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 [Ca2 i1 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 tCa2 i1 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 [Ca2 i1 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 [Ca2 i1 and to determine the effect of agonist 7.3) (7). The tissue was gently triturated, and the undispersed tissue stimulation on the tCa2 11 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 ments) and a dichroic mirror (DM400; Nikon Instruments). Measure- mM): 125 NaCI, 5.0 KCI, 1.0 MgCl-,, 10.0 glucose, 20.0 HEPES, 0.1 ments of fluorescence intensity were collected at 5 data points/s, and CaCI,. and 0. 1 1 1 g/l BSA. All solutions were prepared using highly the data were collected and analyzed with the aid of the P11 software. purified water deionized by a Milli-Q 1 reverse osmosis water pu- Calibration of the fluorescence data was accomplished in vitro ac- rification system (Millipore Corp., Bedford, MA). The pH of the low cording to the method of Grynkiewicz et al. (26). The Rmjn calibration Ca2 p5 was adjusted to 7.35 with NaOH. After the kidneys were solution contained 1.0 jtM fura 2 pentapotassium salt in a solution of rinsed of blood, the perfusate was changed to a similar solution (in mM) 1 15 KCI, 20.0 NaCl, 10.0 MOPS, 1.1 MgCl2, 10.0 EGTA, containing iron oxide (25 mg/mI). and with the pH adjusted to 7.05. The Rniax solution was identical to The kidneys were resected from the animal, decapsulated, and the the solution except that saturating CaCl2 was added to yield a renal medullary tissue was removed. The cortical tissue was gently free Ca2 concentration of 1 .0 mM. pressed through a 1 80- .tm mesh sieve. The pressed renal tissue For determination of [Ca2 ]1, suspensions of freshly isolated renal retained on the surface of the mesh was washed several times with microvascular cells were loaded with the Ca2tsensitive fluorescent ice-cold low Ca2 P55. This was performed to rinse tubular tissue and probe, fura 2 (Molecular Probes, Eugene, OR). The cells were loaded glomeruli through the sieve while retaining the vascular and micro- at room temperature with 10 M fura 2 acetoxymethyl ester (fura vascular tissue on the sieve surface. The sieve retentate was washed 2/AM) for 45 mm in darkness and then kept on ice until used. An vigorously until the majority of the tubular tissue had been removed, aliquot of cell suspension was transferred to the perfusion chamber and the vascular tissue was then transferred to a dish containing (Warner Instrument Corporation, Hamden, CT), and the cells were ice-cold, low Ca2 P55. The vascular tissue was microdissected using allowed to adhere for approximately 30 mm. The perfusion chamber a Nikon series Z stereoscope (Nikon Inc., Tokyo, Japan). Segments of was sealed and mounted to the stage of a Nikon Diaphot inverted interlobular artery with attached afferent arterioles were isolated and microscope. The chamber was attached to a peristaltic perfusion transferred to a 25-mi flask containing ice-cold, low Ca2 P55. The pump, and the cells were continuously superfused with a normal Ca2 low Ca2 PSS was decanted from the vascular segments and replaced PSS solution (PSS) of the following composition (in mM): 125 NaCl, with an enzyme solution of the following composition: 0.15% colla- 5.0 KC1, 1.0 MgCl2, 10.0 glucose, 20.0 HEPES, 1.0 CaCl7, and 0.111
genase (Sigma Type IV: Sigma Chemical Co., St. Louis, MO), g/l BSA. The pump rate was set at 900 .d/min, which replaced the 0.006% elastase (type 2-A; Sigma), 0.05% soybean trypsin inhibitor chamber volume nine times/mm. Cells identified for study were (type l-S; Sigma), and 0.05% BSA (fraction V; Calbiochem, San isolated in the optical field by positioning the adjustable sampling Microvascular Smooth Muscle Cell Ca2 ‘ 57 I window directly over the cell of interest. Neighboring cells and debris were thus excluded from the sampling field. All fluorescence mea- surements were obtained with background subtraction. The effect of cell depolarization on cytosolic calcium concentration was determined by exposing single cells to PSS solutions containing KC1 concentrations of 10, 30, 50, or 90 mM. KC1 was substituted for NaCI on an equimolar basis. Other studies were performed to deter- mine the role of extracellular calcium on the increase in cytosolic calcium induced by potassium depolarization. Cells were sequentially challenged with a 90 mM K in normal PSS and then again in a “Ca2 free” solution (Ca2 free P55), which resembled the P55 solutions except that no CaC1, was added. This reduced extracellular calcium to a concentration below 200 nM as measured using an ion-selective calcium electrode (Ciba/Corning Diagnostics, Halstead Essex, England). No EGTA was added to the solution, because it sometimes has deleterious effects on the ability of the cells to regulate resting cytosolic calcium concentration in a stable fashion. The role of L-type calcium channels in the calcium response to depolarization . ... . . I , . #{149}. was accessed using 10 MM diltiazem. Additional studies were performed to determine the role of potas- sium channels on the increase in cytosolic calcium induced by a high-potassium bath solution. The change in cytosolic calcium con- , * centration evoked by high extracellular potassium was accessed with and without exposure to 100 nM charybdotoxin (CTX; Bachem Inc., Torrance, CA) or 3.0 mM tetraethylammonium chloride (TEA: Sigma). These potassium-channel antagonists were applied both be- IrL . .. fore and during introduction of the high-potassium bath solution. :
