Role of receptor cycling in the regulation of angiotensin II surface receptor number and angiotensin II uptake in rat vascular smooth muscle cells.

In vivo data on the factors controlling angiotensin II (AII) cell surface binding are conflicting. We studied the specific effects of AII on AII binding in rat mesenteric artery vascular smooth muscle cells in culture. Incubation with unlabeled AII at 21 degrees C resulted in time- and concentration-dependent decreases in AII surface binding at 4 degrees C, with a 30% reduction after exposure to 300 nM AII for 15 min. Reductions in cell surface binding were due to decrements in receptor number rather than changes in binding affinity. Loss of surface receptors was mediated by receptor internalization as maneuvers that blocked ligand internalization (cold temperature and phenylarsine oxide [PAO]) attenuated AII-induced loss of surface receptors. After removal of AII, recovery of surface binding was rapid (t1/2 = 15 min) and was mediated by reinsertion of a preexisting pool of receptors into the surface membrane rather than by new receptor synthesis. To determine the role of receptor cycling on AII-induced surface receptor loss, cells were incubated with the endosomal inhibitor chloroquine during exposure to AII at 21 degrees C. Incubation with AII plus chloroquine resulted in a 70% greater loss of surface binding than after incubation with AII alone. To determine the role of receptor cycling on uptake of ligand, cells were incubated with PAO or endosomal inhibitors during exposure to AII at 4 and 21 degrees C. Compared with buffer these agents did not alter AII uptake at 4 degrees C, but decreased uptake by 12-50% at 21 degrees C. These results indicate that after binding AII receptors cycle and that receptor cycling attenuates AII-induced losses of surface receptors and enhances ligand uptake by providing a continuous source of receptors to the cell surface.

Potentiation of angiotensin II action by corticosteroids in vascular tissue.

OBJECTIVES: Studies were performed to determine if corticosteroids act directly on the vasculature to potentiate the vasoconstrictor action of angiotensin II and to determine whether corticosteroids upregulate angiotensin II receptors by receptor redistribution or by synthesis of new receptors. METHODS: Aortic rings from normal Sprague-Dawley rats were incubated ex vivo with corticosteroids in aerated Krebs-Henseleit buffer to avoid secondary systemic effects prior to stimulated contraction. In cultured vascular smooth muscle cells, these experimental techniques were used: colchicine (blocker of microtubule assembly), chloroquine (inhibitor of endosomal pH gradients), measuring surface-bound 125I-Ang II internalization rate, immunoblotting of angiotensin AT1 receptor protein, and incorporation of [35S]methionine into AT1 receptor protein. RESULTS: Contractions to 100 nM angiotensin II in rings incubated with 1 microM aldosterone or dexamethasone for 10 min ex vivo were not different from contractions in control rings. However, angiotensin II-stimulated (but not KCl-stimulated) contractions were enhanced by almost 100% if ex vivo incubation with aldosterone (or corticosterone) lasted for 24 h. Endothelium-dependent relaxation was not significantly reduced by aldosterone pre-incubation. Incubation of cultured vascular smooth muscle cells with a number of corticosteroids for > 8 h resulted in concentration-dependent upregulation of angiotensin II receptor binding and was reversible upon removal of the corticosteroid. Aldosterone did not affect the rate of internalization of surface-bound angiotensin II. In addition, concomitant incubation of colchicine or chloroquine with aldosterone did not hamper angiotensin II receptor upregulation. Incubation of cells with various concentrations of aldosterone for 24 h resulted in concentration-dependent increases in total cell angiotensin II receptor protein content and increases in [35S]methionine incorporation into immunoprecipitated AT1 receptor protein. CONCLUSIONS: At least a portion of the enhancement of angiotensin II action by corticosteroids is via direct interaction of corticosteroids with the vasculature. Corticosteroids appear to upregulate angiotensin II receptors by synthesis of new receptor protein rather than by alterations in receptor trafficking.

Aldosterone enhances angiotensin II receptor binding and inositol phosphate responses.

