INTRODUCTION

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ENDOTHELIN-1 (ET-1) may be synthesized by glomerular endothelial cells (22), epithelial cells (36), and mesangial cells (31). ET-1 stimulates the contraction of mesangial cells via ET_A receptors (32, 35), regulating glomerular capillary surface area and filtration rate (2). Mesangial cells also proliferate in response to ET-1 (10), suggesting a potential role in glomerular disease.

Contraction of both vascular smooth muscle and mesangial cells involves the interplay of many signal transduction pathways (26). After binding to its G protein-coupled ET_A receptor, ET-1 stimulates phospholipase C (PLC) hydrolysis of phosphatidylinositol bisphosphate (PIP₂), generating the two second messengers inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG). IP₃ causes release of intracellular Ca²⁺ while receptor occupancy stimulates Ca²⁺ influx via both receptor- and voltage-operated channels. The resultant increase in Ca²⁺ activates Ca²⁺/calmodulin-dependent kinases, including myosin light chain kinase, which phosphorylates the regulatory myosin light chain required for contraction. DAG activates protein kinase C (PKC) (21). In addition to the above pathway, DAG may be generated directly by PLC or PLD hydrolysis of phosphatidylcholine (6). There is strong evidence that ET-1 may activate both PLC and PLD to generate DAG in smooth muscle (44) and mesangial cells (3), thus implicating PKC in ET-1-stimulated contraction. The independent roles of Ca²⁺ and specific PKC isoforms in regulating the mesangial cell contractile response to ET-1 are incompletely understood.

PKC is comprised of a family of 12 different gene products that are classified into Ca²⁺-dependent or -independent groups according to their structure and function (25). PKC is present in the cell cytoplasm and upon agonist stimulation, rapidly translocates to the particulate or membrane fraction observed by Western immunoblot analysis and immunofluorescence studies (4, 8). Agonist-stimulated PKC translocation occurs coincidentally with Ca²⁺ release from intracellular stores (20), but the specific role of increased cytosolic Ca²⁺ in PKC activation is not known. Cultured rat mesangial cells express mainly DAG-sensitive/Ca²⁺-dependent PKC-, although PKC-I and - are also reported (9), and the DAG-sensitive/Ca²⁺-independent PKC- and - and DAG-insensitive PKC- isoforms (27). In lower passage (T5-10), growth-arrested mesangial cells, Western blot analysis and confocal immunostaining techniques in our laboratory using monoclonal antibodies have consistently identified the expression of PKC-, -, -, and - isoforms (16, 46). Recent in vivo studies from our laboratory, using both immunogold staining and immunoblotting, have identified the abundant expression of PKC-, -II, -, and - in all cells of isolated rat glomeruli, including mesangial cells (1).

A central role for PKC in smooth muscle and mesangial cell contraction is proposed from studies in which phorbol ester stimulates a slow sustained contractile response (33, 39). PKC is implicated in the phosphorylation of myosin light chain, which promotes both actin-activated myosin Mg²⁺-ATPase activity and cross-bridging required for cell motility or contraction. Peptide mapping studies show that phosphorylation of myosin light chain serine-1 or serine-2 and threonine-9 is PKC dependent, whereas the Ca²⁺/calmodulin-dependent myosin light chain kinase phosphorylates threonine-18 and serine-19 (40). The most direct evidence for PKC-mediated contraction is provided by studies where active PKC- or PKC- was injected into saponin-permeabilized ferret aorta smooth muscle cells (11). Active PKC-, but not PKC-, stimulated ferret aorta contraction, identical to phorbol ester-stimulated contraction, which was reversed by a PKC pseudosubstrate inhibitor. An earlier study revealed that Ca²⁺-independent contraction is preceded by translocation of PKC- from the cytosol to the plasma membrane (18). The specific DAG-sensitive PKC isoform response to ET-1 in mesangial cells is unknown.

