Isolated rat glomerular cells demonstrate L-type Ca2+-channel activity

Cd (;aldm (1993)14, 307-390 OLcng~nOmupUKLtd1983

Isolated rat glomerular cells demonstrate L-type Ca*+-channel activity G.F. MCDERMOTT, R.D. HURST and CA. WHITESIDE MRC Group in Membrane Biology, Department of Medicine, University of Toronto, Toronto, Ontario, Canada Abstract - The presence of L-type calcium (Ca2p-channels and the effects of &+-channel antagonists on ceils of rat gfomeruli were investfgated. Gfomerufi were isolated by graded sieving and after preincubatfon (10 min) in zero Ca2+, the uptake of &Ca2’ by gfomerufar ceils was measured. Degfarfftfon wfth KCI (50 mM) or the dihydropyrfdine agonist Bay K 8644 (10 PM) stfmufated Ca uptake by 13% and 24%, respectively, above control (lOO%), which was inhibited by nffedipine (Nif, 10 f&l), P < 0.05, and by both S and R isomers of verapamif (Ver, 10 separate experimental preparation, isolated gfomeruli were preioaded (46 Following a 46 mfn pettfusion (37-C, CaCf2 1.26 mM, in the absence of &Ca2 both KCI (50 mM) and Bay K 8644 (i0 fl) induced cellular &Ca2+ efffux with peak values aljove control of 11% and 1546, respectively, (P < 0.05). Exposure to Bay K 8644 preceded by depolarization with KCI resufted in enhanced &Ca2’ efffux identifying the presence of voltage-dependent Ca2+-channel actfvfty. Cultured rat mesangiaf ceils grown to confluence on coverslips were prefoaded wfth Fura- and cytosofic Ca2+ was measured by r$crotfuorometry. KCI (50 mM), gramfcidin (2 f&l) and/or Bay K 8644 (6 f,rM) stimulated Ca2+ influx which was inhibfted by Ver (lop). Ver did not after endothefinstimulated Ca signafffng. We conclude that L-type Ca ’ channels are present on both rat gfomerufar (endothefiaf and/or mesangiaf) ceils in vivo and on cultured mesangiaf ceils, and their activation may be hormone specific.

In the renal microcirculation. glomemlar mesangial and arteriolar smooth-muscle cell vasoactivity is regulated by hormones such as angiotensin II, endothelin and vasopmssin [l-3]. Pharmacological agents such as angiotensin converting enzyme inhibitors and Ca2’ antagonists decrease the vasoconstrictor response of renal arteriolar smooth-muscle and glomerular mesangial cells [4-6]. In the renal microvasculature, the exact mechanism(s) whereby an iotensin II and endothelin stimulate sustained St Ca influx responsible for sustained mesangial and

smooth-muscle cell contraction, is not entirely clear. Recently, the importance of Nat/Ca2’ exchan e in glomemlar cells contributing to sustained Ca& influx following vasoconstrictor hormone stimulation has been reported 171. In cultured mesangial cells, there is evidence for [8] and against [9] the presence of vasoactive hormone stimulated, voltage operated, L-type Ca2’-channel activity. These conflicting data may reflect alteration of mesangial cell phenotype in cullme where the passage of dedifferentiated cell lines may not accurately reflect Ca2+-channel 387

