Renovascular hypertension impairs the activation of small- and big-condutance calcium-activated potassium channels during the relaxation induced by C-type natriuretic peptide (CNP) on rat aortic rings

L Pernomian1, CBA Restini2, LM Bendhack3. 1School of Medicine from Ribeirão Preto - University of São Paulo, Department of Pharmacology, 14.040-903, Brazil, 2Department of Medicine - University of Ribeirão Preto, Department of Medicine, Brazil, 3Faculty of Pharmaceutical Sciences from Ribeirão Preto – University of São Paulo, Department of Physics and Chemistry, 14.040-903, Brazil

Introduction: C-type natriuretic peptide (CNP) is a relaxing factor of several vascular beds, whose effects involve the activation of calcium-activated potassium (KCa+) channels (Chauhan et al. 2003; Simon et al. 2009). Since the vascular potassium channels activity is reduced during 2K1C hypertension (Callera et al. 2000; Callera et al. 2004), we hypothesized that the relaxation induced by CNP in 2K-1C rat aorta would be impaired. This study aimed to evaluate the consequences of 2K-1C hypertension on the activation of KCa+ channels and the subsequent intracellular calcium mobilization during CNP-induced relaxation in rat aorta. Methods: Male Wistar rats (200g - Ethics Committee Proc.No.071/2009) underwent surgery to the implantation of a silver clip in the renal artery (2K-1C) and control rats were subjected to laparatomy (2K). Six weeks after the surgery, systolic arterial pressure (SAP) was measured by tail-cuff and rats were considered hypertensive when SAP>160mmHg. 2K and 2K-1C rat thoracic aorta was isolated and used on isometric registration of tension in vascular reactivity or fluorescent assays. Cumulative concentration-response curves for CNP (1 pM-0.5 μM, in 5% acetic acid) were performed in endothelium-intact aortic rings, after the pre-contraction with Phenylephrine (PE, 0.1μM, in water) or potassium solution (KCl 45mM), in the absence or presence of Apamin (SKCa selective blocker, 1μM, in water) or Paxilline (BKCa selective blocker, 1μM, in water), added 30 min prior the CNP curve. Pharmacologic parameters analyzed were the agonist maximum effect (Emax, g) and potency (pD2). Aortic endothelial cells or rings were loaded with the calcium fluorescent probe, FLUO-3AM (10μM, 20 min, in Dimethylsulfoxide), and immediately or later (30 min) stimulated with CNP (1μM), to measure the fluorescence intensity (FI) in confocal microscopy or flow cytometry assays. Statistical analysis used was One-way Anova, with Bonferroni pos-hoc (P<0.05). Results: In 2K rat aorta, CNP induced a maximum relaxation of 1.57±0.14g, with a pD2 value equal to 8.16±0.29 (n=13). The CNP-induced relaxation in 2K-1C rat aorta (Emax=1.10±0.10g; pD2=7.67±0.08, n=11) was not different from 2K rat aorta. Under KCl pre-contraction, CNP Emax was reduced in 2K rat aorta (0.75±0.16g, n=6) when compared to PE-pre-contracted 2K rat aorta. Also, the CNP potency was reduced in both 2K and 2K-1C rat aortas (pD2 2K KCl=6.18±0.44, n=6; 2K1C KCl=5.66±0.18, n=6) when compared to the respective PE-pre-contracted aorta. Paxilline or Apamin reduced the CNP pD2 values in PE pre-contracted 2K rat aorta (pD2 2K Paxilline=7.17±0.31, n=7; 2K Apamin=6.66±0.52, n=8), but not in 2K-1C. The immediate CNP stimulus reduced the endothelial or muscular FI from 2K or 2K-1C rat aortic rings. In endothelial cells from 2K or 2K-1C rat aorta, CNP stimulus (30 min) increased the FI, in a similar manner. Conclusion: CNP-induced relaxation involves the activation of small- and big-condutance KCa+ channels in 2K rat aorta. The activation of these potassium channels during CNP-induced relaxation in rat aorta is impaired by the renovascular hypertension. The intracellular calcium mobilization induced by CNP in 2K or 2K-1C rat aorta is a biphasic response, characterized by an initial decrease followed by a later increase in the intracellular calcium levels. This increase in the intracellular calcium levels is sufficient to induce the activation of some isoforms of KCa+ channels in 2K, but not in 2K-1C rat aortas.