In this study, we tested the hypothesis that a significant portion of mesangial cell contraction in response to ET-1 is via a PKC-dependent mechanism. The purpose of this study was to identify the independent roles of Ca²⁺ and PKC contributing to the mesangial cell contractile response and to determine which DAG-sensitive PKC isoforms respond to ET-1. PKC was either inhibited with chelerythrine or downregulated with prolonged phorbol ester treatment. Ionomycin was used to selectively increase intracellular Ca²⁺, independent of ET-1 signal transduction or PKC activation. In response to ET-1 or ionomycin, we assessed the following: change in mesangial planar area as a measure of contraction; altered intracellular Ca²⁺ concentration measured by spectrofluorometry in fura 2-loaded cells; and distribution of DAG-sensitive PKC isoforms by immunoblot and by immunofluorescence confocal imaging. PKC activity was measured by in situ [³²P]ATP phosphorylation of a specific pseudosubstrate peptide. The cellular responses to either ET-1 or ionomycin in the presence or absence of ML-7, a specific myosin light chain kinase inhibitor, were compared to identify the extent of Ca²⁺/calmodulin-dependent contraction. Finally, we determined whether Ca²⁺ signaling in response to ET-1 or ionomycin was altered by PKC inhibition or downregulation.

	MATERIALS AND METHODS

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Mesangial cell culture. Mesangial cells were cultured from collagenase-treated glomeruli obtained by sieving of kidney cortex of 150- to 200-g, male, Sprague-Dawley rats (Charles River, Quebec, Canada) as previously described (12). The mesangial cells were maintained in DMEM (GIBCO Laboratories, Burlington, Ontario, Canada), pH 7.4, supplemented with 20% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10 mM HEPES. Mesangial cells were characterized by their spindle or stellate shape, and immunofluorescence staining was positive for the presence of desmin and vimentin and negative for cytokeratin and factor VIII to differentiate them from glomerular endothelial and epithelial cells (5). Low passage cells (T5-10) growth-arrested for 24 h in 0.5% FBS-DMEM containing (5.6 mM) D-glucose were used for all experiments (7).

Mesangial cell planar area measurements. Mesangial cells (100,000 cells/dish) were detached by transient exposure to 0.5 g/l trypsin in 0.2 g/l EDTA, followed by deactivation of the trypsin by adding excess (8-10 ml) DMEM and centrifugation at 1,000 rpm for 5 min, and were subcultured for 24 h on small 35-mm petri dishes (Falcon; Becton-Dickinson Laboratories, Lincoln Park, NJ) in 2 ml 0.5% FBS-DMEM. On the day of the experiment, the dishes were transferred to the heated (30°C) stage of an inverted phase-contrast light microscope (Bausch & Lomb, New York, NY) and maintained in 5% CO₂ in air at 30°C. The images were captured by a Hitachi KP-113 solid-state television camera (Hitachi Denshi, Tokyo, Japan). Following visualization, the image was digitized to represent time 0 and either ET-1, phorbol 12-myristate 13-acetate (PMA), or ionomycin was added directly to the medium to achieve a final concentration of 0.1 µM of each agonist, in the absence or presence of the PKC inhibitor, 10 µM chelerythrine (1 h pretreatment), PMA (24 h pretreatment), or the myosin light chain kinase inhibitor, 10 µM ML-7 (1 h pretreatment). The same group of cells (n = 10-20/group) was digitized serially for the next 60 min. Images were captured on a 486 DX PC and digitized with Sigma Scan morphometric software (Jandel Scientific, San Rafael, CA). Only those cells with a clearly defined border perimeter were used for planar surface area measurement. Changes in individual cell surface planar area were calculated for all of the cells at each time point.

Measurement of PKC activity. The measurement of PKC activity was determined using the in situ ³²P phosphorylation assay of the pseudosubstrate peptide. This assay provides a measure of "total" PKC activity in living cultured cells and is considered to give the most accurate measure of activation (43).

