Proceedings of The Physiological Society

University of Edinburgh (2011) Proc Physiol Soc 25, PC22

Poster Communications

Mechanisms of attenuation of P2X-mediated [Ca2+]i transients in renal microvascular myocytes in primary hypertension

D. V. Gordienko1,2, O. Povstyan1,3, M. Harhun3, V. Lehen'kyi4, M. Raphael4, N. Prevarskaya4

1. Molecular Pharmacology and Biophysics of Cell Signalling, Bogomoletz Institute of Physiology, Kiev, Ukraine. 2. State Key Laboratory of Molecular and Cell Biology, Kiev, Ukraine. 3. Biomedical Sciences, St. George's University of London, London, United Kingdom. 4. Inserm U1003, Equipe labellis+

Rise of [Ca2+]i in renal vascular smooth muscle cells (RVSMCs) following P2X receptor (P2XR) activation results from Ca2+ entry via P2XRs and voltage-gated Ca2+ channels (VGCCs), and ryanodine receptor (RyR)- and inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release from the sarcoplasmic reticulum (SR) [1]. Here we analysed how this Ca2+ signalling system is altered in primary hypertension by comparison of: (i) expression of genes encoding P2XRs, VGCCs, RyRs and IP3Rs using real-time PCR analysis, (ii) P2XR-mediated currents (IP2X) using perforated-patch tight-seal recording, (iii) αβ-meATP-induced [Ca2+]i transients in fluo-3-loaded myocytes using confocal imaging, (iv) the SR calcium load using caffeine as Ca2+-liberating agent, in RVSMCs from spontaneously hypertensive rats (SHR) and their normotensive control, Wistar Kyoto (WKY) rats. Ca2+ signal calibration was performed using the method modified from Kao et al. [2]. Data are presented as mean ± S.E.M. and compared using unpaired Student’s t-test. We found that in SHR RVSMCs P2rx1 was downregulated 17.9±5.5-fold (p<0.001), Cacna1C and Ryr2 were upregulated 30.3±13.8- and 24.5±8.6-fold, respectively (p<0.01), while there was no significant difference in P2rx4 and Itpr1 expression (p=0.368 and p=0.165, respectively). The peak of αβ-meATP-induced [Ca2+]i transients was reduced in SHR RVSMCs (p<0.001) from 746±58 nM (WKY, n=76) to 270±23 nM (SHR, n=55). The peak density of IP2X was reduced in SHR RVSMCs (p<0.01) from 101±12 pA/pF (WKY, n=25) to 57±7 pA/pF (SHR, n=18). Ca2+ entry via P2XRs, estimated from the fraction of the fluo-3 response remaining after depletion of Ca2+ stores and block of VGCCs, was reduced in SHR RVSMCs (p<0.01) from 29±3 % (WKY, n=20) to 15±2 % (SHR, n=11). Ca2+ entry via VGCCs, estimated from nicardipine-sensitive fraction of the fluo-3 response to αβ-meATP in RVSMCs with depleted SR, was increased in SHR RVSMCs (p<0.01) from 35±5 % (WKY, n=7) to 62±4 % (SHR, n=6). Ryanodine-sensitive fraction of the fluo-3 response to αβ-meATP increased in SHR RVSMCs (p<0.01) from 19±4 % (WKY, n=7) to 42±5 % (SHR, n=8). The peak of caffeine-induced [Ca2+]i transients was reduced in SHR RVSMCs (p<0.001) from 688±32 nM (WKY, n=47) to 246±19 nM (SHR, n=21). Thus, both, reduced Ca2+ entry via downregulated P2X1 and decreased Ca2+ release from the SR with lower Ca2+ load (resulting from an enhanced RyR-mediated Ca2+ leak) contribute to attenuation of [Ca2+]i transients in SHR RVSMCs and may underlie impairment of sympathetically driven and autoregulatory responses in renal vasculature [3] leading to glomerular damage in hypertension.

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