Mechanism of hyperreninemia in the potassium-depleted rat.

Although dietary potassium deficiency (KD) results in an increase in plasma renin activity (PRA), the mechanism of this effect has not been elucidated. In the present study, isolated kidneys from normal rats or from rats made KD by diet were perfused at constant pressure (120 mm Hg) with a Krebs-Ringer-Bicarbonate medium containing albumin. KD led to an increase in PRA (3.6 vs. 1.1 ng angiotensin I ml per h, P less than 0.01), which was associated with a decrease in macula densa (MD) fluid delivery as estimated by urine flow (70 vs. 166 microliters/min per g, P less than 0.005), and an increase in renal vascular resistance (RVR) as perfusion flow rate was decreased from 34 to 24 ml/min per g, P less than 0.005. The increase in PRA was independent of the MD because PRA could not be suppressed when macula densa delivery was increased by perfusing KD kidneys with hypooncotic albumin. Moreover, when kidneys were made nonfiltering by perfusing with hyperconcotic albumin, PRA remained increased in KD kidneys (8.1 vs. 3.5 ng angiotensin I ml per h, P less than 0.01) despite the absence of MD delivery. Because the increase in PRA in both filtering and nonfiltering KD kidneys was associated with an increase in RVR, filtering and nonfiltering kidneys were perfused with the vasodilator papaverine. Despite lower tissue K levels in KD kidneys (278 vs. 357 mu eq/g, P less than 0.01), RVR and PRA were normalized in both filtering and nonfiltering KD kidneys perfused with papaverine. In conclusion, PRA is increased in the KD isolated perfused kidney. This increase occurs independently of both the MD and of tissue K levels and is mediated by the renal vascular receptor.

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.

Mechanism of decreased vascular reactivity to angiotensin II in conscious, potassium-deficient rats.

Chronic potassium deficiency in the rat results in a decrease in the pressor sensitivity to exogenous angiotensin II (AII). To define the mechanism of this resistance to AII, studies were performed in conscious rats after 14-21 d of dietary potassium deficiency. The pressor response to graded doses of AII was 50% less in potassium-deficient than control animals. In contrast, the pressor response to graded doses of norepinephrine was preserved in potassium-deficient rats; therefore, the decreased response to AII was not due to a generalized defect in vascular reactivity. Pretreatment with either the converting enzyme inhibitor, teprotide, or the prostaglandin synthesis inhibitor, indomethacin, failed to normalize the response to AII. Thus, neither prior receptor occupancy with endogenous AII nor the presence of vasodilatory prostaglandins caused the decreased AII response in potassium deficiency. Since the pressor response to AII involves angiotensin interaction with its vascular receptor, binding studies of mesenteric artery and uterine smooth muscle AII receptors were performed. Scatchard analysis showed that potassium deficiency resulted in a decrease in binding affinity (50% increase in Kd) in both uterine (6.00 vs. 3.82 nM; P less than 0.05) and vascular (1.39 vs. 0.973 nM; P less than 0.005) smooth muscle. Furthermore, despite increased circulating AII, there was an increase in AII receptor number in potassium-deficient uterine (308 vs. 147 fmol/mg protein; P less than 0.005) and vascular (470 vs. 316 fmol/mg protein; 0.05 less than P less than 0.1) smooth muscle. Although potassium deficiency resulted in alterations in receptor-binding parameters, the changes in binding affinity and number were directionally opposite, so that in potassium deficiency there was either no change or an increase in total AII binding. We conclude that the decrease in angiotensin pressor sensitivity in potassium-deficient rats is mediated by a postreceptor defect since it occurs subsequent to the binding of AII to its vascular smooth muscle receptor.

Cytoskeleton-dependent endocytosis is required for apical type 1 angiotensin II receptor-mediated phospholipase C activation in cultured rat proximal tubule cells.