Cells were seeded (~20,000/well) onto plastic 96-well microtiter plates (Sarstedt, Newton, NC), maintained in DMEM containing 20% FBS for 3-4 days until confluent, then incubated in 0.5% FBS-DMEM for 24 h. ET-1, at a final concentration of 0.1 µM, was added for 2, 5, 10, 20, 40, and 60 min. In addition, cells were either stimulated with 0.1 µM PMA for 10 min or pretreated with 10 µM chelerythrine for 1 h or 0.1 µM PMA for 24 h. Then, the test media was aspirated, and 40 µl of a reaction buffer were added containing (in mM) 137 NaCl, 5.4 KCl, 10 MgCl₂, 0.3 sodium phosphate, 0.4 potassium phosphate, 25 -glycerophosphate, 5.6 D-glucose, 5 EGTA, 1 CaCl₂, and 20 HEPES. The buffer also contained (in µM) 30 pseudosubstrate peptide, 100 [³²P]ATP (~1,000 cpm/pmol), and 10 digitonin. The reaction was allowed to proceed for 10 min at 30°C before termination of the assay by the addition of 50 µl of ice-cold trichloroacetic acid (final TCA concentration of 5%). Forty-five microliter aliquots of the acidified reaction mixture were then spotted onto 2.1-cm P-81 phosphocellulose disks (Whatman, Clifton, NJ), where the basic pseudosubstrate was retained, and washed batch-wise in three washes of 75 mM phosphoric acid (10 ml/disk, 2 min/wash). The phosphocellulose was then given a final wash in 75 mM sodium phosphate, pH 7.5, dried, and then placed in plastic 20-ml vials containing 10 ml scintillant (Ready Protein; Beckman Instruments, Fullerton, CA) and counted in a Beckman LSC5 liquid scintillation counter. Nonspecific background phosphorylation was assessed using reaction buffer minus peptide substrate and was always <0.05% of added cpm. PKC activity was expressed as picomoles per minute per milligram mesangial cell protein.

Cell fractionation and PKC immunoblots. PKC isoforms were probed in cytosolic, membrane, and particulate fractions of 24 h growth-arrested mesangial cells at 30°C. Cells were washed twice with ice-cold PBS and harvested in 100 µl/100-mm plate buffer A containing (in mM) 1 NaHCO₃, 5 MgCl₂ · 6H₂O, 50 Tris · HCl, 10 EGTA, 2 EDTA, 1 DTT, and 1 phenylmethylsulfonyl fluoride, as well as 25 µg/ml leupeptin and 10 µM benzamidine. The cells were passed though a 26-gauge needle three times and incubated on ice for 30 min. Homogenates were centrifuged at 100,000 g for 1 h at 4°C, and the supernatants were retained as the cytosolic fraction. The pellet was subsequently dissolved in 100 µl buffer B (buffer A with 1% Triton X-100), passed through a 26-gauge needle, and centrifuged again for 1 h at 100,000 g at 4°C. The supernatant was collected and used as the membrane fraction. The remaining Triton X-insoluble pellet was solubilized in 100 µl of 10% SDS, boiled for 10 min, and served as the particulate fraction. Protein concentration was determined using a modified Lowry microassay (Bio-Rad, Hercules, CA). Aliquots were then denatured in 4× SDS sample buffer, boiled for 5 min, and loaded onto 9% polyacrylamide gels. Samples were electrophoresed for 2 h at 100 V, at room temperature. After an overnight transfer, the polyvinylidene difluoride membranes were blocked for 1 h at room temperature in a 25 mM Tris buffer containing 5% nonfat milk and 0.05% Tween 20. Membranes were then exposed to polyclonal anti-PKC-, -, -, or - antibodies (Sigma Chemical, St. Louis, MO) for 1 h at 1:40,000 dilution for PKC- and 1:10,000 for PKC -, -, and -. This was followed by a 20-min incubation with a horseradish peroxidase-conjugated affinity-purified goat anti-rabbit IgG antibody (Jackson Immunochemicals, West Grove, PA) diluted 1:5,000. The blots were rinsed in Tris-saline between each of the preceding steps. The secondary antibody was detected by chemiluminescence (Kirkegaard & Perry, Gaithersburg, MD), and the membranes were developed on Kodak X-Omat AR film (Eastman Kodak, Rochester, NY). Densitometry was performed using NIH Image 1.62 analysis software (NIH, Bethesda, MD). The specificity of primary antibody interaction with sample was eliminated in the presence of peptide that was used to generate the antibody.

