Androgen modulation of adrenal angiotensin receptors.

Several polar androgens increased the binding of angiotensin and its stimulation of aldosteronogenesis in bovine adrenal glomerulosa cells. The effect was seen only if the steroids were applied to the cells and then washed away. This phenomenon and the technique for demonstrating it may have implications for studies of receptor modulation and for clinical states in which responsiveness to angiotensin is increased.

Heparin, lipoproteins, and oxygenated fatty acids in blood: a cautionary note.

We measured 16 nonesterified oxygenated fatty acid derivatives (oxylipids) in plasmas from seven human subjects. Two arterial samples from each subject were analyzed, drawn approximately 2h apart. We observed a marked increase in levels of most oxylipids in the second sample, as high as 470-fold. Between the first and second samples, subjects received approximately 800-1000 IU of heparin to prevent clotting in intravascular catheters. We postulate that heparin activated lipoprotein lipases, which, in turn, released oxylipids from triglycerides and phospholipids in plasma lipoproteins. Some of that lipolysis may have occurred during sample storage. Measurements of nonesterified lipids in human plasma may be distorted if heparin is administered to subjects before blood is drawn and if lipase inhibitors are omitted from stored samples.

Angiotensin and bradykinin interactions with phospholipids.

Reversible interactions were demonstrated between some phospholipids and some polypeptides related to angiotensin and bradykinin. The extent of the interaction was dependent on the structures of the lipid and peptide. The naturally occurring compounds that interacted most avidly were cardiolipin and (des-Asp1)-angiotensins. The apparent dissociation constant of this complex in chloroform was 10(-5) M. The complex contained more than one cardiolipin molecule/molecule of peptide. Kinins interacted most strongly with lecithin. The phospholipids altered the chromatographic behavior of radioiodinated derivatives of the polypeptides, and solubilized radioactive and unlabeled polypeptides in chloroform. In aqueous media, cardiolipin suspensions preferentially bound (des-Asp1)-angiotensin II, and inhibited its binding by antibody. The interactions were sensitive to pH and cations in the aqueous phase, and were reversed by some reagents added to the organic phase. These interactions have direct implications for binding reactions of peptides in vitro, and may bear upon the actions of the hormones in vivo.

Mechanism of fatty acid inhibition of aldosterone synthesis by bovine adrenal glomerulosa cells.

Previous work from this laboratory has suggested that the adrenal glomerulosa is under tonic inhibition by fatty acids. The purpose of the present work was to define the mechanism by which fatty acids inhibit aldosterone synthesis. Experiments with isolated bovine adrenocortical cells showed the following. 1) Fatty acids inhibited angiotensin-II-stimulated and (Bu)2cAMP-stimulated aldosterone synthesis with similar potencies. 2) Inhibition of aldosterone synthesis was highly dependent on fatty acid chain length and degree of unsaturation as well as on configuration of double bonds. Oleic acid was the most potent inhibitor among fatty acids prominent in plasma. 3) Cortisol synthesis was less sensitive to oleic acid inhibition than was aldosterone synthesis. 4) Pregnenolone synthesis by angiotensin-II-stimulated adrenal glomerulosa cells was relatively insensitive to oleic acid. 5) For both glomerulosa and fasciculata cells, cortisol synthesis from 21-deoxycortisol, which requires the participation of P450(21), was relatively insensitive to fatty acids. Cortisol synthesis from corticosterone by fasciculata cells, which requires the participation of P450(17) alpha, was also insensitive to oleic acid. These are microsomal enzymes. 6) In glomerulosa cells, aldosterone synthesis from added corticosterone, which requires the 18-oxidase function of P450(11) beta, a mitochondrial enzyme, was potently inhibited by fatty acids; cortisol synthesis from 11-deoxycortisol by glomerulosa cells, which requires P450(11) beta, was less sensitive to inhibition, and cortisol synthesis from 11-deoxycortisol by fasciculata cells was even less sensitive. 7) Aldosterone synthesis from exogenous 18-hydroxycorticosterone was potently inhibited by oleic acid. Thus, fatty acids are potent inhibitors of the 18-oxidase function of the mitochondrial enzyme P450(11) beta, whereas nonmitochondrial steroidogenic enzymes and the 11-hydroxylase function of P450(11) beta are relatively insensitive to fatty acids. The special sensitivity of aldosterone synthesis to fatty acid inhibition appears to result from the unusual susceptibility of the 18-oxidase function of the mitochondrial steroidogenic enzyme P450(11) beta. This mechanism would allow differential regulation of aldosterone vs. cortisol production by unesterified fatty acids.