Renal proximal tubule sodium reabsorption is enhanced by apical or basolateral angiotensin II (AII). Although AII activates phospholipase C (PLC) in other tissues, AII coupling to PLC on either apical or basolateral surfaces of proximal tubule cells is unclear. To determine if AII causes PLC activation, and the differences between apical and basolateral AII receptor function, receptors were unilaterally activated in rat proximal tubule cells cultured on permeable, collagen-coated supports. Apical AII incubation resulted in concentration- and time-dependent inositol trisphosphate (IP3) formation. Basolateral AII caused greater IP3 responses. Apical AII-induced IP3 generation was inhibited by DuP 753, suggesting that the type 1 AII receptor subtype mediated proximal tubule PLC activation. Apical AII signaling did not result from paracellular ligand leak to basolateral receptors since AII-induced PLC activation occurred when basolateral AII receptors were occupied by Sar-Leu AII or DuP 753. Inhibition of endocytosis with phenylarsine oxide prevented apical (but not basolateral) AII-induced IP3 formation. Cytoskeletal disruption with colchicine or cytochalasin D also prevented apical AII-induced IP3 generation. These results demonstrate that in cultured rat proximal tubule cells, AII is coupled to PLC via type 1 AII receptors and cytoskeleton-dependent endocytosis is required for apical (but not basolateral) AII receptor-mediated PLC activation.

On the mechanism of polyuria in potassium depletion. The role of polydipsia.

The association of potassium (K) depletion with polyuria and a concentrating defect is established, but the extent to which these defects could be secondary to an effect of low K on water intake has not been systematically investigated. To determine whether hypokalemia has a primary effect to increase thirst and whether any resultant polyuria and polydipsia contribute to the concentrating defect, we studied three groups of rats kept in metabolic cages for 15 days. The groups were set up as follows: group 1, normal diets and ad lib. fluids (n = 12); group 2, K-deficient diet on ad lib. fluids (n = 12); and group 3, K-deficient diet and fluid intake matched to group 1 (n = 14). Daily urine flow and urinary osmolality of groups 1 and 3 were not significantly different throughout the study. In contrast, as of day 6, group 2 rats consistently had a higher fluid intake (P < 0.0025), higher urine flow (P < 0.001), and lower urinary osmolality (P < 0.001) than the other two groups. These alterations in fluid intake and urine flow preceded a defect in maximal concentrating ability. On day 7, maximal urinary osmolality was 2,599+/-138 msmol/kg in rats on K-deficient intake and 2,567+/-142 msmol/kg in controls. To determine whether this primary polydipsia is itself responsible for the development of the concentrating defect, the three groups of rats were dehydrated on day 15. Despite different levels of fluid intake, maximal urinary osmolality was impaired equally in groups 2 and 3 (1,703 and 1,511 msmol/kg, respectively), as compared to rats in group 1 (2,414 msmol/kg), P < 0.001. We therefore conclude that K depletion stimulates thirst, and the resultant increase in water intake is largely responsible for the observed polyuria. After 15 days of a K-deficient diet, the impaired maximal urinary concentration in hypokalemia, however, was not related to increased water intake, since fluid restriction did not abolish the renal concentrating defect.

Potassium depletion ameliorates hypertension in spontaneously hypertensive rats.

