Agonist-induced calcium regulation in freshly isolated renal microvascular smooth muscle cells

Abstract

The studies presented here were performed to determine the effect of agonist stimulation on the cytosolic free Ca2+ concentration ([Ca2+](i)) in single smooth muscle cells, freshly isolated from afferent arterioles and interlobular arteries averaging between 10 to 40 μm in diameter. Microvessels were obtained from male Sprague-Dawley rats using an iron oxide collection technique followed by collagenase digestion. Freshly isolated microvascular smooth muscle cells (MVSMC) were loaded with fura 2 and studied using fluorescence photometry techniques. The resting [Ca2+](i) averaged 67 ± 3 nM (N = 82 cells). Increasing the extracellular K+ concentration significantly increased [Ca2+](i) dose-dependently (P < 0.05). Involvement of extracellular Ca2+ in the response to KCl-induced depolarization was also evaluated. Resting [Ca2+](i) increased approximately 132% from 40 ± 5 nM to 93 ± 26 nM in response to 90 mM extracellular KCl. This change was abolished in nominally Ca2+-free conditions and markedly attenuated by diltiazem. Inhibition of K+ channels with charybdotoxin or tetraethylammonium chloride produced a modest transient increase in [Ca2+](i) during the response to 30 mM K+ and had no detectable effect on responses to 90 mM K+. Studies were also performed to establish whether freshly isolated renal MVSMC exhibit appropriate responses to receptor-dependent physiological agonists. Angiotensin II (100 nM) increased cell Ca2+ from 97 ± 10 nM to 265 ± 47 nM (N = 12 cells). Similarly, 100 μM ATP increased MVSMC [Ca2+] from a control level of 71 ± 14 nM to 251 ± 47 nM (N = 11 cells). Norepinephrine administration caused [Ca2+](i) to increase from 63 ± 4 nM to 212 ± 47 nM (N = six cells), and vasopressin increased [Ca2+](i) from 86 ± 10 nM to 352 ± 79 nM (N = five cells). These data demonstrate that receptor dependent and -independent vasoconstrictor agonists increase [Ca2+](i) in MVSMC, freshly isolated from rat preglomerular vessels. Furthermore, the ability to measure [Ca2+]i in responses to physiological stimuli in these single cells permits investigation of signal transduction mechanisms involved in regulating renal microvascular resistance.