Bovine adrenal glomerulosa and fasciculata cells exhibit 28.5-kilodalton proteins sensitive to angiotensin, other agonists, and atrial natriuretic peptide.

Protein synthesis by bovine adrenal glomerulosa and fasciculata cells in response to various modulators of steroid synthesis was examined using [35S]methionine labeling and two-dimensional gel electrophoresis. Both cell types responded to steroidogenic stimuli with rapid changes in a family of 28- to 30-kilodalton (kDa) proteins similar to those described in rat fasciculata by Epstein and Orme-Johnson. In glomerulosa, angiotensin-II (AII), potassium, and (Bu)2cAMP stimulated the appearance of two 28.5-kDa proteins (no. 3 and 4) with pI values of 6.44 and 6.33 and decreased labeling of two other 28.5-kDa proteins (no. 1 and 2) with pI values of 6.9 and 6.59. The rank order of potency on aldosterone synthesis and that on proteins 1-4 were the same: (Bu)2cAMP > AII > potassium. Atrial natriuretic peptide blocked the effects of AII on all four proteins and on aldosterone synthesis. Adrenal secretagogues also affected labeling of four slightly larger (30 kDa) proteins (no. 5-8). Corresponding proteins in each quartet are separated by the same difference in isoelectric points. These eight proteins may represent a core protein systematically modified in a number of ways. Aldosterone synthesis in glomerulosa, like glucocorticoid synthesis in fasciculata, requires ongoing protein synthesis. The 28- to 30-kDa proteins increased by steroidogenic stimuli in both cells and decreased by atrial natriuretic peptide in glomerulosa may be the proteins whose synthesis is crucial to acute control of steroidogenesis. Our results indicate that these proteins are made in response to calcium- or calcium/phosphoinositide-dependent mechanisms as well as by cAMP.

Effects of ouabain and potassium on protein synthesis and angiotensin-stimulated aldosterone synthesis in bovine adrenal glomerulosa cells.

Ouabain (1 microM and below) inhibited both basal and angiotensin II-stimulated aldosterone synthesis in bovine adrenal glomerulosa cells. Ouabain had no effect on binding of 125I-labeled angiotensin, on angiotensin's effects on 45Ca2+ fluxes, or on 32PO4 incorporation into phosphatidylinositol. This spectrum of activities resembles that of the protein synthesis inhibitor cycloheximide, which also blocks aldosterone synthesis. Ouabain was, therefore, tested for its effect on protein synthesis, as measured by uptake of [3H]leucine into acid-precipitable material. Ouabain inhibited protein synthesis at concentrations similar to those that depressed aldosterone synthesis, but did not block uptake of the nonmetabolized amino acid [carboxyl-14C]aminocyclopentane-1-carboxylic acid, nor the entrance of [3H]leucine into cells. When cells previously loaded with 86Rb+ were treated with 1 microM ouabain, they lost approximately half of the accumulated radioactivity in 30 min. When cells were incubated in potassium-free buffer, both protein and aldosterone synthesis were severely inhibited. Increased extracellular potassium reversed ouabain's inhibition of protein and aldosterone synthesis in parallel. Pregnenolone synthesis was inhibited by ouabain, and elevated potassium overcame that blockade. Ouabain did not block aldosterone synthesis from exogenous progesterone. These data fit a model in which ouabain causes loss of cell potassium, which, in turn, depresses protein synthesis. Since protein synthesis is necessary for angiotensin II stimulation of cholesterol side-chain cleavage, ouabain depresses that step, pregnenolone synthesis, and thus, aldosterone synthesis.

Fatty acids are potential endogenous regulators of aldosterone secretion.