Intracellular calcium measurements. Intracellular calcium was measured by the ratiometric method in fura 2-loaded cells (14). Cells were grown to 80% confluence on glass coverslips in DMEM supplemented with 20% FBS for 2-3 days. Cells were then growth arrested for 24 h in DMEM with 0.5% FBS, then loaded with 2 µM of the acetoxymethyl ester of fura 2 (fura 2-AM; Molecular Probes, Eugene, OR) for 40 min at 30°C in DMEM with 1 mg/ml BSA and 1% Pluronic F-127. The loaded cells were washed 3× with Krebs-Henseleit buffer, then placed in DMEM containing 1 mg/ml BSA and allowed to equilibrate for 30 min at room temperature in the dark. The calcium concentration of the DMEM was 1.8 mM. Each coverslip was mounted in a chamber heated to 30°C filled with 1.5 ml of modified Krebs-Henseleit buffer in the sample compartment of a Nikon Diaphot-TMD inverted microscope. ET-1, PMA, or ionomycin was infused with a 21-gauge × 3/4-inch needle to yield a final concentration of 0.1 µM. Fluorescence intensity was measured with alternating excitation at 340 and 380 nm in a Perkin-Elmer LS-5 spectrofluorometer, where R was the measured ratio 340/380. Calibration at the end of each experiment was performed by adding 20 µM ionomycin plus 10 mM CaCl₂, to saturate the dye to give maximum (R_max) fluorescence. Then, to obtain minimal (R_min) fluorescence, 400 mM EGTA plus 3 M Tris · HCl was added to release Ca²⁺ from fura 2. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was calculated as where K_d for fura 2 was 224 nM. The R_min and R_max represent the fluorescence ratios measured with excitation at 340/380 nm under minimum free Ca²⁺ (bound with EGTA) and maximum Ca²⁺ (ionomycin plus 10 mM CaCl₂). S_b2 was the fluorescence at saturating Ca²⁺ levels. S_f2 was the fluorescence at 380-nm excitation in the absence of Ca²⁺. Autofluorescence, measured by quenching fura 2 with MnCl₂, was subtracted from the R_max and R_min values (14).

Immunofluorescence labeling of PKC isoforms and confocal microscopy. Mesangial cells were cultured on glass coverslips, under conditions identical to those described above. The cells were fixed with 3.7% formaldehyde for 15 min at room temperature followed by plasma membrane and nuclear membrane permeabilization with 100% methanol at 20°C for 10 min. After washing three times with PBS, the cell proteins were blocked with 1% goat serum plus 0.1% BSA in PBS for 60 min at room temperature. Rabbit polyclonal anti-PKC-, -, -, or - was diluted to 1:100 in blocking solution and added to each coverslip for 60 min at 37°C. After washing three times with PBS, an FITC-conjugated anti-rabbit secondary antibody diluted 1:160 in blocking solution was added for 60 min at 37°C. The coverslips were mounted on glass slides with Aqua-Poly-mount (Polysciences, Warrington, PA) and SlowFade antifade reagent. The following controls were performed: 1) incubation with FITC-conjugated secondary antibody alone, which demonstrated no significant labeling; 2) preincubation of the primary antibody with specific PKC peptide, which prevented significant fluorescence labeling of these PKC isoforms.

The fluorescence intensities and spatial configurations of the FITC-labeled PKC isoforms were imaged using a confocal laser-scanning image system (LSM 410, Zeiss) with FITC excitation and emission wavelengths of 488 and 520 nm, respectively. The cells were visualized with an oil-immersion inverted objective lens (Axiovert 100, ×63), and the image pixel resolution was 512 × 512 with a gray level scale of 0 (minimum) to 255 (maximum) intensity. To standardize the fluorescence intensity for all the experimental preparations, the confocal image contrast and brightness levels were adjusted optimally for each PKC isoform and then kept constant. The pinhole (size = 20), scanning time (0.546 s), zoom = 1, and magnification (63 × 1.4) of the confocal scanning system were constant for all image analysis.