Statistical Analyses Data are presented as representative traces in some cases and also jC$ r# as grouped data presented as the group mean ± SE. Differences within groups were analyzed by analysis of variance for repeated #{149}::.: measures or a paired a’test. Differences between groups were analyzed by one-way analysis of variance. Post hoc tests were performed using . . . .. . . ! #{149} #{149} the Newman-Keuls multiple range test. Statistical probabilities less than 0.05 (P < 0.05) were considered significantly different. Figure 1. Photographs of a freshly isolated microvascular smooth muscle cell before (upper panel) and after (lower panel) exposure to Results 140 mM KCI. Photographs were taken at the same magnification. using phase-contrast microscopy. Calibration of the image was ac- Figure 1 contains photographs of a typical freshly isolated complished using a stage micrometer. (Key: 50 m is equal to 28 MVSMC before and after exposure to KC1. As can be seen in mm.) panel A, these cells exhibit the fusiform shape typical of vascular smooth muscle cells and exhibit a marked shape change consistent with smooth muscle contraction when ex- tration-dependent increase in the fura 2 fluorescence ratio and posed to a contractile stimulus. Under control conditions, this the calculated [Ca2 ]1 in a single MVSMC (Figure 2). The cell measured approximately 46.4 jtm in length and 4.5 p.m in ability of these cells to respond to and recover from depolar- width across the center, and the sarcolemma had a smooth izing stimuli repeatedly. as well as the reproducibility of the appearance. This cell is again pictured in panel B shortly after responses. is clearly demonstrated in Figure 2. This cell was exposure to a bathing solution containing 140 mM KC1. The challenged a total of seven times over the 60-mm study period cell responded with a rapid “contraction,” which reduced the and yielded consistent and dose-dependent increases in cell length by approximately 37% to 29 .tm and increased the [Ca2 ]1. Longer application of KC1 resulted in a biphasic . . . . width to approximately 8 sm. Cell viability was confirmed by fluorescence profile characterized by a rapid rise in [Ca ] trypan blue exclusion. followed by a lower but stable plateau phase (Figure 2). Av- The first series of experiments were performed to determine erage changes in the K -induced increase in [Ca2 ] are pre- the ability of depolarizing concentrations of KCI to increase sented in Figure 4. K concentrations of 10, 30. 50, or 90 mM [Ca2 J1 reversibly in freshly isolated renal MVSMC. The ef- significantly increased [Ca2 ]1 by 6 ± 3, 42 ± 10, 72 ± 13, fect of increasing KC1 concentration on fura 2 fluorescence in and 121 ± 13%, respectively (P < 0.05 versus the resting single renal MVSMC was examined in 48 cells. Representative [Ca2 in the control buffer containing 3 mM K4). experiments are presented in Figures 2 and 3. Increasing the Elevation of the extracellular K concentration in the bath- K concentration of the bathing medium resulted in a concen- ing solutions was accomplished by an equimolar substitution of 572 Journal of the American Society of Nephrology
I .0 150 80 r 90mM ‘ #{149} 90mM ‘ 90mM ‘ 90mM C KG! KCI KCI KCI C) C’, 70 0 c I I 60 0 0.8 1000 < I- A 050 1i w m - 40 C) z z -I w 0 0.6 030 50 ._ I-. (I) w . 520 0 1 H * -J 10mM 30mM 50mM Li KCI KCI KCI ! ;10
0.4 0 0 10 20 30 40 50 60 n=11 n=9 n=11 n=42 I TIME (MINUTES) 0 10 30 50 90 Figure 2. Effect of increasing KCI concentration on the fura 2 fluo- KCI (mM) rescence ratio and [Ca2 ]1 of a single renal microvascular smooth Figure 4. Dose-response curve depicting the average effect of increas- muscle cell. Single smooth muscle cells exhibited a concentration- ing the KC1 concentration on the [Ca2 ]1. Smooth muscle cells ex- dependent increase in the fura 2 fluorescence ratio when the super- hibited a concentration-dependent increase in [Ca2 j1 when the bath fusion solution was alternated between the control buffer and buffers solution was changed to solutions containing 10. 30. 50, or 90 mM containing 10, 30, 50, or 90 mM KCI as indicated. The above trace is KC1 as indicated. N number of cells studied at each KC1 concen- a representative record selected from 42 cells studied. The break in the = tration. represents a significant (P < 0.05) increase in [Ca2 ]1, fluorescence record at approximately 2000 s is illustrated in real time compared with the resting concentration in those cells. and represents a short interruption for data storage.