Clinical states in which angiotensin II is increased are often associated with increases in mineralocorticoids. To determine the effects of mineralocorticoids on angiotensin II action, we examined the effects of aldosterone on angiotensin II receptor expression and function in cultured rat vascular smooth muscle cells. Incubation with aldosterone resulted in concentration- and time-dependent increases in angiotensin II receptor number, without changes in binding affinity. For example, incubation with 1 microM aldosterone for 40 hours resulted in 59% increases in angiotensin II receptor number. Increases in angiotensin II receptors were dependent on protein synthesis as evidenced by the time dependency of upregulation and inhibition by cycloheximide. Incubation with aldosterone resulted in enhanced angiotensin II-stimulated phospholipase C activation, as demonstrated by increases in angiotensin II-induced inositol phosphate responses in proportion to the increases in receptor number. In addition, aldosterone prevented angiotensin II-induced downregulation of angiotensin II surface receptors and angiotensin II desensitization of inositol phosphate formation. In summary, aldosterone 1) directly increased angiotensin II receptor number, 2) increased angiotensin II-stimulated inositol phosphate responses, and 3) prevented angiotensin II-induced downregulation and desensitization. In conclusion, aldosterone may potentiate the pressor responses of angiotensin II via effects on angiotensin II receptors.

Role of citrate synthase in aldosterone-mediated sodium reabsorption.

Aldosterone and other mineralocorticoids increase citrate synthase activity in the kidney and enhance renal sodium reabsorption, but it is unclear whether the increased citrate synthase activity is involved in renal sodium transport. We used the Wistar-Furth rat, an inbred strain found to be deficient in renal citrate synthase activity, as an experimental model to investigate this issue. We confirmed that renal citrate synthase activity from adrenalectomized Wistar-Furth rats was decreased compared with that from control Wistar rats (by 28%). Similarly, urinary citrate excretion was 23% lower in Wistar-Furth rats. Subnormal citrate formation in Wistar-Furth rats could not be accounted for by differences in systemic pH or circulating potassium levels. Because renal citrate synthase activity was reduced in Wistar-Furth rats, we hypothesized that renal sodium excretory responses to mineralocorticoids would be reduced as well. Four-hour sodium excretion after intraperitoneal injection of 5 microg of aldosterone was reduced by 56% in adrenalectomized Wistar rats and by 52% in adrenalectomized Wistar-Furth rats (both P<0.01 compared with vehicle injection). Similarly, the pattern of urinary sodium excretion in response to subcutaneous injections of deoxycorticosterone acetate over a 2-week period was similar in adrenalectomized Wistar and Wistar-Furth rats. In summary, acute and chronic antinatriuretic responses to mineralocorticoids are maintained in Wistar-Furth rats at the level of Wistar rats, despite the marked reduction in citrate synthase activity. These findings are not consistent with an important role for citrate synthase activity in mineralocorticoid-mediated renal sodium transport.

Carbenoxolone damages endothelium and enhances vasoconstrictor action in aortic rings.

Carbenoxolone causes hypertension indirectly by inhibition of 11beta-hydroxysteroid dehydrogenase and consequent elevation of intracellular glucocorticoid levels and enhancement of vasoconstrictor action. We performed the present study to determine whether carbenoxolone also enhances vascular tone directly by mechanisms independent of glucocorticoids and other systemic influences. Exposure of rat aortic rings to 10 to 100 micromol/L carbenoxolone in aerated Krebs-Henseleit buffer for 24 hours resulted in concentration-dependent increases in angiotensin II (Ang II) (100 nmol/L)-stimulated contractions and significant shifting of the phenylephrine cumulative contraction curve to the left but not increases in KCI (120 mmol/L)-stimulated contractions. Maximal enhancement of Ang II contraction was 39 percent. In contrast, brief (15-minute) exposure to 100 micromol/L carbenoxolone did not alter Ang II contractions. Mechanical denudation of the endothelium obviated enhancement of Ang II contractions by carbenoxolone, suggesting interaction of carbenoxolone with the endothelium. Endothelium-dependent relaxation of precontracted rings to acetylcholine or ATP was reduced by more than 90 percent by 24-hour pretreatment with 100 micromol/L carbenoxolone but not with 100 micromol/L deoxycorticosterone acetate (a mineralocorticoid) or 100 mu mol/L glycyrrhizic acid (a natural 11beta-hydroxysteroid dehydrogenase inhibitor). Vascular smooth muscle relaxation with sodium nitroprusside was not inhibited by carbenoxolone. Incubation of cultured endothelial cells with 100 mu mol/L carbenoxolone for 24 hours did not inhibit nitric oxide synthase activity, as measured by conversion of [3H]L-arginine to [3H]L-citrulline. Electron micrography demonstrated that endothelial cell ultrastructure but not vascular smooth muscle cell ultrastructure was abnormal after incubation of rings for 24 hours with 100 micromol/L carbenoxolone. These studies suggest that carbenoxolone concentrations higher than 10 micromol/L enhance vasoconstrictor action via selective toxicity to the endothelium and elimination of endothelium-dependent relaxation.

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)

Beta-2-microglobulin-associated amyloidosis in chronic hemodialysis patients with carpal tunnel syndrome.