Statistical analyses. All results are expressed as means ± SE. Statistical analysis was performed using an unpaired Student's t-test, Mann-Whitney nonparametric unpaired t-test or ANOVA with Dunnett's post test correction as appropriate using InStat 2.01 statistics software (Graph Pad, Sacramento, CA). Differences described as significant represent P < 0.05, unless otherwise stated.

	RESULTS

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Effect of ET-1, ionomycin, and phorbol ester on mesangial cell contraction. Figure 1 demonstrates the change in planar surface area of mesangial cells (n = 30-55) from three to five separate experiments stimulated with ET-1, PMA, or ionomycin as a percentage of the time 0 value. During stimulation with ET-1 or PMA, surface area decreased over 60 min to 49 ± 3% and 56 ± 3% of the original surface area (P < 0.05 vs. time 0). In comparison, cells treated with ionomycin demonstrated a decrease in planar area to only 78 ± 2% of original (P < 0.01 vs. ET-1).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Change in mesangial cell planar area in response to 0.1 µM endothelin-1 (ET-1), 0.1 µM ionomycin (IONO), or 0.1 µM phorbol ester (PMA). Each point represents the mean ± SE of area measurements of 30-55 cells from 5 separate experiments. * P < 0.01 vs. ET-1.

To downregulate or inhibit PKC before ET-1 stimulation, growth-arrested cells were pretreated with 0.1 µM PMA for 24 h or 10 µM chelerythrine for 1 h. As illustrated in Fig. 2, the cells did not contract in response to ET-1 following downregulation of PKC (105 ± 8% of original surface area). Pretreatment with chelerythrine markedly attenuated the response of ET-1 (92 ± 4% of original area), although contraction was not entirely abolished. Chelerythrine completely inhibited the decrease in planar area seen with acute PMA (105 ± 6% of original area). Downregulation of PKC did not affect the decrease in surface area in response to ionomycin (75 ± 3% of original area).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Protein kinase C (PKC) downregulation with 0.1 µM PMA for 24 h or PKC inhibition with 10 µM chelerythrine (CHEL) on ET-1 or ionomycin-induced change in mesangial cell planar area. Each point represents the mean ± SE of area measurements of 20-30 cells from 3 separate experiments. * P < 0.01 vs. 24 h PMA + ET-1.

To determine the involvement of Ca²⁺/calmodulin-activated myosin light chain kinase during ET-1-induced contraction, mesangial cells were pretreated with 10 µM ML-7 for 1 h prior to stimulation. Figure 3 demonstrates that in the presence of ML-7, ET-1 caused a decrease in planar surface area to 85 ± 2% of original area (n = 20 cells), whereas no response to ionomycin (108 ± 3% of original area, n = 25 cells) was observed. ML-7 had no effect on mesangial cell contractile response to PMA (50 ± 5% of original, n = 22), a response identical to that seen in untreated cells (see Fig. 1). The addition of DMSO, a vehicle for all agents used in contraction experiments, had no affect on mesangial cell planar area (100 ± 1% of original area, n = 20 cells).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Effects of 10 µM myosin light chain kinase inhibitor, ML-7, on ET-1 or ionomycin-induced change in mesangial cell planar area. Dimethyl sulfoxide (DMSO) was used as a vehicle for agonist administration. Each point represents the mean ± SE of area measurements from 20-25 cells from 3 separate experiments. * P < 0.01 vs. ML-7 + ionomycin.