The hemodynamic effect of moderate K+ depletion in hypertension is unknown. Since severe K+ depletion reduces systemic vascular resistance in normotensive rats, we determined the effect of K+ depletion on the natural history of hypertension in spontaneously hypertensive rats (SHR). Wistar-Kyoto rats (WKY) and SHR were fed a K+-replete, a moderately K+-depleted, or a severely K+-depleted diet. After 6 weeks, systemic vascular resistance was reduced by 25% in WKY on the severely K+-depleted diet while mean arterial pressure and systemic vascular resistance were comparable in WKY on the other two diets. In SHR on the severely K+-depleted diet for 6 weeks, muscle K+ was reduced by 23% and growth rate by 65%. In SHR on the moderately K+-depleted diet, growth rate was reduced by 23% after 3 weeks. By 6 weeks, however, muscle K+ was reduced by 5 to 6% and growth rate was comparable to that in SHR receiving the K+-replete diet. The administration of either K+-depleted diet prevented the development of hypertension (systolic blood pressure: severely depleted, 116 +/- 4; moderately depleted, 122 +/- 3; K+-replete, 155 +/- 5 mm Hg; p less than 0.001 compared with both K+-depleted groups) and reversed established hypertension (systolic blood pressure: severely depleted, 116 +/- 4; moderately depleted, 128 +/- 3; K+-replete, 171 +/- 5 mm Hg; p less than 0.001 compared with both K+-depleted groups). The protective effect of K+ depletion was mediated by a 40% reduction in systemic vascular resistance. These results suggest that K+ depletion has a potent antihypertensive effect in SHR.(ABSTRACT TRUNCATED AT 250 WORDS)

Parathyroid hormone/adenylate cyclase coupling in vascular smooth muscle cells.

Parathyroid hormone (PTH) has been implicated in hypertension, but PTH infusion results in vasodilation. PTH activates adenylate cyclase in vascular smooth muscle, but little is known about the factors that regulate PTH receptor/adenylate cyclase coupling in vascular cells. To characterize hormone-receptor signaling, we measured cyclic AMP levels in rat arterial smooth muscle cells in culture exposed to PTH (bovine 1-34). PTH yielded time- and concentration-dependent increases in cyclic AMP levels. Compared with isoproterenol, PTH was more potent, with a threshold at 2 x 10(-9) versus 5 x 10(-8) mol/L and half maximal responses at 10(-8) versus 2.4 x 10(-7) mol/L. PTH-induced increases in cyclic AMP were independent of extracellular calcium, cyclooxygenase metabolites, phospholipase C, and protein kinase C because PTH-induced increases in cyclic AMP were not prevented by variations in extracellular calcium, indomethacin, angiotensin II, vasopressin, and protein kinase C activators or inhibitors. PTH/adenylate cyclase coupling was G protein-dependent because increases in cyclic AMP were prevented by preincubation with cholera toxin but not with pertussis toxin. Prolonged exposure to PTH resulted in time- and concentration-dependent homologous desensitization of cyclic AMP responses. Desensitization occurred proximal to G protein/adenylate cyclase because after prolonged PTH, responses to forskolin and cholera toxin remained intact. Desensitization was independent of protein kinase A and receptor sequestration because cyclic AMP responses remained after prolonged exposure to forskolin and pretreatment with phenylarsine oxide, colchicine, and cytochalasin D. We conclude that in vascular smooth muscle cells, PTH is coupled to adenylate cyclase through a cholera toxin-sensitive G protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemodynamic effects of alterations in potassium.

Potassium is the major intracellular cation. Despite this fact, the systemic and renal hemodynamic effects of alterations in either serum K or in total body K are only partially understood. In isolated preparations acute K excess causes vasodilation while acute K deficiency results in vasoconstriction. Although chronic K excess may decrease arterial pressure in experimental models of hypertension, no definitive conclusions can be stated on the effect of K excess in hypertensive patients. In normotensive animals, chronic K depletion is associated with decreased systemic vascular resistance and increased renal vascular resistance. Although a number of studies have shown that K depletion ameliorates experimental hypertension, no definitive conclusions can be stated on the effect of K depletion in hypertensive patients. The vasodilatory effect of K depletion appears to be a direct effect on vascular smooth muscle since it is associated with an increase in total body Na as well as an increase in cardiac output and in renin ane arginine vasopressin levels. Although renin levels are increased in K deficient rats to a value comparable to na-depleted rats, angiotensin antagonism results in a substantially smaller decrease in arterial pressure than in Na-depleted rats (11 +/- 1.6 vs 24 +/- 3.4 mm Hg, p less than 0.01). This relative resistance to the pressor effect of angiotensin also results in a blunted pressor sensitivity to exogenous angiotensin II. Since changes in K balance appear to have a major effect on the control of hemodynamics, further studies are warranted to determine whether alterations in K balance would be useful in the treatment of hypertension.

