Background-Nebivolol and carvedilol are third-generation ␤-adrenoreceptor antagonists, which unlike classic ␤-blockers, have additional endothelium-dependent vasodilating properties specifically related to microcirculation by a molecular mechanism that still remains unclear. We hypothesized that nebivolol and carvedilol stimulate NO release from microvascular endothelial cells by extracellular ATP, which is a well-established potent autocrine and paracrine signaling factor modulating a variety of cellular functions through the activation of P2-purinoceptors. Methods and Results-Contraction and relaxation of renal glomerular vasculature were measured by determination of intracapillary volume with [ 3 H]-inulin. Biologically active NO was measured with highly sensitive porphyrinic NO microsensors in a single glomerular endothelial cell (GEC). Extracellular ATP was measured by a luciferin-luciferase assay. Enzymatic degradation of extracellular ATP by apyrase and blockade of P2Y-purinoceptors by suramin or reactive blue 2 inhibited both ␤-blocker-induced glomerular vasorelaxations and ␤-blocker-stimulated NO release from GECs. Both ␤-blocker-induced vasorelaxations were in the micromolar concentration range identical to that required for the ␤-blocker stimulation of ATP and NO release from GECs. The maximum of NO release for nebivolol and carvedilol was very similar (188Ϯ14 and 226Ϯ17, respectively). Blockade of ATP release by a mechanosensitive ion channel blocker, Gd 3ϩ , inhibited the ␤-blocker-dependent release of ATP and NO from GECs. Conclusions-These results demonstrate for the first time that nebivolol and carvedilol induce relaxation of renal glomerular microvasculature through ATP efflux with consequent stimulation of P2Y-purinoceptor-mediated NO

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The relaxing effect of extracellular ATP on renal glomeruli has been investigated by applying ATP and its analogues to suspensions of angiotensin II‐precontracted rat renal glomeruli. Based on changes of glomerular [3H]inulin space (GIS) the relaxation of glomeruli was analysed in the presence of agonists: ATP, ADP, AMP, UTP, 2‐methylthio‐ATP (P2Y agonist), β,γ‐methylene‐ATP (P2X agonist) and adenosine. ATP, 2‐methylthio‐ATP, ADP and UTP induced concentration‐dependent relaxation whereas AMP, β,γ‐methylene‐ATP and adenosine had no effect. The rank order of relaxation potency was 2‐methylthio‐ATP > ATP > ADP > UTP. An inhibitor of constitutive nitric oxide synthase (NOS), Nω‐nitro‐L‐arginine (NNA) prevented the ATP‐induced increased accumulation of L‐citrulline and the relaxation effect of ATP. An inhibitor of the neuronal isoform of NOS, 7‐nitroindazole, had no effect on the relaxation effect of ATP. The relaxing effect of ATP was prevented in the presence of inhibitors of cyclic guanylyl cyclase: methylene blue (MB) and the more specific inhibitor 1H‐[1,2,4]oxadiazolo‐[4,3‐a]quinoxalin‐1‐one (ODQ). ATP stimulated an accumulation of cGMP that was diminished in the presence of MB. We indicated that extracellular ATP may relax the glomeruli via activation of P2Y receptors with the subsequent activation of the endothelial isoform of nitric oxide synthase and soluble guanylyl cyclase. We suggest that, based on the described mechanism, extracellular ATP may increase the filtration surface which, in turn, may influence the glomerular filtration rate.

It is possible that increased P2X7 receptor reactivity may be involved in the pathogenesis of diabetic nephropathy.

Purinoceptors (adrengeric receptors and P2 receptors) are expressed on the cellular components of the glomerular filtration barrier, and their activation may affect glomerular permeability to albumin, which may ultimately lead to albuminuria, a well-established risk factor for the progression of chronic kidney disease and development of cardiovascular diseases. We investigated the mechanisms underlying the in vitro and in vivo purinergic actions on glomerular filter permeability to albumin by measuring convectional albumin permeability (Palb) in a single isolated rat glomerulus based on the video microscopy method. Primary cultured rat podocytes were used for the analysis of Palb, cGMP accumulation, PKG-Iα dimerization, and immunofluorescence. In vitro, natural nucleotides (ATP, ADP, UTP, and UDP) and nonmetabolized ATP analogs (2-meSATP and ATP-γ-S) increased Palb in a time- and concentration-dependent manner. The effects were dependent on P2 receptor activation, nitric oxide synthase, and cytoplasmic guanylate cyclase. ATP analogs significantly increased Palb, cGMP accumulation, and subcortical actin reorganization in a PKG-dependent but nondimer-mediated route in cultured podocytes. In vivo, 2-meSATP and ATP-γ-S increased Palb but did not significantly affect urinary albumin excretion. Both agonists enhanced the clathrin-mediated endocytosis of albumin in podocytes. A product of adenine nucleotides hydrolysis, adenosine, increased the permeability of the glomerular barrier via adrenergic receptors in a dependent and independent manner. Our results suggest that the extracellular nucleotides that stimulate an increase of glomerular Palb involve nitric oxide synthase and cytoplasmic guanylate cyclase with actin reorganization in podocytes.

Activation of P2X receptors increased renal sodium and water excretion. Mechanistically, P2X agonists increased renal perfusion and inhibited sodium reabsorption via an Na-K-ATPase-dependent mechanism.

1 Diadenosine polyphosphates (P 1 ,P 3 -diadenosine triphosphate, Ap 3 A; P 1 ,P 4 -diadenosine tetraphosphate, Ap 4 A; and P 1 ,P 5 -diadenosine pentaphosphate, Ap 5 A) are vasoactive molecules. The experimental model of isolated rat renal glomeruli was used to investigate their effects on glomerular vasculature. We measured the changes of glomerular inulin space (GIS) as a marker of glomeruli contractility. 2 Ap 4 A and Ap 5 A induced concentration-and time-dependent reduction of GIS whereas Ap 3 A had no effect. The effects of Ap 4 A and Ap 5 A (both at 1 mM) were prevented by a nonselective P2 receptor antagonist, that is, suramin (10 mM) and P2Y receptor antagonist -reactive blue 2 (50 mM). However, the antagonist of P1 receptor, that is, theophylline (1 mM) and A 1 receptor 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 10 mM) did not affect the responses of glomeruli to Ap 4 A or Ap 5 A. 3 Ap 3 A, in contrast to Ap 4 A and Ap 5 A, prevented angiotensin II-induced reduction of GIS in a concentration-and time-dependent manner. This effect was partially prevented by suramin and markedly reduced by reactive blue 2 and the specific antagonist of P2Y 1 receptor -MRS 2179 (10 mM). However, theophylline and the specific antagonist of A 2 receptor -3,7-dimethyl-1-propargylxanthine (DMPX; 10 mM) -did not affect Ap 3 A action. Abbreviations: Ap n As, diadenosine polyphosphates; Ap 3 A, P 1 ,P 3 -diadenosine triphosphate; Ap 4 A, P 1 ,P 4 -diadenosine tetraphosphate; Ap 5 A, P 1 ,P 5 -diadenosine pentaphosphate; DMPX, 3,7-dimethyl-1-propargylxanthine; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; GIS, glomerular inulin space; MRS 2179, 2 0 -deoxy-N 6 -methyl adenosine 3 0 5 0 -diphosphate diammonium salt; PBS, phosphate-buffered saline