Ca²⁺ signaling and effects of PKC. To determine whether the lack of contractile response to ET-1 seen with PKC downregulation was due to altered Ca²⁺ signaling, [Ca²⁺]_i was measured. Typical tracings of cytosolic free [Ca²⁺]_i in response to 0.1 µM ionomycin, or 0.1 µM ET-1 in the absence and presence of 0.1 µM PMA for 24 h, are illustrated in Fig. 4. Stimulation with either ionomycin (n = 4) or ET-1 (n = 7) caused a rapid initial rise in [Ca²⁺]_i from a basal 80 ± 7 to 220 ± 30 nM and 97 ± 10 to 192 ± 10 nM, respectively, followed by a sustained second phase approaching 130 ± 12 and 136 ± 10 nM, respectively, which returned to baseline after 3 min. Figure 4B illustrates that acute PMA stimulation caused no change in [Ca²⁺]_i. When the cells were preincubated with 0.1 µM PMA for 10 min, no response to ET-1 was observed. As shown in Fig. 4C, chelerythrine pretreatment for 1 h did not affect the ionomycin response but completely abolished the ET-1 rise in [Ca²⁺]_i. In Fig. 4D, PMA pretreatment for 24 h lowered both the basal and peak [Ca²⁺]_i response to ET-1 (60 ± 7 and 139 ± 6 nM, respectively, n = 5) but had no effect on the basal and peak response to ionomycin (100 ± 17 and 200 ± 12 nM, respectively, n = 4).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   A: typical intracellular calcium ([Ca2+]i) responses to 0.1 µM ionomycin or 0.1 µM ET-1. B: tracings in response to either acute 0.1 µM PMA alone or pretreatment with 0.1 µM PMA for 10 min followed by ET-1. C: effects of pretreatment with 10 µM chelerythrine for 1 h. D: effects of PKC downregulation with 0.1 µM PMA for 24 h on ionomycin or ET-1-stimulated calcium responsiveness.

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View larger version (14K): [in this window] [in a new window]   Fig. 5.   In situ PKC activity in response to 0.1 µM ET-1, 0.1 µM ionomycin, or 0.1 µM PMA. Each group represents the mean ± SE of quintuplicate measurements from 3 separate experiments. * P < 0.05 vs. basal.

View larger version (43K): [in this window] [in a new window]   Fig. 6.   Mesangial cell subcellular distribution of PKC- in response to 0.1 µM ET-1, 0.1 µM ionomycin, or 0.1 µM PMA in cytosol (CYT), membrane (MEM), and particulate (PART) fractions. In the representative immunoblot (as in Figs. 7, 8, and 10), "+" represents a positive rat brain control. Each group represents the mean ± SE of 6 separate experiments. * P < 0.05 and  P < 0.01 vs. basal.

View larger version (45K): [in this window] [in a new window]   Fig. 7.   Mesangial cell subcellular distribution of PKC- in response to 0.1 µM ET-1, 0.1 µM ionomycin, or 0.1 µM PMA in cytosol, membrane, and particulate fractions. Each group represents the mean ± SE of 6 separate experiments. * P < 0.05 and  P < 0.01 vs. basal.

View larger version (42K): [in this window] [in a new window]   Fig. 8.   Mesangial cell subcellular distribution of PKC- in response to 0.1 µM ET-1, 0.1 µM ionomycin, or 0.1 µM PMA in cytosol, membrane, and particulate fractions. Each group represents the mean ± SE of 6 separate experiments. * P < 0.05 and  P < 0.01 vs. basal.

View larger version (103K): [in this window] [in a new window]   Fig. 9.   Confocal immunofluorescence imaging of PKC-, -, and - in mesangial cells. A-C: PKC- in the basal state and following 0.1 µM ET-1 stimulation without and with PMA pretreatment for 24 h, respectively. D-F: PKC- under the same treatment conditions. G-I: PKC- under the same treatment conditions.

View larger version (102K): [in this window] [in a new window]   Fig. 10.   Confocal immunofluorescence imaging of PKC- in mesangial cells. A: PKC- in the basal state. B: mesangial cells following 0.1 µM ET-1. C: mesangial cell subcellular distribution of PKC- in response to 0.1 µM ET-1, 0.1 µM ionomycin, or 0.1 µM PMA in cytosol, membrane, and particulate fractions.