500 250 : 450 C 400 z 0 350 i 200 300 I- z w 250 0 150 z 200 z 0 0 0 150 0 100 LOW SODIUM :D 100 : 0 0 -I 50 -J 90m 00 0 50 KCl
100 200 300 400 0 200 400 600
TIME (SECONDS) TIME (SECONDS)
Figure 3. Effect ofprolonged exposure to 90 mM KC1 on the [Ca211 Figure 5. Effect of a low Na bathing solution on [Ca2 ]4 in a single of a single renal microvascular smooth muscle cell. KC1 administra- microvascular smooth muscle cell. Extracellular Na sodium concen- tion is indicated by the solid horizontal bar. tration is reduced by 90 mM for the period indicated by the solid horizontal bar. Responsiveness to KC1 (90 mM) depolarization is confirmed in the same cell, as indicated by the arrows.
KC1 for NaCI. Thus, the increase in [Ca2 4i evoked by the Ktsubstituted solutions could have resulted from the acutely was reduced by equimolar substitution with 90 mM N-meth- decreased extracellular sodium concentration. Therefore, we ylglucamine chloride. As shown in Figure 5, acute, isosmotic determined the response of freshly isolated MVSMC to an reduction of the extracellular Na concentration had no detect- )± ‘+ acute decrease in extracellular sodium concentration. Cells able effect on [Ca } . The [Ca } measured approximately 68 were monitored while being bathed with a solution containing nM and 65 nM during normal and low Na conditions, respec- physiological concentrations of Na and K and while being tively. Increasing the extracellular K concentration as de- exposed to a similar solution in which the NaCl concentration scribed caused [Ca2 ]1 to increase rapidly to approximately Microvascular Smooth Muscle Cell Ca2 573
255 nM. These changes are reflected in the averaged responses I20 of six cells. Resting [Ca2 ]1 averaged 56 ± 14 nM and 48 ± 110 C 12 nM during exposure to normal and low Na solutions, I00 respectively. Subsequent exposure of the cells to 90 mM KC1 : 90 sharply increased the [Ca2 ]1 to 161.03 ± 26.39 nM. 0 80 -J We evaluated the role of Ca2 influx in the Ktinduced 70 increase in [Ca2 4i1 in single MVSMC. The response of single 0 60 0 cells to a solution containing 90 mM KC1 in the absence of 50 -J extracellular Ca2 was bracketed between control and recovery 0 40 C,, responses to 90 mM KC1 obtained in the presence of 1 .0 mM 0 30 extracellular Ca2 . A representative example of these experi- 20 ments is presented in Figure 6, and averaged data are presented 0 10 in Figure 7. Analysis of the results of five such experiments z 0 revealed that 90 mM KC1 evoked marked and reproducible LU 0 elevations of [Ca2 ]1 in the presence of 1.0 mM Ca2 (Figures z 1.0mM calcium 1 .0 mM calcium+ 6 and 7). Resting [Ca2 ]1 increased by approximately 132% calcium free calcium diltiazem I from 40 ± 5 nM to 93 ± 26 nM in response to 90 mM KC1 and 0 9OmMKCI 9OmMKCI rapidly returned to a concentration of 36 ± 5 nM during the ensuing recovery period. Intracellular Ca2 concentration re- Figure 7. Effect of increasing KCI concentration on the peak change mained relatively stable during the acute removal of Ca2 from in [Ca2 ]4 exhibited by renal microvascular smooth muscle cells. The the external solution; however, the rise in Ca2 in response to average peak increase in [Ca2 ]1 is presented for all of the cells KCI was completely abolished (Figures 6 and 7). [Ca2 ]1 exposed to 90 mM KCI in the presence of 1 .0 mM Ca2 to 90 mM averaged 34 ± 5 nM during exposure to 90 mM KCI in the KCI under Ca2’-free conditions (left side) and to 90 mM KCI in the presence of 1.0 mM Ca2 plus 10 sM diltiazem (right side). N the absence of extracellular Ca2 . Restoration of the extracellular = number of cells studied in each group; ‘ P < 0.05 versus baseline Ca2 concentration to 1 .0 mM restored the normal [Ca2 1I [Ca2’11; t - P < 0.05 versus control response to 90 mM KCI. response evoked by KC1 (Figure 6). The increase in [Ca2 i1 induced by high concentrations of extracellular K presumably results from the depolarization of the sarcolemma and the opening of voltage-dependent Ca2 350 channels. Therefore, we determined the effect of blocking voltage-dependent Ca2 influx with the benzothiazapine de- 300 rivative, diltiazem, which is a selective antagonist of voltage- z 0 dependent L-type Ca2 channels. The results of these studies i 250
F- z 200 w 0 10pM DILTIAZEM C 150 z 0 0 150 100 .11 0 -J z 100 I I LU 0 90mM 90mM 90mM 0 KCI KCI KCI z 0 0 CALCIUM FREE 0 500 1000 1500 0 50 TIME (SECONDS)