CELL CALCIUM

388

activity in vivo. Another explanation may be that certain experimental CODditiOllStempOrarily alter Ca2+-channelfunction in vitro. It is conceivable that the three important components of the glomemlar autoregulatory microvasculature, i.e. the afferent arteriole, the efferent arteriole and the mesangial cells, may express unique populations of Ca2+-channels,explaining their differential response to Ca2+antagonists [2, lo]. A specific response to Ca2+ antagonists at each location may be relevant, particularly where altered contractility contributes to the pathophysiology of glomerular disease states 141. Bakris et al. 1111have reported an important differential effect of the Ca2+-channelantagonists nifedipine (Nif) versus diltiazem on the glomemlar filtration rate and urinary protein excretion in insulin-dependent diabetes mellitus atients with progressive glomemlopathy. Also Ca2?-thannel antagonists have been shown to impede the develo ment of atherosclerosis [ll] suggesting that Ca2P entry through L-type channels of vascular endotbelial and/or smooth muscle cells contribute to the pathogenesis. The fact that L-type Ca2+-channel blockade inhibits cultured mesangial cell proliferation [13] implies that Ca2+ influx dimctly or indirectly regulates mitogenesis. The purpose of this study was to identity whether cells of freshly isolated normal rat glomemli and mesangial cells in culture display L-type Ca2+-channelactivity. Isolated glomeruli were used to analyze cellular 45Ca2+influx and efflux in re sponse to depolarization and the Gtype channel agonist Bay K 8644. In cultured mesangial cells microtluorometry measurements of raised cytosolic Ca2+following L-type Ca2+channel activation were compared in cells on coverslips versus suspended cells recently exposed to trypsin. The Ca2+-channel antagonists Nif and Verapamil (Ver) (including the S and R isomers) were used to identify L-type Ca2’channel activity in both isolated glomeruli and cultured cells.

MO, USA) and tap water ad libittun. 2 rats per experiment were used. Bay K 8644 was obtained from Calbiochem Corporation (CA, USA). Ver hydrochloride (soluble in 25% ethyl alcohol), Nif (soluble in 25% acetone) and gramicidin were obtained from Sigma Chemical Company (MO, USA). Ver racemic mixture (1:l) and the S and R isomers were gifts from Searle Canada Inc. (Ontario, Canada). Thapsigargin (Thaps) was obtained from L-C Services Corporation (MA, USA). The following materials were obtained from Gibco Laboratories (NY, USA): trypsin-EDTA (0.5% trypsin + 5.3 mM EDTA); fetal bovine serum (FBS); penicillin-stmptomycin (5000 units penicillin + 5000 pg streptomycin/ml), fungixone (250 p.g amphoteracin B + 205 pg sodium desoxycholat&nl); Minimal Essential Medium (MEM) with D-valine; Dulbecco’s Modified Eagle’s Medium @ME&I). Glomerularcell 45C~t uptake

For 45Ca2t efflux experiments, glomemli were isolated Tom finely chopped kidney cortex at mom temperature (22°C) by a graded sieving and centrifugation method in Hank’8 balanced salt solution (HBSS, pH 7.4; Gibco Laboratories, Ont, Canada) containing CaC12,1.26 mM 1141. Specifically, the fmely chopped tissue was passed through a 250 pm sieve and the collected material was then centrifuged at 2000 rpm at 22’C. The pellets were resuspended in HBSS. drawn up through a 20 G needle and centrifuged briefly at 2000 rpm, 22°C. The tissue pellets were resuspended, drawn up through a 20 G needle, placed on a 106 pm sieve and gently sieved using a spatula and collected on a 75 pm sieve. This glomcrular preparation was 95% tubule-free on examination by light microscopy. Using this isolation technique, the outer visceral epithelium was damaged as previously reported 1151, but the remaining glomerular end0thelia.l and mesangial cells were intact and have been used successfully to study glomerular cell function, such as Nat-dependent myo-inositol transxnt [15,161. Materials and methods Studies on glomerular cell Ca2+ uptake were performed by using a method modified from that deMaterials scribed by Wollheim et al. [18]. Following isolaAdult male Sprague-Dawley rats (375-400 g) were tion, glomeruli were allowed to stabilize for 10 min maintained on Purina rat chow (Ralston-Purina, in HBSS containing CaClz (1.26 mM) at pH 7.4.