Adrenal glomerulosa cells washed with delipidated albumin produced increased amounts of aldosterone in response to angiotensin-II (AII) or (Bu)2cAMP. Albumin treatment also increased binding of 125I-labeled AII to high affinity binding sites on adrenal cells. Lipid extracts of albumin solutions that were used to wash cells inhibited AII binding and aldosterone responses by washed glomerulosa cells. Chromatographic fractionation and mass spectroscopic analysis indicated that the inhibitors removed from cells by albumin were long chain fatty acids. Exogenous fatty acids not only inhibited AII binding, but they inhibited basal aldosterone production and increments in aldosterone caused by AII or dbcAMP, suggesting an effect on postreceptor steps in aldosteronogenesis. The most potent and most abundant fatty acids removed from adrenal cells were oleic, linoleic, and arachidonic. These fatty acids inhibited at micromolar concentrations in the absence of albumin and at somewhat higher concentrations in its presence. Cells that had been washed, then inhibited by exogenous oleic acid in vitro, were restored to their enhanced responsiveness by a second albumin wash, making it unlikely that cell damage is the mechanism of inhibition by fatty acids. Responses of fasciculata cells were not potentiated by albumin washes, and cortisol production was less sensitive than aldosterone production to exogenous fatty acids. Binding of ANP to glomerulosa cells was not affected by albumin or fatty acids. These results combined with clinical correlations make it plausible that unesterified fatty acids are naturally occurring regulators of the adrenal glomerulosa. Insulin's ability to lower plasma levels of fatty acids may be one way that it causes sodium retention.

Angiotensin-responsive adrenal glomerulosa cell proteins: characterization by protease mapping, species comparison, and specific angiotensin receptor antagonists.

Angiotensin II (AngII)-stimulated aldosterone synthesis is mediated by the AngII type 1 (AT1) receptor and requires ongoing protein synthesis. Hormonally-stimulated turnover of a family of 28- to 30-kDa proteins (p30, or steroidogenic acute regulatory proteins) has been linked to enhanced steroid synthesis in several tissues. Our previous work showed that AngII, dibutyryl cAMP, potassium, and atrial natriuretic peptide affected labeling of a group of eight proteins (four of 28 kDa and four of 30 kDa) in bovine adrenal glomerulosa cells. This report extends our findings in three ways: 1) The eight [35S]-methionine-labeled p30 proteins in bovine cells were compared with each other by chymotryptic peptide mapping. Similarity in maps indicated that the eight proteins share a common primary structure. 2) Dibutyryl cAMP treatment of rat adrenal glomerulosa cells affected the levels of four 28-kDa proteins and one 35-kDa protein, whereas AngII affected two of the 28-kDa proteins. There were no responsive 30-kDa proteins in rats comparable with those seen in bovine cells. These results indicate a species difference in the affected proteins. 3) The AT1 receptor antagonist, losartan, inhibited the effects of AngII on aldosterone synthesis and turnover of the p30 proteins in bovine adrenal glomerulosa cells. PD123319, an antagonist specific for the AngII type 2 receptor, did not block AngII-stimulated aldosterone synthesis and had much less effect on p30 protein labeling than did losartan. These results add to the growing body of evidence that this family of p30 or steroidogenic acute regulatory proteins plays a role in the acute regulation of steroidogenesis by a wide variety of stimulatory hormones in several tissues and species. In addition, losartan's inhibition of AngII's effects on the p30 proteins is consistent with a key role for these proteins in processes linking occupation of the AT1 receptor to stimulation of aldosterone synthesis.

Angiotensin effects on calcium and steroidogenesis in adrenal glomerulosa cells.

We investigated the role of cellular calcium pools in angiotensin II-stimulated aldosterone synthesis in bovine adrenal glomerulosa cells. Angiotensin II decreased the size of the exchangeable cell calcium pool by 34%, consistent with previous observations that angiotensin II causes decreased uptake of 45Ca+2 into cells and increased efflux of 45Ca+2 from preloaded cells. Atomic absorption spectroscopy showed that angiotension II caused a decrease of 21% in total cellular calcium. Angiotensin II caused efflux of 45Ca+2 in the presence of EGTA and retarded uptake of 45Ca+2 when choline was substituted for sodium, suggesting that hormone effects on calcium pools do not involve influx of trigger calcium or sodium. Cells incubated in calcium-free buffer and 0.1 mM or 0.5 mM EGTA synthesized reduced (but still significant) amounts of the steroid in response to hormone. Cells incubated in increasing concentrations of extracellular calcium contained increasing amounts of intracellular calcium and synthesized increasing amounts of aldosterone in response to angiotensin II. These results point to the participation of intracellular calcium pools in angiotensin II-stimulated steroidogenesis and the importance of extracellular calcium in maintaining these pools.

