AGONIST-INDUCED CALCIUM REGULATION IN FRESHLY ISOLATED RENAL MICROVASCULAR SMOOTH-MUSCLE CELLS

The studies presented here were performed to determine the effect of a gonist stimulation on the cytosolic free Ca2+ concentration ([Ca2+](i) ) in single smooth muscle cells, freshly isolated from afferent arteri oles and interlobular arteries averaging between 10 to 40 mu m in diam eter. Microvessels were obtained from male Sprague-Dawley rats using a n iron oxide collection technique followed by collagenase digestion. F reshly 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 respo nse to KCl-induced depolarization was also evaluated. Resting [Ca2+](i ) increased approximately 132% from 40 +/- 5 nM to 93 +/- 26 nM in res ponse to 90 mM extracellular KCl. This change was abolished in nominal ly Ca2+-free conditions and markedly attenuated by diltiazem. Inhibiti on of K+ channels with charybdotoxin or tetraethylammonium chloride pr oduced 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+. Studi es were also performed to establish whether freshly isolated renal MVS MC exhibit appropriate responses to receptor-dependent physiological a gonists. Angiotensin II (100 nM) increased cell Ca2+ from 97 +/- 10 nM to 265 +/- 47 nM (N = 12 cells). Similarly, 100 mu M ATP increased MV SMC [Ca2+](i) from a control level of 71 +/- 14 nM to 251 +/- 47 nM (N = 11 cells). Norepinephrine administration caused [Ca2+](i) to increa se 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 vas oconstrictor agonists increase [Ca2+](i) in MVSMC, freshly isolated fr om rat preglomerular vessels. Furthermore, the ability to measure [Ca2 +](i) in responses to physiological stimuli in these single cells perm its investigation of signal transduction mechanisms involved in regula ting renal microvascular resistance.