Agonist-Induced Calcium Regulation in Freshly Isolated Renal Microvascular Smooth Muscle Cells

Agonist-Induced Calcium Regulation in Freshly Isolated Renal Microvascular Smooth Muscle Cells EDWARD W. INSCHO,* MICHAEL J. MASON,* ALAN C. SCHROEDER,t PAUL C. DEICHMANN,* KARL D. STIEGLER, and JOHN D. IMIG* Departments of *physiology and tSurgerv, tTulane University School of Medicine, New Orleans, Louisiana. Abstract. The studies presented here were performed to deter- modest transient increase in [Ca211 during the response to 30 mine the effect of agonist stimulation on the cytosolic free mM K and had no detectable effect on responses to 90 mM Ca2 concentration ([Ca2]1) in single smooth muscle cells, K. Studies were also performed to establish whether freshly freshly isolated from afferent arterioles and interlobular arter- isolated renal MVSMC exhibit appropriate responses to recep- ies averaging between 10 to 40 m in diameter. Microvessels tor-dependent physiological agonists. Angiotensin II (100 nM) were obtained from male Sprague-Dawley rats using an iron increased cell Ca2 from 97 ± 10 nM to 265 ± 47 nM (N = oxide collection technique followed by collagenase digestion. 12 cells). Similarly, 100 j.M ATP increased MVSMC 1Ca2]1 Freshly isolated microvascular smooth muscle cells (MVSMC) from a control level of7l ± 14 nM to 251 ± 47 nM (N =11 were loaded with fura 2 and studied using fluorescence pho- cells). Norepinephrine administration caused [Ca2]1 to in- tometry techniques. The resting [Ca2]1 averaged 67 ± 3 nM crease from 63 ± 4 nM to 212 ± 47 nM (N =six cells), and (N =82 cells). Increasing the extracellular K concentration vasopressin increased [Ca2i1 from 86 ± 10 nM to 352 ± 79 significantly increased [Ca2]1 dose-dependently (P < 0.05). nM (N =five cells). These data demonstrate that receptor- Involvement of extracellular Ca2 in the response to KC1- dependent and -independent vasoconstrictor agonists increase induced depolarization was also evaluated. Resting [Ca2]1 [Ca2]1 in MVSMC, freshly isolated from rat preglomerular increased approximately 132% from 40 ± 5 nM to 93 ± 26 nM vessels. Furthermore, the ability to measure tCa2i1 in re- in response to 90 mM extracellular KC1. This change was sponses to physiological stimuli in these single cells permits abolished in nominally Ca2-free conditions and markedly investigation of signal transduction mechanisms involved in attenuated by diltiazem. Inhibition of K channels with regulating renal microvascular resistance. (J Am Soc Nephrol charybdotoxin or tetraethylammonium chloride produced a 8: 569-579, 1997) Control of renal hemodynamics, glomerular capillary pressure, such as angiotensin II or ATP, or receptor-independent vaso- and GFR is achieved through the regulation of interlobular constrictor stimuli, such as KC1, stimulate afferent arteriolar arterial and afferent arteriolar tone (1 ,2). Active tension devel- vasoconstriction through activation of L-type Ca2 channels opment in the renal microvasculature is a function of complex leading to voltage-dependent Ca2 influx (6,8 - 12,14,15,17). agonistlreceptor interactions, which are communicated to con- Vasoconstriction of afferent arterioles by angiotensin II, ATP, tractile proteins through generation of intracellular second or KC1 can be blocked with L-type Ca2 channel blockers such messengers. One of the more prominent second messengers as diltiazem, verapamil, or nifedipine (8-10,12,14). influenced by agonist/receptor interactions is intracellular Specific studies into the cellular mechanisms of renal mi- 2± ‘± Ca ([Ca ]) (3-5). Smooth muscle cells making up the crovascular control have been hampered by the inaccessibility preglomerular microvasculature are equipped with ion chan- of renal microvascular tissue and the difficulty in obtaining nels capable of translocating extracellular Ca2 into the cell pure preparations of intrarenal microvascular segments for interior (6-16). Increasing [Ca2i1 in this way represents an study. Several different approaches have been utilized in an important mechanism by which preglomerular tone and thus effort to unfold the mechanisms involved in the regulation of glomerular capillary pressure is regulated. renal microvascular function, including micropuncture (18), A major pathway by which afferent arterioles increase the blood perfused juxtamedullary nephron technique (19,20), [Ca2]1 is through activation of voltage-dependent Ca2 chan- the hydronephrotic kidney technique ( 14- 16), and isolated nels (6,8 -1 2, 14, 15). Receptor-dependent vasoactive agonists, afferent and efferent arterioles (8,9, 1 1 ,2 1); however, each of these techniques suffers from the disadvantage of being a multicellular preparation of varying complexity. The multicel- Received October 14, 1996. Accepted December 19, 1996. lular nature of these preparations makes it difficult to assess Correspondence to Dr. Edward W. Inscho, Department of Physiology SL#39. specific vascular smooth muscle cell responses to vasoactive Tulane University School of Medicine. 