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L-TYPE Ca2+ ACI’IVITY IN RAT GLOMERULAR CELLS

Aliquots (350 pl) of glomeruli were then individually resuspended in Ca2’-free HBSS at 37°C and 60 pl amounts immediately placed in polyethylene micro tubes (Micro Tube, 0.4 ml, Sarstedt, Que., Canada) so that an air space of approximately 60 u.l was formed above 200 pl of an oil mixture (10: 3 dibutylphthalate:olive oil). Immediately, 30dga;! HBSS, with no CaC12, containing 1 pCi (11.1 pCi/ruh 8.4 mCi/mg; ICN Biomedicals, CA, USA) and 2 pCi of the extracellular space marker [3H]-mannitol (22.2 @i/ml, 164.7 mCi/mg; Du Pont Canada Inc., Ont., Canada) with test agents (ionomycin 10 @I, KCl 50 mM, or Bay K 8644 10 pM) were added with mixing. Followin a 10 min $ incubation at 37°C glomerular uptake of 5Ca2t was terminated by high speed cen%gation for 30 s through the oil layer (Microfuge 11, Beckman Instruments, Ont, Canada). The bottom of the micro tube containing the pellet of tissue was carefully removed and placed in 200 p.l of 20% perchloric acid iu a scintillation vial. Disruption of glomeruli was assisted by vigorous shaking. Following disruption, 10 ml of scintillation fluid (Read Protein, Beckmau Instruments) was added and 4YCa2’ and [3Hl-mannitol content determined usin& liquid scintillation speclrometry. The extent of Ca + uptake was calculated, corrected for the extracellular trapping of [3H]-mannitol, and expressed as % of control (non-stimulated). 45Ca2t effluxporn preloaded glomeruli Studies on the kinetics of 4sCa2+ efflux from preloaded glomeruli were performed by a modification of the original method described by Fraukel et al. [ 171 for isolated rat pancreatic islets of Langerhans. This technique is based on the rationale that a sudden increase in intracellular Ca2’ will be reflected by a corresponding efflux of Ca2’ from the cell, via plasma membrane Ca2’-ATPase, into the external medium Following glomemlar isolation, 45Ca2t (300 i.NXul, 8.4 mCi/mg) and KC1 (50 r&I) were added to the suspension and incubated for 45 min at 37°C. The final Ca2’ concentration of this incubation medium was 6.0 mM. Following incubation, the glomeruli were resuspended in HBSS with 1.26 mM CaCl2. centrifuged at 1000 rpm and resuspended in 1 ml of HBSS. Glomeruli (900 pg

glomemlar protein) were transferred to glass wool plugs in small plastic chambers of 300 pl volume. They were placed in a constant perifusion system which consisted of a reservoir of HBSS (PH 7.4, CaClz 1.26 mM, in the absence of extracelhrlar 45Ca2t) and attached to a peristaltic pump via polyethylene tubing (id. 1.0 mm, Pharmacia Inc., Baie d’Urfe. Que., Canada). Care was taken to eliminate air bubbles from the system. Glomeruli were perifused at 1 ml/ruin for 45 ruin at 37°C prior to the introduction of any test agent. Test agents were introduced by direct injection from a 5 ml syringe on an infusion pump at a rate of 50 p.l/min into the flow of medium. Immediately following injection, total effluent medium was collected over successive 1 min periods (total 15 min) throughout the perifirsion. Scintillation fhdd (10 ml, Ready Safe) was added to 500 pl aliquots of each sample and 45Ca2t counts determined by liquid scintillation spectrometry (Beckman model LS75000 fi-counter). Amounts of 45Ca2+ efflux were calculated relative to and reported above the corresponding control point Each experiment consisted of two test channels and one control (no test agent). Each experiment was repeated at least 3 times (n = 3). Primarymesangial cell cultures For primary culture of mesangial cells, glomemli were isolated from male Sprague-Dawley rats (200 g) under completely sterile conditions. Kidney cortex was removed, finely chopped and passed through a 250 pm pore size sterile sieve. Glomeruli were then rinsed through a nylon mesh of 105 pm pore size (Thompson, Ont., Canada) and collected on another nylon mesh of 75 pm pore size. After washin in HBSS (3x), glomeruli were cultured in ! 75 cm flasks in MEM with D-Vabe containing 20% FBS at 37°C with 5% CGa. MEM with Dvaline eradicates the growth of fibroblasts, since these cells are unable to convert D- to L-valine [19]. Within 5-7 days, glomeruli attached themselves to the culture flasks. MEM with D-Valise was changed at intervals of 7 days until mesangial cells became confluent and were the most prominent cell evident (5-6 weeks). Mesaugial cells were identified by their Ca2+ signalling responsiveness to vasopressin and positive cytoskeletal immunofluorescent stain-