Fatty acids enhance vascular alpha-adrenergic sensitivity.

Hypertensive patients are heavier and have a more centralized body fat distribution, which is associated with risk factor clustering and resistance to insulin's actions, including suppression of plasma nonesterified fatty acids. We postulated that abnormalities of nonesterified fatty acids contribute to the increased vascular alpha-adrenergic reactivity and tone observed in our previous studies of obese hypertensive subjects. To test this hypothesis, in two separate protocols 10% Intralipid was infused into a dorsal hand vein with heparin to activate lipoprotein lipase and raise fatty acid levels locally. In protocol 1, the effects of Intralipid/heparin compared with those of 5% dextrose/heparin on dorsal hand vein sensitivity to phenylephrine were assessed by use of the linear variable differential transformer technique in 8 normotensive subjects. In protocol 2, the effects of Intralipid/heparin were compared with those of saline/heparin on hand vein responses to both phenylephrine and angiotensin II in 11 normotensive African American women. Intralipid/heparin reduced the dose of phenylephrine required to produce 50% of the maximal venoconstrictor response from 582 to 137 ng/min (compared with dextrose/heparin, P < .01) in protocol 1 and from 293 to 137 ng/min (compared with saline/heparin, P < .01) in protocol 2. Intralipid/heparin did not significantly alter hand vein responses to angiotensin compared with saline/heparin. These data suggest that abnormalities of nonesterified fatty acids in obese hypertensive patients with risk factor clustering may contribute to their increased neurovascular tone.

Lead increases aldosterone production by rat adrenal cells.

Exposure to lead has been postulated to contribute to elevated blood pressure in humans and has been shown to raise blood pressure in animals. The mechanism of action of lead on blood pressure is unknown. We fed lead to rats in their drinking water and then examined the production of aldosterone by their adrenal cells in vitro. We also measured excretion of aldosterone and corticosterone by intact rats stimulated with corticotropin, with and without lead treatment. At a dose (273 ppm) that raised blood levels to 30 to 40 micrograms/dL, comparable to blood levels in exposed humans, lead induced increased aldosterone secretion in vitro and in vivo. The effect of lead was most evident when cells or animals were stimulated with aldosterone secretagogues. Experiments in vitro indicate that exposure to lead in vivo increases activity of one or more steps in the late pathway of aldosterone biosynthesis. The results suggest that the hypertensive effect of lead involves relative hyperaldosteronism and may be most evident when secretion of this hormone is stimulated.

Relationships among plasma aldosterone, high-density lipoprotein cholesterol, and insulin in humans.

To investigate the pathogenesis of hypertension in patients with obesity and insulin resistance and to explore the role of plasma lipids, we studied 30 subjects at the end of 7 days of low (20 mEq/d) then high (200 mEq/d) sodium diets. Glucose and insulin tolerance tests were performed at the end of each week and blood and urine collected for measurements of plasma aldosterone, renin activity, electrolytes, insulin, and lipoproteins. There was a strong negative correlation between plasma aldosterone and high-density lipoprotein cholesterol during both diets. There were weaker positive correlations between plasma aldosterone and insulin or triglycerides. When the aldosterone-renin ratio was the dependent variable and the correlation controlled for serum potassium, the inverse relationship with high-density lipoprotein cholesterol and the positive correlation with insulin remained, but only during the high salt diet. Subjects were divided into three groups based on high-density lipoprotein cholesterol. Subjects with the lowest high-density lipoprotein cholesterol levels showed the highest aldosterone, plasma triglycerides, body mass index, and waist-to-hip ratio. Those subjects also demonstrated the greatest resistance to insulin action on glucose and plasma unesterified fatty acids. There was a weak direct correlation between plasma aldosterone and systolic blood pressure during the high salt diet. These data suggest that high aldosterone levels may be a link between dyslipidemia, insulin resistance, and hypertension, a relationship made more evident by high salt intake.