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.

Dimethylthiourea prevents hydrogen peroxide and neutrophil mediated damage to lung endothelial cells in vitro and disappears in the process.

Dimethylthiourea (DMTU) progressively disappeared following reaction with increasing amounts of hydrogen peroxide (H2O2) in vitro. DMTU disappearance following reaction with H2O2 was inhibited by addition of catalase, but not aminotriazole-inactivated catalase (AMT-catalase), superoxide dismutase (SOD), mannitol, benzoate or dimethyl sulfoxide (DMSO) in vitro. By comparison, DMTU disappearance did not occur following addition of histamine, oleic acid, elastase, trypsin or leukotrienes in vitro. Addition of DMTU also decreased H2O2-mediated injury to bovine pulmonary artery endothelial cells (as reflected by LDH release) and DMTU disappeared according to both added amounts of H2O2 and corresponding degrees of injury. DMTU disappearance was also relatively specific for reaction with H2O2 in suspensions of endothelial cells where it was prevented by addition of catalase, but not AMT-catalase or SOD and did not occur following sonication or treatment with elastase, trypsin or leukotrienes. Addition of washed human erythrocytes (RBC) also prevented both H2O2 mediated injury and corresponding DMTU decreases in suspensions of endothelial cells. In addition, phorbol myristate acetate (PMA) and normal neutrophils, but not O2 metabolite deficient neutrophils from patients with chronic granulomatous disease (CGD), caused DMTU disappearance in vitro which was decreased by simultaneous addition of catalase, but not SOD, sodium benzoate or DMSO. Finally, addition of normal neutrophils (but not CGD neutrophils) and PMA caused DMTU disappearance and increased the concentrations of the stable prostacyclin derivative (PGF1 alpha) in supernatants of endothelial cell suspensions. In parallel, DMTU also decreased PMA and neutrophil-mediated PGF1 alpha increases in supernatants from endothelial cell monolayers. Our results indicate that DMTU can decrease H2O2 or neutrophil mediated injury to endothelial cells and that simultaneous measurement of DMTU disappearance can be used to improve assessment of the presence and toxicity of H2O2 as well as the H2O2 inactivating ability of scavengers, such as RBC, in biological systems.

Functional role of a novel cis-acting element (GAGA box) in human type-1 angiotensin II receptor gene transcription.

GH/growth factors have been shown to increase angiotensin type 1 receptor expression. In the present study we determined the cis-acting regulatory region controlling GH-induced transcription of the human type-1 angiotensin receptor (hAT(1)). In human proximal tubule cells transfected with a chloramphenicol acetyl transferase (CAT) reporter plasmid under the control of the hAT(1) promoter, GH induced CAT activity. Serial deletions of the hAT(1) promoter region indicated that an area between -314 bp and -70 bp upstream of the 5'-end of the cDNA sequence was essential for this activation to occur. Although sequence analysis identified putative multiple nuclear protein binding sites in this region, we determined that a 12 bp sequence (5'-GAGAGGGAGGAG-3', GAGA box) located between -161 bp and -149 bp was important for GH-mediated activation. Using mobility shift assays we demonstrated increased DNA binding activity to the labeled GAGA box in nuclear extracts treated with GH, suggesting this sequence is a GH response element. Southwestern analysis identified an 18 kDa GAGA box-binding protein (GAGA-BP). GH-induced activity of the GAGA-BP occurred within 2.5 min and reached a maximum at 5 min. Activation did not require de novo protein synthesis. Removal of the GAGA box abolished GH-induced transcription as well as basal transcription of the hAT(1) gene. Additional studies demonstrated that epidermal growth factor, platelet-derived growth factor and insulin activate the GAGA-BP, suggesting these growth factors can also regulate the transcription of the hAT(1) gene through the GAGA box. Our data show that the GAGA-BP acts as a trans-acting factor binding to the cis-acting regulatory element in the hAT(1) promoter, which is necessary for the basal and growth factor(s)-mediated transcriptional activation of the hAT(1) gene.

