypertension itself to these changes is unclear. Accordingly, we investigated RBC ion transport and plasma ouabain-like factor (OLF) in four- to five-week old normotensive Dahl salt-sensitive (DS) and salt-resistant (DR) rats on low salt diet. Although both strains were normotensive, systolic blood pressure (SBP) of DS (123 +/- 2 mm Hg) was higher than that of DR (116 +/- 1 mm Hg). No interstrain difference was evident in RBC pump activity measured as ouabain-sensitive 86rubidium (86Rb) uptake (DS = 0.277 +/- .030 and DR = 0.271 +/- .029 mumol/10(9)RBC/h) even though RBC Nai was greater in DS than DR (14.9 +/- 2.0 v 10.7 +/- 1.0 mEq/L; P less than 0.05). Plasma OLF was higher in DS than DR (28.9 +/- 4.7 v 16.5 +/- 2.3 pmol/mL; P less than 0.05), but did not correlate with RBC pump activity in either strain. RBC Nai was directly correlated with pump activity in DS (r = 0.84, P less than 0.01) and demonstrated a trend to correlate in DR (r = 0.71, P = 0.07). RBC Nai was also directly correlated with SBP in DR (r = 0.73, P less than 0.05) and DS (r = 0.70, P = 0.05). We conclude that RBC Nai is genetically determined in Dahl rats and is elevated in normotensive DS who are at risk for hypertension development.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effect of dietary salt on renal growth in Dahl salt-sensitive and salt-resistant rats.

A high salt diet has been shown to increase renal mass of intact rats, although the mechanism by which this occurs has not been investigated. We used Dahl rats that are sensitive (DS) or resistant (DR) to the hypertensinogenic effect of salt to examine changes in renal size and composition caused by a high salt diet. Renal index, deoxyribonucleic acid (DNA), protein, water content, protein/DNA ratio, and cell number and size were measured in age-matched DR and DS on a high salt diet for 7, 14, or 28 days. The results were compared with those obtained from respective rats on a low salt diet. High salt diet elevated renal index and protein in DR and DS rats at each time point. After 7 days of a high salt diet, DNA increased in DS only. Protein/DNA ratio was progressively decreased by a high salt diet in DS and remained unchanged in DR rats. Cell number was increased 35% in DS versus only 13% in DR rats at 4 weeks. Cell size decreased 24% in DS and only 11% in DR rats. These results indicate that renal growth due to hyperplasia accompanies ingestion of a high salt diet in both DR and DS rats, but the rate of growth and the mechanism through which it occurs differ between strains. This difference may be important in delineating salt sensitivity and future development of hypertension.

Adrenal and vascular tyrosine hydroxylase activity in Goldblatt hypertension.

To examine the role of the sympathetic nervous system in hypertension, the in vitro activity of tyrosine hydroxylase was examined in one-kidney, one clip (1K1C) and two-kidney, one clip (2K1C) hypertensive rabbits and their respective controls 2 weeks after surgical procedures. The in vitro activity of tyrosine hydroxylase provides a measure of catecholamine synthesis and serves as a biochemical index of activity of noradrenergic neurons and the adrenal medulla. Mean atrial pressure rose from 91.5 +/- 1.0 to 128.5 +/- 5.6 mm Hg (p less than 0.01) in the 1K1C group and from 91.8 +/- 1.3 to 106.5 +/- 5.0 mm Hg (p less than 0.02) in the 2K1C group, whereas no change in blood pressure was found in their respective controls. Adrenal tyrosine hydroxylase activity was increased 85% in the 1K1C group, as compared with values in one-kidney controls (from 11.8 +/- 1.5 to 21.8 +/- 1.1 pmol CO2/min/mg; p less than 0.0002), and was increased 49% in the 2K1C group, as compared with values in two-kidney controls (from 8.01 +/- 1.2 to 11.9 +/- 1.1 pmol CO2/min/mg; p less than 0.02). In the 1K1C group, proximal mesenteric tyrosine hydroxylase activity was decreased 46% compared with values in one-kidney controls (from 23.5 +/- 5.0 to 12.8 +/- 2.5 pmol CO2/min/mg; p less than 0.03) and distal mesenteric tyrosine hydroxylase activity was decreased 42% (from 7.73 +/- 1.2 to 4.46 +/- 0.8 pmol CO2/min/mg; p less than 0.03). In the 2K1C group, neither proximal nor distal mesenteric tyrosine hydroxylase activity was altered. Tyrosine hydroxylase activity was not detectable in the femoral arteries, or in the thoracic and abdominal aorta.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of catecholamines in the hypothalamus and brainstem in one-kidney, one clip and two-kidney, one clip hypertension in rabbits.