Aspects of angiotensin action in the adrenal. Key roles for calcium and phosphatidyl inositol.

The steps between exposure of bovine adrenal glomerulosa cells to angiotensin and the stimulated increase in aldosterone production were studied in two ways. Binding of angiotensin to receptors, and hormone effects on phosphatidyl inositol turnover, 45Ca2+ fluxes, and aldosterone production were measured directly. Other potential intermediate steps were investigated indirectly by use of inhibitors. Angiotensin slowed calcium influx and accelerated phosphatidyl inositol turnover in proportion to hormone dose. The effects correlated with receptor binding and aldosterone production. None of the inhibitors tested, except saralasin, inhibited angiotensin's effect on phosphatidyl inositol turnover. Altered calcium flux and stimulated aldosterone production were affected by the calmodulin inhibitor trifluoperazine and the intracellular calcium antagonist 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8). Several reagents did not affect angiotensin binding, its effect on phosphatidyl inositol, or 45Ca2+ flux, but severely inhibited steroidogenesis. These included the phospholipase A2 inhibitor mepacrine, the protein synthesis inhibitor cycloheximide, and the Na+/k+-ATPase inhibitor ouabain. Colchicine had very little effect on the processes we measured, suggesting that microtubules play no role in angiotensin action in the adrenal. Based o these observations, we propose that angiotensin II affects the adrenal glomerulosa cell by first interacting with receptors, then increasing phosphatidyl inositol turnover, then altering cellular calcium distribution. Step distal to altered calcium distribution that contribute to increased steroid output include altered phospholipid metabolism, protein synthesis, and Na/k metabolism.

The effect of prolactin on human aldosterone-producing adenomas in vitro.

There is evidence for an unidentified aldosterone-stimulating factor of pituitary origin. We measured the effect of ovine PRL (oPRL) on aldosterone secretion by isolated cell suspensions of human aldosterone-producing adenomas (APAs) and compared it to the effects of angiotensins, ACTH, and potassium (K+). In the first APA, the aldosteronotropic action of large doses of oPRL was double that of angiotensin II (AII); the response to ACTH was triple that to AII, while K+ had a small stimulatory effect. Results with the second APA showed that physiological concentrations of oPRL caused a response nearly double that to AII, but, once again, less than the response to ACTH; K+ was inert. ACTH contamination of the oPRL preparation was too minute to account for these findings. We conclude that oPRL possesses aldosterone-stimulating activity in APAs greater than that of angiotensins and potassium, but less that that of ACTH. These data suggest a role for PRL in aldosterone secretion by aldosterone-producing adenomas.

Intralipid enhances alpha1-adrenergic receptor mediated pressor sensitivity.

The dyslipidemia in obese hypertensive persons may contribute to their increased vascular alpha-adrenergic receptor reactivity and tone. To further examine this notion, we conducted 2 studies of pressor sensitivity to phenylephrine, an alpha1-adrenergic receptor agonist, in lean normotensive subjects. In the first study (n=6), pressor responses to phenylephrine were obtained before and during a saline and heparin infusion. On another day, pressor reactivity to phenylephrine was measured before and during infusion of 20% Intralipid at 0.5 mL . m-2 . min-1 with heparin at 1000 U/h to increase lipoprotein lipase activity and raise nonesterified fatty acids (NEFAs). In the second study (n=8), baseline reactivity to phenylephrine was obtained on 2 separate days and repeated after raising NEFAs and triglycerides either with 0.8 mL . m-2 . min-1 of 20% Intralipid alone or together with heparin. The infusion of saline and heparin did not significantly change plasma NEFAs from baseline (516+/-90 versus 512+/-108 micromol/L, respectively; P=NS) or the dose of phenylephrine required to raise mean blood pressure by 20 mm Hg ([PD20PE]; 1.00+/-0.14 versus 0. 95+/-0.10 microg . kg-1 . min-1, respectively, P=NS). Intralipid at 0.5 mL . m-2 . min-1 with heparin raised plasma NEFAs to 793+/-30 micromol/L per liter (P<0.05 versus baseline) and reduced PD20PE from 1.01+/-0.10 to 0.80+/-0.09 microg . kg-1 . min-1 (P<0.05). Compared with baseline, Intralipid alone increased plasma NEFAs to 946+/-80 micromol/L (P<0.05), and NEFAs increased further with the addition of heparin to 2990+/-254 micromol/L (P<0.01). Despite an apparently greater increase of plasma NEFAs with Intralipid and heparin, Intralipid alone and together with heparin similarly reduced PD20PE. Across all study conditions, changes in levels of triglycerides and NEFAs correlated with changes in mean arterial pressure responses to phenylephrine, especially at the 0.4- microg . kg-1 . min-1 infusion rate of phenylephrine (r=0.64, P<0.01 and r=0. 54, P<0.01, respectively). These data suggest that raising levels of plasma NEFAs and/or triglycerides enhances alpha1-adrenoceptor mediated pressor sensitivity. The findings suggest that lipid abnormalities in obese hypertensives, which include elevated NEFAs and triglycerides, contribute to greater vascular alpha1-adrenergic reactivity.