	DISCUSSION

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Low passage, growth-arrested rat mesangial cells contract in response to ET-1, PMA, and ionomycin. ET-1 elicits the strongest contractile response. The response pattern to PMA was similar to ET-1. Furthermore, inhibition of PKC activity prevents both Ca²⁺ signaling and the contraction in response to ET-1, indicating a key role for PKC in these events. Mesangial cells express the DAG-sensitive PKC isoforms-, -, and -, as well as atypical PKC-, but ET-1 stimulates translocation of PKC- and - from the cytosol to membrane and particulate (cytoskeleton-rich) fractions. Acute PMA causes translocation of PKC-, -, and - to the membrane and particulate fractions as demonstrated by immunoblot. Fluorescence imaging demonstrates translocation of PKC- to the nucleus and cell periphery in response to ET-1. Increased PKC activity in response to ET-1 and PMA but not ionomycin is confirmed by in situ ³²P phosphorylation of the pseudosubstrate peptide. Thus we are the first to report that in mesangial cells, ET-1-induced contraction is associated with the translocation and activation of PKC- and - isoforms, and to a lesser extent PKC-, but not PKC-.

Our measurements of planar area were comparable to earlier studies that reported a mean decrease of 25-40% in mesangial cell surface area at 30 min in response to ET-1 (34). In our previous work, it was determined that the ET-1-induced change in planar area is not due to detachment or rounding up of cells, but is attributable to true contraction, preceded by phosphorylation of myosin light chain (24). A decrease in glomerular volume in response to ET-1 is seen in isolated glomeruli (14), presumably due to mesangial cell contraction. In the presence of 10 µM KT-5926, a less specific myosin light chain kinase inhibitor than ML-7, ET-1 no longer stimulates myosin light chain phosphorylation or change in planar area (24). In our current study, ML-7 prevented contraction even in response to 2 µM ionomycin (data not shown) but did not completely abolish ET-1-induced contraction. Since downregulation of PKC with chronic PMA completely abolished ET-1-stimulated mesangial cell contraction, the earlier findings with KT-5926 (24) may be due to nonspecific inhibition of PKC (30).

As observed in Fig. 4, ionomycin and ET-1 stimulated intracellular Ca²⁺ to similar levels observed previously using single cell fluorescence imaging (42). To determine whether PKC activation altered Ca²⁺ signaling in response to ET-1, mesangial cells were preincubated with the PKC inhibitor chelerythrine, which attenuated both contraction and abolished Ca²⁺ signaling in response to ET-1. Furthermore, acute PMA exposure 10 min prior to ET-1 stimulation similarly abolished the Ca²⁺ response. Pretreatment with chelerythrine or acute PMA had no effect on ionomycin-stimulated Ca²⁺ responses. Taken together, these results suggest that during ET-1 signal transduction, acute activation of one or more DAG-sensitive PKC isoforms is required for stimulation of the complete Ca²⁺ response. Since acute activation of PKC with PMA did not raise intracellular Ca²⁺, it is unlikely that PKC causes release of Ca²⁺ from intracellular stores or directly opens plasma membrane Ca²⁺ channels. Instead, we postulate that activated PKC indirectly facilitates ET-1-stimulated Ca²⁺ flux either through plasma membrane or intracellular membrane channels or both. PKC regulation of calcium channel function maybe at the level of the cytoskeleton (29) or receptor G protein coupling (45). PMA for 24 h blunted the Ca²⁺ signal seen with ET-1, an effect also observed in rat vascular smooth muscle cells (44). This finding is in keeping with a role for PKC in the longer term regulation of signaling components required for mesangial cell response to ET-1. PKC may regulate the ET-1 receptor and/or receptor-coupling mechanism. Indeed, treatment of vascular smooth muscle cells with a phorbol ester initiates ET-1 receptor internalization and downregulation within 60 min of exposure (28). Furthermore, downregulation of vascular smooth muscle cell PKC blocks the sustained phase of the Ca²⁺ response dependent on L-type Ca²⁺ channels (44). Whether L-type Ca²⁺ channels in vascular smooth muscle or mesangial cells are regulated by PKC-dependent phosphorylation is not known.