In order to examine the role of central catecholaminergic neurons in hypertension, the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of catecholamines, was studied in the hypothalamus, midbrain, pons-upper medulla, mid-medulla and lower medulla of one-kidney, one clip (1-K,1C) and two-kidney, one clip (2-K,1C) hypertensive rabbits and their respective operated controls (1-K,1 Cc and 2-K,1 Cc). Comparing the 1-K,1 C group to the 1-K, 1 Cc group, the activity of TH was increased by 79% in the hypothalamus (P less than 0.02), 37% in the mid-medulla region (P less than 0.02) and was unchanged in the midbrain, pons-upper medulla and the lower medulla. Comparing the 2-K,1 C group to the 2-K,1 Cc group, the activity of TH was increased by 89% in the mid-medulla (P less than 0.01), decreased by 36% in the pons-upper medulla (P less than 0.01) and unchanged in the hypothalamus, midbrain and lower medulla. These results indicate that similarities and differences exist in the contribution of central catecholaminergic neurons to the pathophysiology of 1-K,1 C and 2-K,1 C hypertension in rabbits.

Lysophospholipids are natriuretic and diuretic in rats.

The effects of lysophosphatidylcholine (LPC) and some of its analogues on renal sodium (UNaV) and water excretion (V) were examined in the pentobarbital sodium-anesthetized rat. LPC caused the increase in UNaV and V with a steep dose response. The natriuretic effect began within 20 min after a bolus injection and lasted for 7 h. The natriuresis and diuresis were not affected when the phospholipid was given as an LPC-albumin complex. Natriuretic activity was eliminated by substituting unsaturated bonds in the 1-acyl group and by removing the choline group on position 3. Natriuretic activity was not affected by substitution of 1-alkyl for 1-acyl groups and was stereospecific. These results demonstrate a unique, previously unrecognized effect of lysophospholipids and suggest that they may account for some of the previously demonstrated natriuretic activity of plasma.

Tissue distribution of an endogenous ligand to the Na, K ATPase molecule.

A variety of evidence indicates the presence of a circulating ligand to the Na, K ATPase molecule that is involved in the regulation of extracellular sodium metabolism. To examine the potential role of endogenous ligands to the Na, K ATPase molecule in the regulation of intracellular sodium metabolism, the tissue distribution of digitalis-like activity was quantitated in several brain regions and peripheral organs. The digitalis-like activity of desalted and delipidated extracts of tissue was widely distributed and produced a displacement of tritiated ouabain that was parallel to the displacement produced by cold ouabain. These results suggest that tissue contains an endogenous ligand to the Na, K ATPase molecule and that this ligand may regulate intracellular sodium metabolism in an autocoid-like manner.

Characterization of a competitive and reversible ligand to E2 conformation of Na, K ATPase molecule.

The binding of cardiac glycosides to the E2 conformation of Na, K ATPase suggests that this conformation may represent a potential regulatory site for an endogenous ligand. To examine this hypothesis, delipidated and desalted, ethanolic extracts were prepared from plasma. Plasma extracts produced displacement of 3H-ouabain from Type I and II high affinity binding sites that was parallel to the displacement by cold ouabain. Plasma extracts shifted the binding curve of 3H-ouabain to the right indicating a competitive and reversible displacement. Extracts also produced a dose-dependent inhibition of Na, K ATPase and K-stimulated, p-nitrophenyl phosphatase activity. These results indicate that this ligand may function as an endogenous ligand to the E2 conformation of the Na, K ATPase molecule and be involved in its regulation.

Nephrectomy-induced alterations in the synthesis of catecholamines in the sympathetic nervous system and central nervous system.

Reduction in renal function is a key factor to the development of salt-dependent hypertension; however, the mechanism is obscure. To examine the role of the sympathetic nervous system (SNS) and central catecholaminergic neurons in this predisposition to the development of hypertension, the activity of tyrosine hydroxylase (TH) was determined in SNS and in several brain regions. In another group of unilaterally nephrectomized rabbits, cardiovascular responsiveness to norepinephrine was determined. A unilateral nephrectomy increased the activity of TH in the midmedulla, a brain region important in the baroreflex regulation of blood pressure, and in the adrenal gland, the major source of circulating catecholamines. The activity of TH was decreased in the pons-upper medulla region. No alterations were found in the proximal and distal mesenteric arteries, lower medulla, midbrain or hypothalamus. No alteration in blood pressure or cardiovascular responsiveness to norepinephrine was found. This study indicates that a unilateral nephrectomy produces long-lasting effects on central catecholaminergic neurons and the sympathetic nervous system without an effect on blood pressure or cardiovascular responsiveness.

