Alpha2-adrenoceptors enhance angiotensin II-induced renal vasoconstriction: role for NADPH oxidase and RhoA.

Alpha(2)-adrenoceptors potentiate renal vascular responses to angiotensin II via coincident signaling at phospholipase C. This leads to increased activation of the phospholipase C/protein kinase C/c-src pathway. Studies suggest that c-src activates the reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase/superoxide system, and reactive oxygen species stimulate the RhoA/Rho kinase pathway. Therefore, we hypothesized that NADPH oxidase/superoxide and RhoA/Rho kinase are downstream components of the signal transduction pathway that mediate the interaction between alpha(2)-adrenoceptors and angiotensin II on renal vascular resistance. In rat kidneys, both in vivo and in vitro, intrarenal infusions of angiotensin II increased renal vascular resistance, and UK14,304 (alpha(2)-adrenoceptor agonist) enhanced this response. Intrarenal Tempol (superoxide dismutase mimetic) or Y27632 (Rho kinase inhibitor) abolished the interaction between UK14,304 and angiotensin II both in vivo and in vitro. The interaction was also blocked by inhibitors of NADPH oxidase (in vivo using chronic gp91ds-tat administration and in vitro with diphenyleneiodonium). In cultured preglomerular vascular smooth muscle cells, UK14,304 enhanced angiotensin II-induced intracellular superoxide (2-hydroxyethidium production) and potentiated activation of RhoA (Western blot of activated RhoA bound to the binding domain of rhotekin). The interaction between angiotensin II and UK14,304 on superoxide generation and RhoA activation was blocked by inhibitors of phospholipase C (U73312), protein kinase C (GF109203X), c-src (PP1), NADPH oxidase (diphenyleneiodonium), or superoxide (Tempol). We conclude that NADPH oxidase/superoxide and RhoA/Rho kinase are involved in the interaction between alpha(2)-adrenoceptors and angiotensin II on renal vascular resistance by mediating signaling events downstream of the phospholipase C/protein kinase C/c-src pathway.

cAMP-adenosine pathway in the proximal tubule.

The "extracellular cAMP-adenosine pathway" refers to the conversion of cAMP to AMP by ecto-phosphodiesterase, followed by metabolism of AMP to adenosine by ecto-5'-nucleotidase, with all the steps occurring in the extracellular compartment. This study investigated whether the extracellular cAMP-adenosine pathway exists in proximal tubules. Freshly isolated proximal tubules rapidly converted basolaterally administered cAMP to AMP and adenosine. Proximal tubular cells in culture (first passage) rapidly converted apically administered cAMP to AMP and adenosine. In both freshly isolated proximal tubules and cultured proximal tubular cells, conversion of cAMP to AMP and adenosine was affected by a broad-spectrum phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine), an ecto-phosphodiesterase inhibitor (1,3-dipropyl-8-p-sulfophenylxanthine), and a blocker of ecto-5'-nucleotidase (alpha,beta-methyleneadenosine-5'-diphosphate) in a manner consistent with exogenous cAMP being processed by the extracellular cAMP-adenosine pathway. In cultured proximal tubular cells, but not freshly isolated proximal tubules, stimulation of adenylyl cyclase increased extracellular concentrations of cAMP, AMP, and adenosine plus inosine, and these changes were also modulated by the inhibitors in a manner consistent with the extracellular cAMP-adenosine pathway. Conversion of renal interstitial (basolateral) cAMP and AMP to adenosine in vivo was shown by microdialysis coupled with ion trap mass spectrometry. Western blot analysis showed A1, A2A, and A3 receptors on both apical and basolateral proximal tubular membranes, with A1 and A2A receptors more highly expressed on basolateral compared with apical membranes. We conclude that cAMP that reaches either the apical or basolateral membranes of proximal tubular cells is converted in part to adenosine that has ready access to adenosine receptors.

Pancreatic polypeptide-fold peptide receptors and angiotensin II-induced renal vasoconstriction.

The Gi pathway augments renal vasoconstriction induced by angiotensin II in spontaneously hypertensive but not normotensive Wistar-Kyoto rats. Because the Gi-coupled pancreatic polypeptide (PP)-fold peptide receptors Y1 and Y2 are expressed in kidneys and are activated by endogenous PP-fold peptides, we tested the hypothesis that these receptors regulate angiotensin II-induced renal vasoconstriction in kidneys from hypertensive but not normotensive rats. A selective Y1-receptor agonist [(Leu31,Pro34)-neuropeptide Y; 6 to 10 nmol/L] greatly potentiated angiotensin II-induced changes in perfusion pressure in isolated, perfused kidneys from hypertensive but not normotensive rats. A selective Y2-receptor agonist (peptide YY(3-36); 6 nM) only slightly potentiated angiotensin II-induced renal vasoconstriction and only in kidneys from hypertensive rats. Neither the Y1-receptor nor the Y2-receptor agonist increased basal perfusion pressure. BIBP3226 (1 micromol/L, highly selective Y1-receptor antagonist) and BIIE0246 (1 micromol/L, highly selective Y2-receptor antagonist) completely abolished potentiation by (Leu31,Pro34)-neuropeptide Y and peptide YY(3-36), respectively. Y1-receptor and Y2-receptor mRNA and protein levels were expressed in renal microvessels and whole kidneys, but the abundance was similar in kidneys from hypertensive and normotensive rats. Both Y1-receptor-induced and Y2-receptor-induced potentiation of angiotensin II-mediated renal vasoconstriction was completely abolished by pretreatment with pertussis toxin (30 microg/kg IV, blocks Gi proteins). These data indicate that, in kidneys from genetically hypertensive but not normotensive rats, Y1-receptor activation markedly enhances angiotensin II-mediated renal vasoconstriction by a mechanism involving Gi. Although Y2 receptors can also potentiate angiotensin II-mediated renal vasoconstriction via Gi, the effect is modest compared with Y1 receptors. These findings may have important implications for the etiology of genetic hypertension.