Water intoxication in a psychotic patient with normal renal water excretion.

A psychotic patient with hyponatremia and obtundation following short-term ingestion of large amounts of water was found to have maximally dilute urine, and underwent brisk diuresis until the serum osmolality returned to normal. This is the first report of normal renal diluting capacity as documented by measurement of urine osmolality in a water-intoxicated, schizophrenic patient, and demonstrates that normal renal diluting mechanisms may on occasion be overwhelmed by massive water ingestion.

The role of neutrophils in acute renal failure.

The role of neutrophils in acute renal failure is controversial. Acute renal failure can clearly occur in the absence of neutrophils. However, recent studies using specific neutrophil markers indicate that neutrophils accumulate in postischemic kidneys. Moreover, reperfusion of ischemic kidneys with neutrophils worsens ischemic injury and causes kidney neutrophil retention. Neutrophil retention is dependent on the state of neutrophil activation and the duration of renal ischemia. This interaction could account for the high frequency of acute renal failure in conditions associated with prolonged prerenal asotemia and neutrophil priming such as the adult respiratory distress syndrome, or sepsis. Neutrophil retention is mediated by interaction of neutrophil integrins and endothelial cell ICAM-1 because maneuvers reducing the expression and/or function of these adhesion molecules is protective in experimental models of ischemia. Nitric oxide is a key modulator of neutrophil worsening of ischemic injury because maneuvers that decrease nitric oxide production worsen and those which increase nitric oxide protect ischemic kidneys from neutrophil effects. The clinical significance of neutrophils may relate to the observation that bioincompatible membranes activate complement, and retard recovery from acute renal failure. In conclusion, neutrophils are an important contributor to ischemic acute renal failure. It remains to be determined whether decreasing neutrophil function accelerates recovery in acute renal failure.

Interleukin-1 treatment increases neutrophils but not antioxidant enzyme activity or resistance to ischemia-reperfusion injury in rat kidneys.

Hearts from rats treated with interleukin-1 (IL-1) intraperitoneally developed a rapid (6 h after IL-1), transient increase in neutrophils, tissue hydrogen peroxide (H2O2), and oxidized glutathione (GSSG) levels, and a subsequent (36 h after IL-1) increase in myocardial glucose-6-phosphate dehydrogenase (G6PD) activity and tolerance to ischemia-reperfusion. In the present investigation, we found that rats treated similarly with IL-1 had increased numbers of neutrophils in their kidneys, which were comparable to myocardial neutrophil increases, but did not develop increased renal tissue H2O2 or GSSG levels acutely (6 h after IL-1) or increased G6PD activity or resistance to ischemia-reperfusion injury later (36 h after IL-1). Our findings indicate that IL-1 treatment increased neutrophil accumulation in rat kidneys but did not increase oxidative stress, antioxidant enzyme activity, or resistance to ischemia-reperfusion injury. We conclude that organ-to-organ differences exist with respect to IL-1-induced tolerance.

Potassium: weighing the evidence for supplementation.