0 Figure 8. Effect of the voltage-gated Ca2 channel blocker diltiazem -J <0 I on the response of a single microvascular smooth muscle cell to 0 90mM 90mM 90mM KC1-induced depolarization. The response to 90 mM KC1 in the KCI KCI KCI presence of 10 jiM diltiazem is bracketed between control and recov- 0 500 1000 1500 2000 ery responses obtained in the absence of diltiazem. TIME (SECONDS)
Figure 6. Effect of a Ca2 -free bathing solution on the response of a single microvascular smooth muscle cell to KC1-induced depolariza- are presented in Figures 7 and 8. The representative trace in tion. The response to 90 mM KCI in the absence of extracellular Ca2 Figure 8 illustrates the response to 90 mM KC1 in the presence is bracketed between control and recovery responses obtained in the of 10 p.M diltiazem bracketed between control and recovery presence of 1.0 mM extracellular Ca2 . responses. In these experiments, resting [Ca2 ]1 averaged 74 ± 574 Journal of the American Society of Nephrology
8 nM (Figure 7) and increased to 166 ± 29 nM in response to In a second series of experiments, cells were stimulated with 90 mM KC1. Exposure of the cells to 10 tM diltiazem did not 30 mM K , and when the response had reached a stable alter [Ca2 ]1 (68 ± 10 nM) and upon exposure to 90 mM KCI, plateau, the bath was changed to one containing an identical [Ca2411 increased modestly but significantly to 82 ± 13 nM. K concentration plus either 100 nM CTX or 3.0 mM TEA. As Removal of diltiazem from the bathing solution completely can be seen in Figure 10 (Panel A), 30 mM KC1 evoked a restored the Ca2 response of these cells to 90 mM KCI stable increase in [Ca2 ]1 throughout the period of exposure. In (Figure 8). nine cells from three different dissociations, resting [Ca2 ]1 Patch clamp studies have shown that preglomerular smooth averaged 74 ± 3 nM and increased significantly to 145 ± 17 muscle cells possess Ca2tactivated potassium channels and 1 1 1 ± 6 nM during the initial and sustained components, (7,24). Therefore, additional studies were performed to deter- respectively. In the TEA series, introduction of 30 mM K to mine the actions of these channels on the intracellular calcium the bathing solution increased [Ca2 ]1 initially by 56 ± 18% to response to potassium depolarization. Single cells were ex- 1 13 ± 9 nM before stabilizing at 92 ± 4 nM during the steady posed to solutions containing either 30 or 90 mM KC1 before state (see Figure 10, Panel B, for an example). Addition of or during exposure to CTX or TEA. In one series of experi- TEA to the bathing solution transiently increased the [Ca2 11 ments, the response to 90 mM K was determined under by 15 ± 2% (P < 0.01) before returning to the pre-TEA control conditions and again after approximately 2 mm of concentration (93 ± S nM). Similar results were obtained using exposure to 100 nM CTX. Figure 9 presents a representative the Ca2tactivated potassium channel blocker CTX. In seven example of the response to high K before and during CTX cells from three dissociations, resting [Ca2 ]1 averaged 99 ± treatment. The initial 200-s exposure to 90 mM K caused a 13 nM (refer to Figure 10, Panel C, for an example trace). sharp increase in [Ca2 i1 which was completely reversible. Introduction of 30 mM K to the bathing solution increased CTX treatment did not significantly alter resting [Ca2 ]1 nor [Ca2411 initially by 88 ± 21% to 199 ± 49 nM before stabi- did it alter the magnitude or time course of the calcium re- lizing at 148 ± 25 nM during the steady-state. Subsequent sponse to the subsequent exposure to high Kt Resting [Ca2 ]1 addition of CTX to the bathing solution caused a transient 22 ± averaged 84 ± 9 nM in I I cells obtained from five different 10% increase in [Ca241 (P < 0.05) before returning toward the dissociations. High K caused this concentration to increase pre-CTX concentration (149 ± 27 nM) within approximately 1 significantly to 346 ± 84 and I 87 ± 44 nM at the peak and mm. The transient increase in [Ca2 ]1 induced by CTX or TEA sustained phases, respectively. Intracellular calcium concentra- appears to be evident only when the cells are subjected to tion returned to 88 ± 1 1 nM in the recovery period and was not moderate depolarizing stimuli, because this response was not significantly changed in the presence of CTX (83.5 ± 1 1 nM). observed when the cells were depolarized to a greater extent The second KC1 treatment increased [Ca2411 to approximately with 90 triM KCI (data not shown). In addition, increasing the 304 ± 105 and I 52 ± 26 nM for the initial and sustained duration of exposure to CTX or TEA, either alone or in phases, respectively. These changes are not significantly dif- combination, did not significantly alter resting [Ca2 11 or the ferent from the changes observed in the first exposure to KC1. cellular calcium response to increases in extracellular K . The response of these cells to elevation of extracellular K represents a receptor-independent stimulus. Renal microvascu- ,-.. 