Hyperinsulinemia and the aldosterone and pressor responses to angiotensin II.

To determine whether hyperinsulinemia alters angiotensin II-mediated aldosterone secretion, the increase in plasma aldosterone after intravenous angiotensin II (5, 10, and 20 ng/kg/min for 15 minutes each) was measured before and after euglycemic hyperinsulinemia in seven chronically instrumented dogs. In a random sequence on 4 successive days, dogs received either 0, 2, 4, or 8 milliunits/kg/min insulin. Euglycemic hyperinsulinemia, at all insulin doses, resulted in a significantly greater (p less than 0.01) change in the angiotensin II-stimulated increments of plasma aldosterone than was observed when angiotensin II was administered alone. However, there was no dose-dependence of insulin's effect on angiotensin II-stimulated aldosterone. The effect of weight gain on the angiotensin II response was also evaluated in five dogs. After weight gain, euglycemic hyperinsulinemia augmented angiotensin II-stimulated aldosterone to the same magnitude that was observed before weight gain. Possible mechanisms whereby insulin could increase angiotensin II-stimulated aldosterone production include: increased intracellular potassium, reduced plasma free fatty acids, and a direct action of insulin to induce increased adrenal steroidogenesis. In addition to altering the angiotensin II-aldosterone dose-response curve, hyperinsulinemia also increased the pressor action of angiotensin II. In contrast to the angiotensin II-aldosterone response, progressive hyperinsulinemia resulted in a progressive increase in the pressor response to angiotensin II. The increased pressor response is probably due to an increased activation of the sympathetic nervous system by insulin.

The possible biological role of aldosterone metabolites.

Following the incubation of aldosterone with microsomes from liver of adrenalectomized male rates, a previously unidentified polar neutral metabolite of aldosterone, designated "peak A" material, was isolated and purified using high pressure liquid chromatography. This peak A material, which contains a reduced hydroxylated metabolite of aldosterone, was shown to possess 2% to 3.5% of the mineralocorticoid activity relative to aldosterone. When bovine adrenal glomerulosa cells were incubated with peak A material (3 and 10 micrograms/ml), the binding of 125I-angiotensin II was inhibited by 20% and 36%, respectively. The mineralocorticoid activity of the six possible ring A reduced metabolites was tested. The 5 alpha-reduced metabolites were more potent than the 5 beta-, and the 3 alpha- were more potent than the 3 beta-reduced metabolites. The renal and extrarenal transformations of aldosterone to the polar and nonpolar (reduced) metabolites and their possible role in the accepted mechanism of action of aldosterone is discussed.

Salt loads raise plasma fatty acids and lower insulin.

