title: 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 microns 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 Ca(2+)-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 microM ATP increased MVSMC [Ca2+]i 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.
authors.affiliation: Department of Physiology, Tulane University School of Medicine, New Orleans, Louisiana 70112, USA.
authors.forename: E W
authors.initials: EW
authors.lastname: Inscho
authors.forename: M J
authors.initials: MJ
authors.lastname: Mason
authors.forename: A C
authors.initials: AC
authors.lastname: Schroeder
authors.forename: P C
authors.initials: PC
authors.lastname: Deichmann
authors.forename: K D
authors.initials: KD
authors.lastname: Stiegler
authors.forename: J D
authors.initials: JD
authors.lastname: Imig
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D018999:Charybdotoxin / Q000494:pharmacology; D004110:Diltiazem / Q000494:pharmacology; D066298:In Vitro Techniques; D008297:Male; D008833:Microcirculation; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009638:Norepinephrine / Q000378:metabolism; D011188:Potassium / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012079:Renal Circulation; D013050:Spectrometry, Fluorescence; D019789:Tetraethylammonium / Q000494:pharmacology; D014667:Vasopressins / Q000378:metabolism
chemical_list: D002121:Calcium Channel Blockers; D014667:Vasopressins; D000804:Angiotensin II; D018999:Charybdotoxin; D019789:Tetraethylammonium; D000255:Adenosine Triphosphate; D004110:Diltiazem; D011188:Potassium; D002118:Calcium; D009638:Norepinephrine