240 lar smooth muscle function is influenced by a multitude of -i - - -- - I : 220 circulating and locally generated vasoactive agonists, which lOOnM CTX bind to their respective receptors. Isolation of these MVSMC 200 involves gentle enzymatic digestion, which could potentially damage membrane-bound receptor proteins and prevent recep- t;; 180 tor-dependent, agonist-induced responses. Preservation of the 160 z functional integrity of such receptors is critical if these cells are LU 140 to be used to study physiological responses. Therefore, we 0 z 120 evaluated the ability of these cells to respond to receptor- 0 dependent agonists that are known to influence renal micro- 0 100 vascular function in vivo and in vitro. We evaluated the effect : 80 of exposure to angiotensin II, ATP, norepinephrine, and vaso- 0 -I pressin on [Ca2 ]1. As shown in Figure 1 1, stimulation of these 90mM K 90mM K 0 40 cells with either 100 nM angiotensin II (Panel A) or 100 j.tM ATP (Panel B) resulted in a rapid increase in [Ca2 i1. This 1000 1200 0 200 400 600 800 increase was biphasic, and included a rapid initial phase fol- TIME (SECONDS) lowed by a lower but sustained plateau phase. Angiotensin II (100 nM) increased [Ca2 ]1 from a control of 97 ± 10 nM to Figure 9. Effect of the potassium channel blocker CTX on the response of a single microvascular smooth muscle cell to KCI-induced 266 ± 47 nM (N = I 2 cells; P < 0.05 versus control). depolarization. The response to 90 mM KCI before and during expo- Similarly, 100 p.M ATP increased MVSMC [Ca2 1 from a sure of the cell to 100 nM CTX is illustrated. Administration of KC1 control level of 71 ± 14 nM to 251 ± 47 nM (N = 11 cells; and CTX is indicated by the solid horizontal bars. P < 0.05 versus control). Representative responses to vaso- Microvascular Smooth Muscle Cell Ca2 575
I I I I C C z 200 A z 250 0 0 F- I 80 A F-
I- I 60 z LU I 40 z 0 LU z 0 I 20 z 0 0 I 00 0 50 100nMANGII 30mM K + 0 80 : C) 0 0 100 200 300 400 500 600 -J 100 200 300 400 500 600 0 TI ME (SECONDS) TIME (SECONDS)
I 1 I I I I C 130 S .- - 600 z B 0 120 500 110 ck 400 F- z 100 LU 300 90 0 0 z 200 0 0 80 0 100 3mM TEA D 70 + 30mM K 0 : 0
-I 60 I I I I I I 0 -J 400 500 600 700 0 0 100 200 300 400 500 600 0 TI ME (SE CONDS) TIME (SECONDS) Figure 11. The lCa2 1 responses of representative single renal mi- crovascular smooth muscle cell exposed to 100 nM angiotensin II I I I I C I (Panel A) or 100 ,.tM ATP (Panel B). Angiotensin and ATP were z I 30 administered as indicated by the solid horizontal bar. I 20 I 10 pressin (1.0 jtM) and norepinephrine (100 nM) are shown in I 00 Figure 12. Norepinephrine treatment (Panel A) increased 90 [Ca2 11 from 63 ± 4 nM under control conditions to 213 ± 47 nM (N = 6 cells; P < 0.05 versus control). Similarly, vaso- 80 pressin administration (Figure l2B) increased [Ca2 i1 from lOOnM CTX 70 30mM K 86.0 ± 10 nM to 352 ± 79 nM(N = 5 cells; P < 0.05 versus control). Each of these responses was fully reversible. I I I I I I 0 100 200 300 400 500 600 Discussion TIME (SECONDS) Vascular smooth muscle function at the single cell level has been intensely investigated for some time now; however, the Figure 10. Effect of the potassium channel blockers TEA and CTX on the response of a single microvascular smooth muscle cell to KCI- majority of these efforts have utilized cells obtained from large induced depolarization. Representative traces of a control response to arteries such as the aorta or mesenteric artery. More recently, 30 mM KC1 (Panel A), 30 mM KC1 plus 3.0 mM TEA (Panel B), and investigators have recognized the fact that regional blood flow 30 mM KC1 plus 100 nM CTX are illustrated. TEA and CTX were is regulated by resistance adjustments made in small-caliber introduced during the plateau phase of the response to KCI. (Panel B) arteries and arterioles and thus have begun to focus on cells The response to 90 mM KC1 before and during exposure of the cell to obtained from resistance-size vessels (7,24,27). Of particular 100 nM CTX is illustrated. Administration of KC1, TEA, and CTX is interest to the regulation of renal hemodynamics is the regu- indicated by the solid horizontal bars. lation of afferent arteriolar caliber, which under pressurized 576 Journal of the American Society of Nephrology