The clinical manifestations of beta-2-microglobulin (beta 2M)-associated amyloidosis in chronic hemodialysis patients with carpal tunnel syndrome from a medical center hospital are presented. The predominant morbidity of beta 2M-amyloid was musculoskeletal, with deposits identified in surgical or biopsy specimens from trigger fingers, carpal tunnels, fractures, and radiolucent bone lesions. Lucent bone lesions were the characteristic radiologic finding of beta 2M-amyloidosis and were most commonly found in carpal bones, humeral heads, and femoral heads. Carpal tunnel syndrome occurred in greater than 20% of our chronic hemodialysis patients. The longer the period of time on chronic hemodialysis the greater the morbidity from beta 2M-amyloid. Although significant amounts of beta 2M-amyloid were detected in the perivascular regions of viscera, clinical compromise of internal organs from this type of amyloid was not documented. In acute studies, beta 2M clearance during hemodialysis was markedly increased using the Fresenius polysulfone dialyzers compared to cuprophane dialyzers. In summary, beta 2M-amyloid is common and causes significant morbidity in chronic hemodialysis patients. Long-term dialysis with highly permeable membranes effects greater beta 2M clearance which may result in less tissue deposition of beta 2M-amyloid, and therefore, fewer clinical complications.

A protocol-based treatment for intradialytic hypotension in hospitalized hemodialysis patients.

Human serum albumin is used in hemodialysis (HD) units as treatment for hypotension despite its high cost and undetermined efficacy. During a 4-month period in 1995, albumin was used in 22% of 1,296 consecutive HD treatments in the HD unit or intensive care units (ICUs) at our tertiary-care hospital. We evaluated the safety and efficacy of a protocol designed to minimize albumin use for treating HD-associated hypotension (HDAH). The protocol consisted of the stepwise use of saline, mannitol, and albumin for the purpose of achieving physician-determined ultrafiltration goals. Patients were exempted from receiving the protocol for age younger than 18 years, freshly declotted angioaccess, or cardiovascular instability. The protocol was evaluated prospectively in 2,559 consecutive dialysis sessions (15% in ICUs) in 442 patients. Hypotension occurred during 608 sessions (24%), and attending nephrologists elected to initiate the protocol in 71% of these cases. Of the 433 instances in which the protocol was begun, reversal of hypotension was achieved without the need for albumin in 91% and with the addition of albumin in an additional 2%. Protocol treatment was not completed because of nursing error in 1% or clotting of filter or angioaccess in 4%. Use of the protocol failed to reverse hypotension in only 2% of the cases in which it was completed. Albumin was administered in only 6% of the 2,559 HD treatments. In summary, our protocol-based approach to HDAH was effective, easy for nurses to use, albumin sparing, and cost reducing.

Tyrosine phosphorylation of phosphatidylinositol 3-kinase and of the thromboxane A2 (TXA2) receptor by the TXA2 mimetic I-BOP in A7r5 cells.

Thromboxane A2 (TXA2) interacts with its G-protein coupled receptor, the TP receptor, to produce contraction and proliferation of vascular smooth muscle cells. We have shown previously that proliferation of primary cultures of vascular smooth muscle cells initiated by [1S-(1alpha, 2beta(5Z), 3alpha(1E, 3R), 4alpha]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxab icyclo-[2.2.1]heptan-2yl]-5'-heptenoic acid (I-BOP), a stable TXA2 mimetic, is mediated by activation of mitogen-activated protein (MAP) kinase. In the present study, we examined further the intracellular mediators involved in TXA2 activation of vascular smooth muscle cells. Transient transfection of the cDNA for the TP receptor into A7r5 vascular smooth muscle cells resulted in expression of TP receptors with a receptor density, Bmax, of 0.7 +/- 0.2 pmol/mg protein and a receptor affinity, Kd, of 0.6 +/- 0.1 nM (N = 7). Mock transfected cells lacked significant receptor expression. In TP receptor transfected cells, I-BOP increased the activation of MAP kinase 2-fold, stimulated tyrosine phosphorylation of cellular proteins of relative molecular mass (Mr) of 140, 85, 60, 56, and 45 kDa, and increased the message for c-jun, a nuclear transcription factor involved in mitogenesis, 2.6-fold. Immunoblot analysis indicated that the 85-kDa protein represented phosphoinositide 3-kinase (PI3-K), while the 60 kDa protein was the TP receptor. The activity of PI3-K was increased 3.5-fold by the addition of I-BOP (0.1 microM). In summary, the present study demonstrated that stimulation of the TP receptor results in tyrosine phosphorylation of the receptor and of PI3-K.