Some fatty acids are potent inhibitors of angiotensin binding and aldosterone production in adrenal glomerulosa cells and thereby may be involved in regulating salt and water balance. To study the possible regulation of fatty acids by salt, we measured the levels of unesterified fatty acids in plasma from patients subjected to extremes of dietary salt intake and saline infusion. Insulin and catecholamines, two known regulators of plasma fatty acids, also were measured. Infusion of 2 l saline over 4 hours caused the levels of most unesterified fatty acids to rise. Total unesterified fatty acids rose 60-100%. A high salt diet caused a smaller rise in total unesterified fatty acids (approximately 33%). In both instances, oleic and palmitoleic acids showed the greatest proportionate increases, whereas stearic acid was relatively unaffected. When salt loads were administered by either intravenous or dietary routes, plasma insulin levels fell by approximately 50%. Plasma norepinephrine increased after saline infusion but not during a high salt diet. Postsaline levels of fatty acids correlated inversely with postsaline levels of aldosterone, supporting a possible role for fatty acids as physiological regulators of the adrenal glomerulosa. A rise in plasma fatty acids and fall in insulin in response to salt loads could act in concert to increase sodium excretion, constituting a physiological mechanism contributing to salt and water balance.

Oleic acid inhibits endothelial nitric oxide synthase by a protein kinase C-independent mechanism.

Many obese hypertensive individuals have a cluster of cardiovascular risk factors. This cluster includes plasma nonesterified fatty acid concentrations and turnover rates that are higher and more resistant to suppression by insulin than in lean and obese normotensive individuals. The higher fatty acids may contribute to cardiovascular risk in these patients by inhibiting endothelial cell nitric oxide synthase activity. To test this hypothesis, we quantified the effects of oleic (18:1[cis]) and other 18-carbon fatty acids on nitric oxide synthase activity in cultured bovine pulmonary artery endothelial cells by measuring the conversion of [3H]L-arginine to [3H]L-citrulline. Oleic acid (from 10 to 100 mumol/L) caused a concentration-dependent decrease in nitric oxide synthase activity at baseline and during ATP and ionomycin (Ca2+ ionophore) stimulation. At 100 mumol/L, linoleic (18:2[cis]) and oleic acids caused similar reductions of nitric oxide synthase activity, whereas elaidic (18:1[trans]) and stearic (18:0) acids had no effect. Oleic acid also inhibited the endothelium-dependent vasodilator response to acetylcholine in rabbit femoral artery rings preconstricted with phenylephrine (P < .05) but had no effect on the response to nitroprusside. The pattern of 18-carbon fatty acid effects on nitric oxide synthase activity in endothelial cells is consistent with activation of protein kinase C. Although oleic acid increased protein kinase C activity in endothelial cells, neither depletion of protein kinase C by 24-hour pretreatment with phorbol 12-myristate 13-acetate nor its inhibition with staurosporine eliminated the inhibitory effect of oleic acid on nitric oxide synthase.(ABSTRACT TRUNCATED AT 250 WORDS)

Obesity hypertension is related more to insulin's fatty acid than glucose action.

Although resistance to insulin-mediated glucose disposal has emerged as a link between abdominal obesity and hypertension, abnormalities of nonesterified fatty acid metabolism may play a greater role. Analyses were performed on existing data from 17 abdominally obese subjects (11 hypertensive, 6 normotensive) to determine whether fatty acid concentration and turnover were related to blood pressure independently of hyperinsulinemia and resistance to insulin-mediated glucose disposal. Glucose utilization, fatty acid concentration, and fatty acid turnover were obtained fasting and during euglycemic hyperinsulinemia at 10 and 40 mU/m/min. Analyses were also performed on another group of 30 subjects with a wide range of risk factors who had blood pressure data as well as glucose and fatty acid measurements during an insulin tolerance test. Fatty acid concentration and turnover were markedly more resistant to suppression by insulin in obese hypertensive than in lean or obese normotensive individuals. In the 17 obese subjects, blood pressure measured at screening, in the laboratory, and over a period of 24 hours correlated significantly with fatty acid concentration and turnover but not with glucose disposal measured during the hyperinsulinemic clamp. These correlations remained significant after fasting insulin, the insulin area under the curve during an oral glucose tolerance test, and glucose disposal during the clamp were controlled for. In the second group of subjects, plasma fatty acids 15 minutes after intravenous insulin also correlated with blood pressure. These correlations remained significant after insulin and an index of sensitivity to insulin-mediated glucose disposal were statistically controlled for. The data indicate that blood pressure is related to the effects of insulin on fatty acid metabolism. The findings raise the possibility that resistance of hormone-sensitive lipase to insulin participates in elevating the blood pressure of abdominally obese hypertensive subjects by increasing fatty acid concentration and turnover.