In our study during ionomycin treatment, raised intracellular Ca²⁺ neither increased PKC activity in situ nor altered the cell compartmental distribution of the PKC isoforms. Although PKC- is "Ca²⁺ dependent," requiring the association of Ca²⁺ with a specific binding site for activation, it appears that raised intracellular Ca²⁺ alone is insufficient to activate PKC-. Nevertheless, Keranen and Newton (17) demonstrated in vitro that PKC- requires 0.8 ± 0.2 µM (average ± SD) Ca²⁺ for half-maximal binding and 1.5 ± 0.4 µM for half-maximal activity. The action of intracellular Ca²⁺ on PKC- activity and translocation during agonist stimulation is unclear, although our data suggest that normal (unstimulated) intracellular Ca²⁺ is sufficient for PKC- stimulation. This is supported by the fact that acute PMA caused marked translocation of PKC- in the presence of normal intracellular Ca²⁺.

ET-1 stimulated translocation of PKC- to the nucleus and periphery of the cell, observed by immunofluorescence. By immunoblot, ET-1 did not stimulate a change in PKC- cytosol content, although membrane-associated PKC- did increase. By contrast, acute PMA exposure dramatically decreased the cytosol and increased both the membrane- and particulate-associated PKC- contents. First, these data indicate that cellular compartmental analysis by immunoblot alone may be insufficient to detect translocation of PKC-. Second, agonist stimulation of PKC isoforms through receptor binding and multiple signal transduction mechanisms may involve interactive and modulating pathways leading to translocation patterns that differ from those observed with simple phorbol ester activation. Thus the pattern of translocation of PKC- stimulated by ET-1 differed remarkably from that caused by PMA, which caused a dramatic decrease in cytosolic content. Finally, the immunofluorescence imaging data suggest that a more detailed analysis of cellular compartments is necessary to analyze translocation by immunoblot. The nuclear pattern of all the PKC isoforms following ET-1 stimulation indicates that immunoblot of isolated nuclear content should be included in future analysis. Translocation to the nucleus may suggest a role for PKC-, -, and - in gene transcription.

Several mechanisms may be involved in PKC regulation of mesangial cell cytoskeletal contraction. Although Ca²⁺/calmodulin activation of myosin light chain kinase and subsequent myosin light chain phosphorylation is the most well-described mechanism described for the contractile response of smooth muscle cells, our data strongly support a principal role for agonist-stimulated PKC. Phosphorylation of vascular smooth muscle myosin light chain is regulated by PKC (40), which may be similar in mesangial cells (38). In vitro, PKC phosphorylates the actin-binding proteins caldesmon and calponin, which may lead to cellular contraction presumably due to cytoskeletal reorganization (12). Both caldesmon and calponin in the nonphosphorylated state inhibit myosin Mg²⁺-ATPase, preventing contraction (12). Once phosphorylated, they are no longer inhibitory, thus favoring contraction. Mesangial cells contain both caldesmon (15) and calponin (37). In mesangial cells, DAG-sensitive PKC isoforms activated by phorbol ester can also cause stimulation of mitogen-activated protein kinase (MAPK) of the Erk1/Erk2 family (41). In permeabilized ferret aorta cells stimulated with the -agonist phenylephrine, MAPK first codistributes with membrane-associated PKC and then targets to the contractile apparatus (19). Recent studies have revealed that MAPK coimmunoprecipitates with calponin, and calponin also coimmunoprecipitates with PKC- (23). However, only caldesmon and not calponin is phosphorylated by MAPK, suggesting that following -agonist activation of PKC-, calponin likely acts in the signaling cascade as an adaptor protein that may facilitate translocation of MAPK to the membrane with PKC- (23). There, MAPK is activated to phosphorylate caldesmon. These molecular mechanisms and the action of specific PKC isoforms require further elucidation in cultured mesangial cells and in vivo.

In summary, ET-1 activation of mesangial cell PKC is an important pathway stimulating the contractile response. PKC is necessary for the normal Ca²⁺ responsiveness of mesangial cells to ET-1. The specific DAG-sensitive PKC isoforms activated by ET-1 include PKC-, -, and -, which are translocated to membrane, cytoskeleton, and nuclear compartments. However, the atypical PKC- is not involved in the ET-1 response. Elucidation of the specific actions of these PKC isoforms regulating mesangial cell cytoskeletal function awaits further investigation.