We have previously reported that dopamine-1 receptor-mediated activation of phospholipase C is diminished in renal cortical slices of adult spontaneously hypertensive rats. To determine the potential consequences of this phenomenon, we performed the present studies in which renal proximal tubule suspensions obtained from spontaneously hypertensive and Wistar-Kyoto rats of 10-12 weeks of age were used. The tubule suspensions were incubated with dopamine in the presence or absence of dopamine receptor antagonists, and sodium, potassium adenosine trisphosphatase (sodium pump) activity was measured as the ouabain-sensitive adenosine trisphosphate hydrolysis. We found that dopamine produced a concentration-related inhibition of sodium pump activity in the normotensive rats but not in the hypertensive rats. Dopamine-induced inhibition of sodium pump activity in the normotensive rats was abolished by the phospholipase C inhibitor U-73122 or the protein kinase C inhibitor sphingosine, suggesting the involvement of a phospholipase C-coupled protein kinase C pathway in this response. Dopamine-induced inhibition in the normotensive rats was attenuated by the dopamine-1 receptor antagonist SCH 23390 but not by the dopamine-2 receptor antagonist domperidone. To identify possible sites of defect in dopamine-1 receptor-coupled signaling pathways in the hypertensive rats, we incubated the proximal tubules with phorbol 12,13-dibutyrate or the synthetic diacylglycerol analogue l-oleoyI-2-acetyl-rac-glycerol. The results showed that both compounds inhibited sodium pump activity as effectively in the hypertensive as in the normotensive rats, suggesting that the protein kinase C-coupled sodium pump pathway was not defective in the hypertensive animals. Failure of dopamine to inhibit sodium pump activity in the hypertensive rats could not be due to a defective dopamine-1 receptor adenylate cyclase coupling, because dopamine was still unable to inhibit sodium pump activity in the presence of dibutyryl cyclic adenosine monophosphate or forskolin. These results show that dopamine failed to inhibit sodium pump activity in the proximal tubules of adult hypertensive rats, which may be mainly due to a defect in the dopamine-1 receptor-mediated signal transduction pathway. The site of this defect is most likely proximal to the activation of protein kinase C and may involve a defect in the dopamine-1 receptor phospholipase C coupling process. (Hypertension 1993;21:364-372) KEY WORDS • receptors, dopamine • dopamine • sodium • phospholipase C • adenosine trisphosphatase, sodium, potassium A bnormal renal sodium handling has been known / \ to be one of the major factors involved in the -Z A . initiation and maintenance of high blood pressure in several models of hypertension, including genetic hypertension. 1 -2 Endogenous kidney dopamine plays an important role in regulation of renal sodium excretion, so it has been proposed that impaired renal sodium handling in spontaneously hypertensive rats (SHR) may partly be due to a malfunction or a d...

Northern blotting studies have demonstrated mRNA for the serotonin 5-HT1A receptor in human neonatal kidney (B. K. Kobilka, T. Frielle, S. Collins, T. Yang-Feng, T. S. Kobilka, U. Francke, R. J. Lefkowitz, and M. G. Caron. Nature Lond. 329: 75-79, 1987). To confirm expression of receptor protein in kidney, we raised antibodies to two peptides derived from the third intracellular loop of the human 5-HT1A receptor. Specific immunoglobulin G (IgG) was purified sequentially on protein A-Sepharose and peptide-Affigel 10 columns. Each IgG was able to: 1) quantitatively immunoprecipitate [3H]8-OH-2-(di-n-propylamino)1,2,3,4-tetrahydronaphthalene ([3H]8-OH-DPAT)-labeled human and rat receptors; 2) immunoblot a new protein in cells transfected with human 5-HT1A receptor DNA; and 3) immunoautoradiographically label areas of rat brain (frontal cortex, hippocampus, and lateral septum) in a highly characteristic pattern similar to that labeled by 125I-Bolton-Hunter-8-methoxy-2-(N-propyl-N-propylamino)Tetralin, a specific 5-HT1A receptor autoradiography ligand. By use of a light microscopic immunoperoxidase labeling technique, incubation of each IgG antibody with sections of rat and human kidney demonstrated an identical pattern of immunoreactivity. Specific labeling of basolateral plasma membranes was detected throughout medullary and cortical thick ascending limbs (TAL), in distal convoluted tubules (DCT), in connecting tubule cells of the connecting tubule, and in principal cells of the initial collecting tubule. There was no labeling in the inner medulla, glomeruli, or blood vessels. The labeling was blocked by preincubation with the corresponding peptide, but not with noncorresponding peptide or carrier protein. There was no labeling with preimmune IgG. Electron microscopic immunoperoxidase labeling confirmed the specific localization of the IgG antibody along the basolateral plasma membrane in all positively staining cells in rat kidney. Radioligand binding studies with the specific 5-HT1A receptor ligand [3H]8-OH-DPAT confirmed the presence of 5-HT1A receptor binding sites in bulk-isolated rat medullary TAL. These studies provide the first evidence that the 5-HT1A receptor is expressed on the basolateral surface of TAL and DCT cells of human and rat kidney. The specific localization to these cells suggests a possible role for the 5-HT1A receptor in the regulation of salt and water transport in mammalian kidney.

Previously, we demonstrated that adenosine (Ado) was released by the medullary thick ascending limb (MTAL) during hypoxia. The present experiments were designed to examine the effects of Ado and adenosine analogues on net chloride (JCl) and bicarbonate (JHCO3) absorption by the isolated, perfused MTAL of the rat. Ado, 10 nM, in the presence or absence of arginine vasopressin (AVP, 10(-10) M) reduced JCl by 50%. The inhibition of Ado was reproduced with the selective A1 agonist, N-6-phenylisopropyladenine (2 nM), and was reversed by 8-cyclopentyl-1,3-dipropylxanthine, an A1-receptor antagonist. Thus the inhibition of JCl is likely mediated through A1 receptors. In contrast, Ado had no effect on (JHCO3) either in the presence or absence of AVP. Ado also had no influence on the effect of AVP to inhibit JHCO3. The lack of effect on JHCO3 suggests that the inhibition of JCl by Ado is unlikely to be mediated through changes in cellular adenosine 3',5'-cyclic monophosphate. These results support the hypothesis that Ado released into the renal medulla during hypoxia may protect the MTAL from ischemic injury by directly inhibiting NaCl absorption and reducing transport-related oxygen consumption.

We studied the effects of norepinephrine on solute transport and oxidative metabolism in proximal tubules. Norepinephrine (10(-6) M) in the bath stimulated fluid absorption (Jv) by proximal convoluted tubules from 0.76 +/- 0.10 to 1.01 +/- 0.11 nl X mm-1 X min-1 (P less than 0.001). Bicarbonate, chloride, and phosphate transport also increased in proportion to the increases in Jv. Norepinephrine increased ouabain-sensitive oxygen consumption (QO2) in suspensions of cortical tubules by 1.3 nmol X mg protein-1 X min-1 and had no effect on ouabain-insensitive QO2 or mitochondrial respiration. Na+-K+-ATPase activity in basolateral membranes prepared from cortical homogenates incubated with norepinephrine increased from 277.1 +/- 34.9 to 411.1 +/- 38.6 nmol Pi X mg protein-1 X min-1 (P less than 0.005). Norepinephrine also increased Na+-K+-ATPase activity of cortical homogenates by 72% but had no effect on Na+-K+-ATPase if added directly to purified basolateral membranes. These studies show that norepinephrine stimulates solute transport in the proximal tubule by increasing Na+-K+-ATPase activity indirectly through some component or components of the adrenergic receptor system.

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