Proceedings of The Physiological Society

University of Cambridge (2008) Proc Physiol Soc 11, PC127

Poster Communications

Patterns of Ca2+ signals induced by purinergic and adrenergic stimulation of smooth muscle cells from rat renal resistance arteries.

O. Povstyan1,2, M. Harhun1, T. B. Bolton1, D. V. Gordienko1,2

1. Ion Channels and Cell Signalling Centre, St. George's University of London, London, SW17 0RE, United Kingdom. 2. Laboratory of Molecular Pharmacology and Biophysics of Cell Signalling, O.O.Bogomoletz Institute of Physiology, Kyiv, Ukraine.

Renal vascular tone, which determines renal blood flow, is controlled by sympathetic nerves via activation of α1-adrenoceptors (α1-ARs) and P2X purinoceptors (P2X-Rs) [1]. Activation of these causes a robust increase of [Ca2+]i. The aim of this work was to visualise with high temporal resolution sub-cellular calcium events evoked by stimulation of α1-ARs or P2X-Rs in renal vascular smooth muscle cells (RVSMCs) and to analyse the dynamics of these events. Experiments were performed on RVSMCs freshly isolated from rat renal resistance arteries, which were separated from the kidney using an iron oxide sieving technique [2]. Analysis of gene expression in single RVSMCs (250 cells were collected with a glass micropipette) using RT-PCR technique confirmed expression of the genes encoding for α1A-ARs and P2X1-Rs, but not for α1B-ARs, P2X3-Rs and P2X5-Rs. Changes of [Ca2+]i induced by stimulation of α1-ARs or P2X-Rs in RVSMCs loaded with the Ca2+ indicator Fluo-4AM were visualised using fast (33-40 Hz) x-y confocal imaging. Data are presented as mean ± S.E.M.; independent Student’s t-test was performed to verify statistical significance of the results. Stimulation of α1-ARs with 10 μM noradrenaline (NA) or P2X-Rs with 10 μM α,β-methylene adenosine 5’-triphosphate (AMP-CPP) evoked a rise in [Ca2+]i, which consisted of transient and sustained components and was initiated by a sub-plasmalemmal [Ca2+]i upstroke (SPCU) [3]. AMP-CPP-evoked SPCUs had a significantly (p<0.0001) faster upstroke (mean time-to-peak 0.72±0.06 s; n=13) than NA-induced ones (mean time-to-peak 1.56±0.15 s; n=12). Time-to-peak of the AMP-CPP-induced global [Ca2+]i rise (0.79±0.07 s; n=19) was also significantly (p<0.0001) shorter than the NA-induced one (1.89±0.18 s; n=17). Both AMP-CPP- and NA-induced [Ca2+]i transients, however, were of similar duration: the full duration at half-maximal amplitude of the transients were 4.08±0.30 s (n=19) and 4.49±0.35 s (n=17), respectively. Simultaneous imaging of AMP-CPP-induced [Ca2+] changes in cytosol and mitochondria ([Ca2+]m) using Fluo-4 and Rhod-2, respectively, revealed that [Ca2+]m started to rise with a delay of 0.34±0.08 s following initiation of the [Ca2+]i transient and reached its peak 1.02±0.17 s later (n=14). Mitochondrial sequestration of Rhod-2 was confirmed by double staining with MitoTracker Green FM (MTG; a molecule that covalently binds to the inner mitochondrial membrane and fluoresces independently of mitochondrial membrane potential and [Ca2+]m). Double-staining of RVSMCs with Brefeldin A BODIPY 558/568 and MTG revealed a tight association between the sarcoplasmic reticulum (SR) elements and mitochondria. The spatial patterns of agonist-induced [Ca2+]i transients were related to the intracellular distribution of SR and mitochondria.

Where applicable, experiments conform with Society ethical requirements