Mechanism of the vascular angiotensin II/alpha2-adrenoceptor interaction.

alpha(2)-Adrenoceptors potentiate vascular responses to angiotensin II. The goal of this study was to test the hypothesis that the phospholipase C (PLC)/protein kinase C (PKC)/c-src/phosphatidylinositol 3-kinase (PI3K) pathway contributes to the vascular angiotensin II/alpha(2)-adrenoceptor interaction. In rats in vivo, intrarenal infusions of angiotensin II (10 ng/kg/min) increased renal vascular resistance by 5.8 +/- 0.5 units, and this response was enhanced (p < 0.05) to 9.1 +/- 1.2 units by UK-14,304 [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine; 3 microg/kg/min; alpha(2)-adrenoceptor agonist]. Intrarenal infusions of U-73122 [1-[6-[[(17beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]-hexyl]-1H-pyrrole-2,5-dione; 3 microg/min; PLC inhibitor], GF109203X [bisindolylmaleimide I; 10 microg/min; PKC inhibitor], CGP77675 [1-(2-{4-[4-amino-5-(3-methoxyphenyl)pyrrolo[2,3-d]pyrimidin-7-yl]phenyl}ethyl)piperidin-4-ol; 5 microg/min; c-src inhibitor], and wortmannin (1 microg/min; PI3K inhibitor) abolished the angiotensin II/alpha(2)-adrenoceptor interaction. In isolated perfused rat kidneys, angiotensin II (0.3, 1, and 3 nM) increased perfusion pressure (by 15 +/- 8, 39 +/- 4, and 93 +/- 9 mm Hg, respectively), and UK-14,304 (1 microM) potentiated these responses (to 36 +/- 4, 67 +/- 7, and 135 +/- 17 mm Hg, respectively). This angiotensin II/alpha(2)-adrenoceptor interaction was abolished by U-73122 (10 microM), GF109203X (3 microM), CGP77675 (5 microM), and wortmannin (0.2 microM). Preglomerular microvascular smooth muscle cells expressed phospholipase (PLC)-beta(2), PLC-beta(3), c-src, phospho(tyrosine 416)-c-src, and PI3K. In these cells, angiotensin II (0.1 microM) and UK-14,304 (1 microM) per se did not increase phospho-c-src; however, the combination of angiotensin II plus UK-14,304 doubled phospho-c-src, and this interaction was abolished by U-73122 (10 microM) and GF109203X (3 microM). In conclusion, the PLC/PKC/c-src/PI3K pathway may contribute importantly to the interaction between alpha(2)-adrenoceptors and angiotensin II on renal vascular resistance.

Enhanced activation of RhoA by angiotensin II in SHR preglomerular microvascular smooth muscle cells.

Angiotensin II causes a greater renal vasoconstriction in spontaneously hypertensive rats (SHR) than in normotensive Wistar Kyoto rats (WKY), and alpha2-adrenoceptor agonists potentiate angiotensin II-induced renal vasoconstriction more in SHR. Because angiotensin II activates RhoA, and RhoA contributes to vasoconstriction, we tested the hypothesis that the ability of angiotensin II to stimulate RhoA in preglomerular vascular smooth muscle cells and the ability of alpha2-adrenoceptor activation to potentiate this response are augmented in cells from SHR. In SHR preglomerular vascular smooth muscle cells, angiotensin II (1 micromol/L) greatly stimulated RhoA activity, and this effect was markedly potentiated by UK 14,304 (1 micromol/L; alpha2-adrenoceptor agonist) (fold increase from vehicle-treated cells: 9.0 +/- 2, 0.8 +/- 0.2, and 13.6 +/- 3.2 in cells treated with angiotensin II, UK 14,304, and angiotensin II + UK 14,304, respectively). In contrast, in WKY cells, angiotensin II only mildly activated RhoA (2.0 +/- 0.50), and this response was not enhanced by UK 14,304. The expression of Galpha12 and Galpha13, G-proteins thought to link G-protein-coupled receptors to RhoA, was not increased in SHR cells. We conclude that angiotensin II-induced activation of RhoA is much more robust in the preglomerular microcirculation of SHR compared with WKY and that this may contribute to the etiology of genetic hypertension.