Plasma volume expansion increases lysophosphatidylcholine and digitalis-like activity in rat plasma.

A circulating factor with digitalis-like activity has been proposed to play a role in the regulation of plasma volume. Lysophosphatidylcholine has been found to be active in many assays for digitalis-like activity. To examine the relationship between plasma digitalis-like activity and plasma lysophosphatidylcholine, the effect of plasma volume expansion with saline on the plasma levels of phospholipids and on the ability of delipidated extracts of plasma to displace tritiated ouabain from the digitalis receptor was determined. Lysophosphatidylcholine was elevated after 15, 30, and 120 minutes of volume expansion but was decreased at 60 minutes. Phosphatidylcholine was decreased at 15, 60, and 120 minutes. Plasma sphingomyelin was not altered at any time point. The ability of plasma to displace tritiated ouabain was increased only at the 60 minute time point. These results indicate that the increase in digitalis-like activity in volume expanded states is mediated by a combination of at least two factors, lysophosphatidylcholine and another factor whose digitalis-like activity is not related to the surfactant actions of a lipid.

Cardiovascular tissue phospholipid metabolism in the Dahl rat: influence of dietary salt.

Cardiac and thoracic aortic tissue were removed from 8-9 week old Dahl salt resistant (DR) and salt sensitive (DS) rats following 2 and 4 weeks of 8% NaCl diets. The tissue was used to determine the pool size and rate of [32P] Pi incorporation into phosphatidylinositol-4-5-bisphosphate (PIP2), phosphatidylinositol-4-phosphate (PIP) and phosphatidic acid (PA). The studies show no strain differences in the pool size of these phospholipids nor any changes in pool size as a consequence of the duration of exposure to the 8% NaCl diet. However, [32P] Pi incorporation into PIP2, PIP and PA was increased in the cardiac tissue isolated from both DR and DS rats exposed to 4 versus 2 weeks of 8% NaCl diet prior to sacrifice. The relative increase was comparable in both strains. Further, the extent of [32P] Pi incorporation into these phospholipids was also increased in the aorta of DR, but not DS, rats exposed to 8% NaCl diets for 4 versus 2 weeks. The present study defines a strain specific difference in aortic tissue response to prolonged 8% NaCl diet exposure.

Plasma levels of an ouabain-like factor associated with an increased incidence of cerebral haemorrhage in one-kidney, one wrap hypertension in rabbits.

To examine the role of an ouabain-like factor (OLF) in the pathogenesis of cardiovascular disease in hypertension, we examined plasma levels of OLF in a malignant form of one-kidney, one wrap (1K,1W) hypertension. Two weeks after the induction of hypertension plasma OLF increased to 282% compared to control animals. Additionally, blood pressure, the cardiac index, incidence of cerebral haemorrhage and vascular aneurysms were increased. Blood pressure was found to be inversely correlated with cardiac hypertrophy. No correlations between plasma OLF and blood pressure, cardiac hypertrophy or vascular aneurysms were found. However, hypertensive animals with cerebral haemorrhage had significantly higher plasma OLF than animals without cerebral haemorrhage. These results suggest that high plasma OLF in hypertensive subject may increase the risk of cerebral haemorrhage.

Alpha 2-adrenergic receptors influence tyrosine hydroxylase activity in retinal dopamine neurons.

Dopamine (DA) is a putative neurotransmitter in a population of interneurons in the mammalian retina that are activated by photic stimulation. Pharmacological studies were conducted to determine if alpha 2-adrenergic receptors influence the activity of retinal tyrosine hydroxylase (TH), a biochemical indicator of changes in the activity of the DA-containing neurons. TH activity was low in dark-adapted retinas and high in light-exposed retinas. Systemic administration of the alpha 2-adrenoceptor antagonists, yohimbine and piperoxane, to dark-adapted rats significantly stimulated TH activity. This effect was apparently mediated locally within the retina because the response could also be elicited by direct injection of yohimbine into the vitreous. The dose-response relationships for the effects of alpha 2-adrenoceptor antagonists on retinal TH activity were similar to those for the effects on brain noradrenergic neurons, where alpha 2-adrenoceptors have been shown to be involved in the autoregulation of neuronal activity. Clonidine, an alpha 2-adrenoceptor agonist, had no effect when administered alone to dark-adapted rats, but it attenuated the stimulatory effect of yohimbine. In contrast, clonidine decreased TH activity of light-exposed retinas, an effect that was reversed by yohimbine. These observations suggest that alpha 2-adrenoceptors influence the activity of retinal DA-containing neurons.