major site of regulation of renal blood flow or renal vascular C resistance. z 200 The studies presented here demonstrate that vascular smooth 0 A muscle cells freshly isolated from rat preglomerular microves- F- sels respond to increased extracellular K with an increase in < I 50 [Ca241. Regulation of [Ca2 ]1 in vascular smooth muscle cells F- z involves a complex balance between multiple Ca2 influx, LU 0 I 00 efflux, and release pathways, as well as sequestration of cyto- z solic free Ca2 into intracellular Ca2 stores (3-5,7,17,28). 0 0 Ca2 influx into vascular smooth muscle cells can occur 50 through either voltage-dependent or voltage-independent path- :: 200nM NOREPINEPHRINE ways (3-5,7,17,28,29). Voltage-independent Ca2 influx relies 0 J0 I I I I I on activation of selective and nonselective ion channels with- 900 I 000 1 100 1200 1300 1400 1500 out requiring an initial change in resting membrane potential TIME SECONDS’ (3,29). In contrast, voltage-dependent Ca2 influx requires ‘ I membrane depolarization before activation of ion channels in the membrane (3,29). Voltage-dependent Ca2 channels in
C vascular smooth muscle are essentially divided into two func-
600 I tionally distinct types (3,4,29,30). T-type channels open tran- 0 siently and then rapidly inactivate, whereas L-type channels 500 I- - open more slowly and remain open for longer periods of time. < Previous studies demonstrated the participation of voltage- I- 400 z dependent Ca2 channels in agonist-mediated vasoconstriction LU 300 0 of preglomerular vessels ( 10, 14, 16, 19) and in isolated arte- z rioles (8,9, 1 1 ). In the studies presented here, the role of volt- 0 200 - 0 age-dependent L-type Ca2 channels is demonstrated by pro- nounced attenuation of the Ktinduced increase in [Ca2 11 in 100 - : the presence of diltiazem. Pretreatment with diltiazem reduced 3 0 - 1.0 iMVASOPRESSIN ______I I the magnitude of the Ktinduced increase in [Ca2411 by ap- < 1400 1450 1 500 proximately 80 to 90%. This observation suggests that Ca2 0 influx through L-type Ca2 channels accounts for the majority TIME (SECONDS) of the increase in cell Ca2 observed during exposure to Kt Figure 12. The [Ca24 I responses of representative single renal mi- Nevertheless, a small increment in [Ca2 ]1 consistently re- crovascular smooth muscle cell exposed to 200 nM norepinephrine mained during diltiazem treatment, which was not present in (Panel A) or I .0 sM vasopressin (Panel B). Norepinephrine and the absence of extracellular Ca2 . The fact that the small vasopressin were administered as indicated by the solid horizontal bar. increase in [Ca2 ]1 observed during diltiazem treatment was totally absent when Ca2 was removed from the extracellular medium demonstrates that the rise in [Ca2 ]1 with diltiazem is conditions averages approximately 20 jtm and accounts for an influx-dependent event. This effect could result from in- approximately 90% of the preglomerular resistance. Furthering complete blockade of L-type channels with diltiazem because our understanding of the mechanisms used by preglomerular benzothiazipines are use-dependent; however, in a few exper- vascular smooth muscle cells to regulate microvascular tone iments, the small increment in [Ca2 ]1 was retained even when and to respond to physiological stimuli represents an important cells received duplicate challenges with 90 mM K while step in our efforts to better understand renal hemodynamic continually exposed to diltiazem. This observation is inconsis- regulation. Unfortunately, whole-kidney preparations are ex- tent with recruitment of previously unblocked L-channels pro- tremely complex systems involving interaction between a mul- viding an influx pathway during diltiazem treatment and sug- titude of vascular and epithelial cell types. Approaches utiliz- gest that some other mechanism may be responsible. Another ing isolated arterioles still suffer from the presence of possible explanation is the activation of other voltage-sensitive endothelial cells, which can markedly alter vascular smooth Ca2 channels such as T-channels. Renal arterial smooth mus- muscle responsiveness to vasoactive agents and stimuli. The cle cells appear to express