L-carnitine administration reverses acute mental status changes in a chronic hemodialysis patient with hepatitis C infection.

A chronic hemodialysis patient presented with elevated serum ammonia concentration (189 micromol/l) and acutely altered mental status. He had been adequately dialyzed over the prior months and had no evidence of liver dysfunction, despite serological evidence for hepatitis C virus infection. His mental status deteriorated to coma despite vitamin replenishment, intensive hemodialysis, lactulose treatment, and blood pressure control over a 3-day period. Blood free L-carnitine concentration was depressed, and total carnitine concentrations was normal. Three hours after a single 2 g dose of L-carnitine was administered intravenously, the mental status reverted to normal. Hyperammonemia resolved over a 5-week period. We suspect that subclinical liver dysfunction and dialysis status in tandem contributed to the carnitine deficiency, hyperammonemia, and confusion and that the L-carnitine administration reversed these biochemical and clinical abnormalities.

Bilateral congenital renal arteriovenous fistulas.

Bilateral renal arteriovenous (AV) fistulas were discovered in a patient with refractory hypertension. The lesion in the right kidney appeared to be congenital, whereas the etiology of the left-sided lesion could not be determined. Ablation of both fistulas effected a significant decrease in blood pressure. The angiographic appearance of a renal AV fistula often reveals its cause. These fistulas can cause significant morbidity. A review of 49 cases of congenital renal AV fistulas reveals that most are found in women and in the right kidney. Bilateral renal AV fistulas have not been previously described. Hypertension commonly develops in patients with renal AV fistulas and may resolve or improve upon fistula ablation. Improvement in blood pressure after fistula ablation occurs more frequently in traumatic fistulas than in congenital ones. Although the pathophysiology of hypertension is felt to be the shunting of the blood flow by the fistula from the renal parenchyma and subsequent stimulation of the renin system, renal vein renin sampling may be of little diagnostic value.

Prerenal azotemia from excessive sweating in an adult with a cystic fibrosis gene mutation.

We present the case of a 58-year-old male with chronic kidney disease who was admitted to the hospital multiple times with extracellular fluid volume depletion and prerenal azotemia. Some episodes were associated with gastrointestinal fluid losses and others with profuse diaphoresis in the absence of gastrointestinal fluid losses. At the age of 57 years, a common cystic fibrosis transmembrane conductance regulator protein mutation and a family history of cystic fibrosis were documented. We hypothesize that the abnormal cystic fibrosis transmembrane conductance regulator resulted in repeated bouts of excessive sweating, extracellular fluid volume depletion, and acute renal failure. This case is unique because of the prolonged period of time over which multiple documented episodes of prerenal acute renal failure occurred and because of the onset of the episodes in adulthood.

Fatty acid inhibition of angiotensin II-stimulated inositol phosphates in smooth muscle cells.

Inositol phosphate (InsP) responses to angiotensin II (ANG II) stimulation were measured in cultured rat vascular smooth muscle cells (VSMC) incubated with and without fatty acids (FA). VSMC were washed after 24 h of FA incubation to achieve cellular incorporation of FA yet eliminate ambient FA. Incubation with eicosapentaenoic acid (EPA)-supplemented medium resulted in concentration-dependent incorporation of EPA and depletion of arachidonic acid in VSMC membranes. Incubation with EPA, but not other FA, resulted in inhibition of ANG II-stimulated InsP formation (29% inhibition with 100 microM EPA). In contrast, InsP formation in response to guanine nucleotide-binding protein stimulation was not affected by EPA. ANG II receptor binding to membranes prepared from EPA-loaded VSMC was 18% lower than binding in membranes from sham-loaded cells. In other studies, VSMC were exposed acutely to FA to avoid cellular incorporation. Exposure to all FA resulted in concentration-dependent reductions in ANG II binding and ANG II-stimulated InsP formation; binding affinity was reduced without changes in receptor density. We conclude that ANG II-stimulated InsP formation is modestly and selectively inhibited by EPA incorporation and more profoundly inhibited by acute exposure to many FA via interference with ANG II receptor binding.

The milk-alkali syndrome in pregnancy. Case report.

We present a case of the milk-alkali syndrome occurring in pregnancy, an association not described in the medical literature. Ingestion of calcium carbonate and calcium-containing food was precipitated by the hyperemesis of pregnancy. Complications--hypercalcemia, dehydration, renal insufficiency, and pancreatitis--resolved within days. Here, the milk-alkali syndrome was the cause of the hypercalcemia of pregnancy. This case illustrates a rare cause of and complications from the milk-alkali syndrome.