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ing for desmin and vimentin. On reaching confluence, mesangial cells were brought into suspension by brief exposure to trypsin-EDTA (2.5 ml, 510 mitt, 37°C). The trypsin-EDTA was inactivated by adding an excess of DMEM. Then the cell suspension was cultumd in DMEM with 20% FBS, penicillin (5000 units/100 ml), streptomycin (5000 p.g/lOOml) and fungizone (250 pg/lOOml) at 37’C with 5% CO;r. Subcultured cells reached confluence in approximately 5-7 days. Hormone signalling was measured by Ca2’ fluorescence in mesangial cells between passages l&20. Fluorescence &termination of mesangial cell qtosolic calcium

Cytosolic Ca2+ was analyzed using two different preparations, cells suspended in a cuvette and cells grown on coverslips. First, mesangial cells were grown to confluence (5-7 days) in DMEM with 20% FBS. Then cells were briefly suspended by ex-

posure to trypsin-EDTA in phosphate buffered saline (PBS) followed by addition of excess DMEM. Cells were allowed to recover in nutrient medium for 30 min. Then they were incubated at 37°C for 40 mm with the cell permeant calcium probe Jndol/AM (2.5 pM). Aliquots of cells (approximately 106) were washed and resuspended in HEPES-buffered NaCl solutions containing 1.0 mM MgCla and 1.5 mM CaCl2. Fhromscence intensity in counts/s were then made on a fluorescence spectrophotometer (SPEX DM30, SPEX Industries Inc., NJ, USA) equipped with a thermostatted cuvette holder (37’C) and a continuous stirring Illechanism An excitation wavelength of 355 nm was used; fluorescence emission, F, was recorded at 400 nm. Second, cells were grown to confluence (> 3 days) on glass coverslips and preloaded with FuraZAM (5 pM) for 40 min at 37°C in DMEM contaming 1 mg/ml bovine serum albumin @H 7.4). After washing twice with PBS, cells were incubated for 40 min to allow cleavage of the intracellular dye. Cells were kept at room temperature until used. The coverslips were mounted in a chamber containing 1.5 ml of HEPES-buffered NaCl solution with 1.0 mM MgCla and 1.5 mM Carla. The chamber was placed in the heated (37°C) sample compartment of a Nikon microscope. Test agents were intxxluced via a rapid infusion set. Fhromscence intensity, F, was measured at alternate excitation of 340 and 380 run wavelengths, with emission set at 500 nm. For calibration, maximum fluorescence (F-) was determined by the addition of Ca2’ (2.5 mM) and the Ca2’ ionophore ionomycin (2.5 pM) at the end of each experiment. Autofluorescence (Fw~o) was analyzed by adding manganese (8 mM) to quench the dye which was subtracted from all sample fluorescence values before calculation. The un uenched fluorescence in the total absence of CaY+ (pmk3 is a constant fraction of the corrected maximum signal (Fmax - Fauto) when the dye is saturatedwith Ca2+. For both experiments Fminwas found to be 0.07 (F- - Fauto). Using these values, cytosolic Ca2+ (nM) concentration was call

culated tiom the fluorescence signal using the formula: [Ca2+l = J&IW - &in) ! (&XX- P)l

391

L-TYF’E Ca2+ ACl’IVlTY IN RAT GLGMERULAR CELLS

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above control, respectively. Ver (10 CLM)inhibited the Bay K 8644 response by 88% (P < 0.005, n = 22) and the KC1 response by 91% (P < 0.05, n = 22) (see Fig. 2). N~s(lOa~) totally inhibited Bay K (1OuM) 8644 stimulated Ca uptake (P < 0.001, n = 20) and inhibited KC1 uptake by 90% (P < 0.05, n = 13) see Fig. 3). Ver (10 pM) alone had no effect on $ ‘Ca2’ uptake, while Nif (10 @VI)increased uptake ** p