Changes in potassium levels clearly have hemodynamic significance. In mechanistic terms, they affect the transmembrane potential of vascular smooth muscle cells. They also influence the levels and activity of pressor hormones and of intracellular messengers involved in vasoconstriction. Furthermore, they alter the body's handling of sodium. As the net result, perhaps, of these phenomena, chronic supplementation of dietary potassium is associated with a small but appreciable decline in blood pressure. In humans, the effect, which could be predicted epidemiologically, has been demonstrated in studies of potassium administration in hypertensive patients. In experimental animals, the effect is most pronounced in salt-sensitive models of hypertension. The studies done to date do not permit firm recommendations about modification of dietary potassium content for hypertensive patients. However, in certain clinical settings, potassium repletion even for mildly depressed levels is vitally important, and in other circumstances, excess potassium clearly is dangerous. Still, indications are emerging that potassium may be valuable in preventing renal damage and stroke, quite apart from any effect on hypertension itself. Continued investigation will be of great value in the effort to arrive at a firm understanding of the precise roles that potassium may play in the treatment of hypertension or the prevention of its sequelae.

Role of prostaglandins in renin secretion in the isolated kidney.

Prostaglandins (PG) stimulate renin secretion through a mechanism that does not require activation of the intrarenal vascular, macula densa (MD), or beta-adrenergic receptors. In the present study the isolated perfused rat kidney was used to study the role of PG as a mediator of renin secretion when extracellular calcium was decreased and after activation of each of the intrarenal receptors. A decrease in extracellular calcium resulted in an increase in renin (from 2.1 to 4.5 ng ANG I/ml, P less than 0.01) and a decrease in PGE2 excretion (from 102 to 44 pg X min-1 X g-1, P less than 0.01). PG synthesis inhibition with indomethacin did not attenuate the increase in renin secretion. Following beta-receptor stimulation with isoproterenol, there was an increase in renin (from 2.1 to 6.6 ng ANG I/ml, P less than 0.01) not associated with changes in PGE2 excretion and not prevented by PG inhibition. When the isolated vascular receptor was stimulated by perfusing either filtering or nonfiltering kidneys below the autoregulatory range of pressure, both renin and PGE2 production were increased, and the increases were prevented by PG inhibition. The MD was activated by three methods: eliminating distal nephron fluid delivery by perfusing with hyperoncotic albumin; perfusing at 60 mmHg in the presence of papaverine; and limiting chloride transport by partially replacing chloride with nitrate in the perfusate. In each circumstance renin and PGE2 production were increased and the increase was prevented by PG inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Beta-adrenergic receptor function in rat proximal tubule epithelial cells in culture.

The adrenergic system is important in regulating proximal tubule sodium reabsorption. Although alpha-adrenergic receptors have been identified in proximal tubules, the presence and function of beta-adrenergic receptors (BAR) in proximal tubules is less certain. The purpose of our study was to determine whether functional BAR are present on apical or basolateral surfaces of proximal tubule epithelial cells (PTEC) of rat kidney. We specifically focused on BAR coupling to adenylate cyclase and on differences between requirements for apical and basolateral receptor coupling to adenylate cyclase. To determine BAR expression and function, primary cultures of rat PTECs were grown on permeable supports. Scatchard analysis of 125I-labeled cyanopindolol binding revealed a single class of receptors on both apical and basolateral surfaces. Apical isoproterenol (ISO) resulted in time- and concentration-dependent increases in adenosine 3',5'-cyclic monophosphate (cAMP) that were 50% of responses after basolateral ISO. Apical BAR-cAMP coupling was mediated by B1-adrenergic receptors (B1AR), since apical cAMP responses were abrogated with apical (but not basolateral) B1 but not B2 antagonists. Apical B1AR required endocytosis prior to adenylate cyclase activation, since increases in cAMP were prevented by phenylarsine oxide or colchicine. B1AR-adenylate cyclase coupling was independent of intra- or extracellular calcium, cyclooxygenase metabolites, and protein kinase C (PKC) and dependent on Gs guanine nucleotide regulatory protein. Prolonged exposure to ISO resulted in time- and concentration-dependent homologous desensitization of cAMP responses. Desensitization was independent of receptor sequestration, PKA, or PKC. We conclude the following: B1AR are present on both apical and basolateral surfaces of rat PTECs.(ABSTRACT TRUNCATED AT 250 WORDS)