a high density of T-type Ca2 development of this preparation of freshly isolated renal channels (7) and T-channels are insensitive to L-type Ca2 MVSMC affords investigators the opportunity to evaluate in- channel antagonists (30). Therefore, depolarization could acti- tracellular signal transduction mechanisms without the con- vate Ca2 influx through T-type channels even in the presence founding influence of nonvascular cell types or the modulating of diltiazem. influence of cell to cell interactions. Additionally, it provides It is interesting to note that in experiments in which cells the opportunity to study the regulation of vascular smooth were bathed in nominally calcium-free medium, the resting muscle cells isolated from true resistance vessels, which are the [Ca2411 remained essentially constant (Figure 6). Additionally, Microvascular Smooth Muscle Cell Ca2 577 the response to K depolarization was abolished, attesting to Possible explanations include a compensatory change in mem- the absence of significant amounts of Ca2 in the extracellular brane potential that would repolarize the cell, Ca2 channel medium. The stability of the resting [Ca2 ]1 differs from other inactivation, or altered enhanced Ca2 efflux, but the validity studies in which [Ca24i usually slowly declines in Ca2 -free of these scenarios remains to be determined. It is interesting to conditions (8,1 1,3 1). The most probable explanation for this is note that neither CTX nor TEA significantly altered basal that we did not add calcium chelators such as EGTA to the cytosolic calcium concentration. Collectively, these data sug- bathing solution to reduce extracellular calcium. Instead, solu- gest that, under conditions in which all membrane channels and tions were prepared using highly purified Milli-Q water and no cellular processes are able to influence intracellular Ca2 1ev- Ca2tbased reagents were added to the solution. This results in els, activation of Ca2tactivated potassium channels partici- a “nominally calcium free” solution that has a free Ca2 pate in the cellular response to depolarization but are not the concentration below 200 nM, as measured by a calcium ion- sole determinate of the response. Indeed, Ca2 -activated po- selective electrode. The absence of a calcium chelator in the tassium channels may play a more prominent role in the extracellular medium of nominally calcium-free solutions may regulation of microvascular function under conditions in which thus prevent the reduction of [Ca2411 by avoiding the “extrac- the vasculature is exposed to physical stresses such as lumenal tion” of cellular calcium by EGTA. Nominally calcium-free sheer or stretch induced by transmural pressure gradients. In solutions prepared in this way result in extracellular Ca2 vitro studies have shown that inhibition of Ca2 tactivated concentrations that are sufficiently low enough to resolve in- potassium channels with TEA results in a decrease in the flux-dependent responses. diameter of afferent arterioles perfused at physiological pres- We also considered the possibility that at least some of the sures (33). Other studies have shown that the number of rise in cell [Ca2 ]1 that occurred upon exposure to high extra- Ca2tactivated potassium channels and mRNA expression for cellular K was the result of the more indirect effect of these channels increases in vasculature of hypertensive animals reducing extracellular Na concentration. Reducing extracel- (34,35). lular Na concentration could impair certain Ca2 efflux path- L-type Ca2 channels are known to play an important role in ways and thus indirectly increase [Ca2 ]1. However, studies the regulation of renal microvascular function (6-16,21,36- examining the effect of reducing extracellular sodium concen- 38). L-type Ca2 channel activation has been shown to partic- tration yielded no detectable increase in fura 2 fluorescence ipate in agonist-induced vasoconstrictor responses (7- 1 1,14- and thus no increase in [Ca2 J1. In addition, impairment of 16,19) as well as in the myogenic (37.38) and tubuloglomerular Ca2 efflux pathways would be expected to increase [Ca2 ] feedback components of RBF autoregulation ( I 3,36). Angio- over a more protracted period of time, compared with the rapid tensin-mediated vasoconstriction of the afferent, but not effer- increase in cell Ca2 that is elicited by increased extracellular ent, arterioles can be blocked by Ca2 channel antagonists potassium. Therefore, the