Adenosine biosynthesis in the collecting duct.

Adenosine regulates tubular transport in collecting ducts (CDs); however, the sources of adenosine that modulate ion transport in CDs are unknown. The extracellular cAMP-adenosine pathway refers to the conversion of cAMP to AMP by ectophosphodiesterase, followed by metabolism of AMP to adenosine by ecto-5'-nucleotidase, with all steps occurring in the extracellular compartment. The goal of this study was to assess whether the extracellular cAMP-adenosine pathway exists in CDs. Studies were conducted in both freshly isolated CDs and in CD cells in culture (first passage) that were derived from isolated CDs. Purity of CDs was confirmed by microscopy, by Western blotting for aquaporin-1, aquaporin-2, bumetanide-sensitive cotransporter type 1, and thiazide-sensitive cotransporter; and by reverse transcription-polymerase chain reaction for adenosine receptors. Both freshly isolated CDs and CD cells in culture converted exogenous cAMP to AMP and adenosine. In both freshly isolated CDs and CD cells in culture, conversion of cAMP to AMP and adenosine was affected by a broad-spectrum phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine), an ectophosphodiesterase inhibitor (1,3-dipropyl-8-p-sulfophenylxanthine), and a blocker of ecto-5'-nucleotidase (alpha,beta-methylene-adenosine-5'-diphosphate) in a manner consistent with exogenous cAMP being processed by the extracellular cAMP-adenosine pathway. In CD cells in culture, stimulation of adenylyl cyclase increased extracellular concentrations of cAMP, AMP, and adenosine, and these changes were also modulated by the aforementioned inhibitors in a manner consistent with the extracellular cAMP-adenosine pathway. In conclusion, the extracellular cAMP-adenosine pathway is an important source of adenosine in CDs.

alpha 2-Adrenoceptors potentiate angiotensin II- and vasopressin-induced renal vasoconstriction in spontaneously hypertensive rats.

Hypertension in spontaneously hypertensive rats (SHRs) is due in part to enhanced effects of vasoactive peptides on the renal vasculature. We hypothesize that the G(i) signal transduction pathway enhances renovascular responses to vasoactive peptides in SHRs more so than in normotensive Wistar-Kyoto (WKY) rats. To test this hypothesis, we examined in isolated perfused kidneys from SHRs and WKY rats the renovascular responses (assessed as changes in renal perfusion pressure in mm Hg) to angiotensin II (10 nM) and vasopressin (3 nM) in the presence and absence of UK-14,304 [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine; an agonist that selectively activates the G(i) pathway by stimulating alpha(2)-adrenoceptors]. In SHR, but not WKY, kidneys, UK-14,304 (10 nM) enhanced (P < 0.05) renovascular responses to angiotensin II (control WKY, 43 +/- 6; UK-14,304-treated WKY, 52 +/- 19; control SHR, 66 +/- 17; UK-14,304-treated SHR, 125 +/- 16) and vasopressin (control WKY, 42 +/- 17; UK-14,304-treated WKY, 36 +/- 11; control SHR, 16 +/- 8; UK-14,304-treated SHR, 83 +/- 17). Pretreatment of SHRs with pertussis toxin (30 microg/kg, intravenously, 3-4 days before study) to inactivate G(i) blocked the effects of UK-14,304. Western blot analysis of receptor expression in whole kidney and preglomerular microvessels revealed similar levels of expression of AT(1), V(1a), and alpha(2A) receptors in SHRs compared with WKY rats. We conclude that activation of alpha(2)-adrenoceptors selectively enhances renovascular responses to angiotensin II and vasopressin in SHRs via an enhanced cross talk between the G(i) signal transduction pathway and signal transduction pathways activated by angiotensin II and vasopressin.

Expression of adenosine receptors in the preglomerular microcirculation.

The purpose of this study was to systematically investigate the abundance of each of the adenosine receptor subtypes in the preglomerular microcirculation vs. other vascular segments and vs. the renal cortex and medulla. Rat preglomerular microvessels (PGMVs) were isolated by iron oxide loading followed by magnetic separation. For comparison, mesenteric microvessels, segments of the aorta (thoracic, middle abdominal, and lower abdominal), renal cortex, and renal medulla were obtained by dissection. Adenosine receptor protein and mRNA expression were examined by Western blotting, Northern blotting, and RT-PCR. Our results indicate that compared with other vascular segments and renal tissues, A1 and A2B receptor protein and mRNA are abundantly expressed in the preglomerular microcirculation, whereas A2A and A3 receptor protein and mRNA are barely detectable or undetectable in PGMVs. We conclude that, relative to other vascular and renal tissues, A1 and A2B receptors are well expressed in PGMVs, whereas A2A and A3 receptors are notably deficient. Thus A1 and A2B receptors, but not A2A or A3 receptors, may importantly regulate the preglomerular microcirculation.