1430 Tulane Avenue, New Orleans, LA 70112. agents without having to consider the potential confounding influence of endothelial or tubular cells in the response. For 1046-6673/0804-0569$03.00/0 Journal of the American Society of Nephrology this reason, we began to prepare suspensions of single vascular Copyright C) 1997 by the American Society of Nephrology smooth muscle cells derived from interlobular arteries and 570 Journal of the American Society of Nephrology afferent arterioles, which are intrarenal microvessels averaging Diego, CA) dissolved in low Ca2 P55. The vascular tissue was between 10 and 40 jtm in diameter. Vessels of this size have incubated in the enzyme solution for 20 mm at 37#{176}Cbefore the tissue ( since been successfully prepared for the evaluation of receptor was gently triturated with a Pasteur pipette. The dissociation flask was placed on a magnet to adhere the iron-containing microvascular binding (22) and biochemical assay analysis (23). More re- segments while the dissociation medium containing tubules, epithelial cently, smooth muscle cells have been isolated from such cells, and cellular debris was decanted. Fresh enzyme solution was microvessels for patch clamp studies directed at K channel added to the flask, and the tissue was incubated at 37#{176}Cforanother 20 activity (24). mm. Iron-containing microvascular segments were washed for 10 mm The purpose of the study presented here was to prepare in an ice-cold, enzyme-free, recovery solution of the following com- freshly isolated microvascular smooth muscle cells (MVSMC) position (in mM): 80.0 KC1, 30.0 KH,P04, 5.0 MgSO4, 20.0 glucose, from rat interlobular arteries and afferent arterioles for the 5.0 Na2ATP, 5.0 phosphocreatine, 3.0 EGTA, and 10.0 MOPS (pH measurement of [Ca2i1 and to determine the effect of agonist 7.3) (7). The tissue was gently triturated, and the undispersed tissue stimulation on the tCa211 in these cells. Receptor-independent was transferred to a new aliquot of fresh buffer solution. This cycle of alterations in [Ca2]1 were measured in response to membrane trituration and transfer was repeated four to five times, after which the depolarization. Receptor-dependent responses were assessed remaining tissue was discarded. Healthy viable cells were most often found in the second and third fractions; therefore, these cells were by measuring changes in [Ca2]1 in response to the established pooled and cleaned of any residual iron by magnetic separation. The vasoconstrictor agonists angiotensin II, ATP, norepinephrine, cells were collected by centrifugation (5,800 Xg) for 30 s, and the and vasopressin. supernatant was discarded. The cell pellet was resuspended in ice-cold medium 199 (Sigma) containing 100 U/ml penicillin and 200 j.g/ml Methods streptomycin and supplemented with 10% (vol/vol) fetal calf serum Tissue Preparation and Renal Microvascular Smooth (M-l99; Whittaker Bioproducts, Walkersville, MD). Cell suspensions Muscle Cell Isolation were stored on ice until used. Studies were performed in accordance with the guidelines and practices put forth by the Tulane University Advisory Committee for Animal Resources. Suspensions of preglomerular microvessels were Fluorescence Measurements in Single Microvascular prepared using a modification of the methods of Chaudhari and Smooth Muscle Cells Kirschenbaum (25), Chatziantoniou and Arendshorst (22), and Ge- Experiments were performed using a monochrometer-based fluo- bremedhin et al. (24). Each male Sprague-Dawley rat (250 to 375 g) rescence spectrophotometer equipped with a 75-watt xenon bulb and was anesthetized with pentobarbital sodium (40 mg/kg; iv) and its chopper wheel (Photon Technology International, South Brunswick, abdominal cavity exposed via a midline incision. The superior mes- NJ). Excitation wavelengths of 340 and 380 nm were delivered to the enteric artery was cannulated, and the cannula tip was advanced to the sample chamber by means of a fiber optic cable attached to the base lumen of the abdominal aorta. Ligatures were placed around the of the microscope, and the emitted light passed through a 5 10 ± 20 abdominal aorta at sites proximal and distal to the left and right renal barrier filter before detection by the photometer (Photon). Slit widths arteries, respectively. The kidneys were cleared of blood by perfusion of 3 nm were set for both excitation monochrometers. The optical path of the isolated aortic segment with an ice-cold, low Ca24 physiolog- included a 40X objective (Nikon Fluor 40, NA = 1.3; Nikon Instru- ical salt solution (low Ca2 P55) of the following composition (in

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