data presented here support the pos- (1 2, 14). L-type Ca2 channel antagonists also block the affer- tulate that the increase in renal microvascular smooth muscle ent arteriolar vasoconstrictor responses evoked by KC1 [Ca2 ] occurs through a direct effect of K -induced activation (10,14,39,40) and extracellular ATP (10). Pressure-induced of voltage-dependent Ca2 influx pathways and that L-type myogenic responses of renal arteries can be blocked by L-type Ca2 channel activity accounts for most, if not all, of that channel antagonists, as can autoregulation of afferent arteriolar response. blood flow (13) and tubuloglomerular feedback responses (36). Studies have shown that preglomerular smooth muscle cells Indeed, using a very similar preparation of renal MVSMC, possess Ca2tactivated potassium channels (7,24,32); how- Gebremedhin et al. (24) recently described the properties of ever, the physiological role of these channels in the regulation several different K channels. In particular, they described a of renal microvascular function remains unclear. In experi- small- and large-conductance K channel that could be impor- ments using inside-out patches excised from preglomerular tant in regulating membrane potential and thus contribute sig- vascular smooth muscle cells, administration of CTX, apamin, nificantly to the voltage-dependent regulation of renal vascular 4-aminopyridine, or TEA inhibited the K currents propagated function. The current studies complement those of Gebremed- through multiple types of Ca2tactivated potassium channels hin et al. (24) by measuring [Ca2 ]1 with fura 2 and demon- (17,24). To the extent that calcium-activated K channels are strating the presence of functional Ca2 channels and the involved in regulating membrane potential in arterial smooth ability of MVSMC to regulate their [Ca2411 in response to muscle, blocking agents such as CTX and TEA would be receptor-dependent agonists. expected to depolarize the membrane further and contribute to Retention of functional membrane receptors is clearly dem- the influx of extracellular Ca2 . Whether this depolarizing onstrated by the responses presented in Figures 1 1 and 12. stimulus would be maintained is unclear. In this study, using Each of the evaluated agents stimulated increases in cytosolic moderately depolarized cells, K a channel inhibition with TEA Ca24 consistent with their being Ca2tdependent vasoconstric- or CTX transiently increased [Ca2 }1 before returning to the tors in the renal microcirculation. The general pattern or time preinhibitor level. This behavior is consistent with a depolar- course of the agonist-induced response appears to be distinct izing influence of CTX and TEA and implicates calcium- for each agent tested. This observation is consistent with the activated K channels as participating in the calcium response suggestion that each agonist utilizes different signal transduc- to depolarization. The mechanisms responsible for returning tion pathways and/or intracellular regulatory pathways to elicit [Ca2 ]1 to the preinhibitor level remain to be determined. a renal microvascular response. Some agonists may stimulate 578 Journal of the American Society of Nephrology rapid Ca2 influx through activation of both voltage-dependent changes in smooth muscle calcium in rat renal arterioles. J Am and voltage-independent channels. Others may require gener- Soc Nephrol 3: 1792-1803, 1993 ation of second messengers through activation of different 9. Conger JD, Falk SA: KCI and angiotensin responses in isolated phospholipases such as phospholipase C and phospholipase rat renal arterioles: Effects of diltiazem and low-calcium me- dium. Am J Phvsiol 264: Fl34-F140, 1993 A2. Alternatively, receptor-mediated increases in [Ca2 ]1 can 10. Inscho EW, Ohishi K, Cook AK, Belott TP, Navar LG: Calcium also involve inhibition of Ca2 extrusion pathways. Further activation mechanisms in the renal microvascular response to studies are currently underway to more completely evaluate the extracellular ATP. Am J Phvsiol 268: F876-F884, 1995 second messenger systems employed by these vasoactive II . Carmines PK, Fowler BC, Bell PD: Segmentally distinct effects agents. of depolarization on intracellular [Ca2 ] in renal arterioles. Am J In summary, the report presented here describes the effect of Physiol 265: F677-F685, 1993 receptor-dependent and -independent vasoconstrictor agonists I 2. 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