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Am J Physiol Renal Physiol 274: F564-F572, 1998;
0363-6127/98 $5.00
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Vol. 274, Issue 3, F564-F572, March 1998

Phosphoinositide signaling in rat inner medullary collecting duct

Chung-Lin Chou, Sonia I. Rapko, and Mark A. Knepper

Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Previous studies in microdissected rat inner medullary collecting duct (IMCD) segments have demonstrated that carbachol, arginine vasopressin (AVP), and the V2 vasopressin receptor agonist 1-desamino-8-D-arginine vasopressin (DDAVP) induce a similar increase in intracellular Ca2+. The present study tested whether these agents activate the phosphoinositide hydrolysis pathway. In intracellular inositol 1,4,5-trisphosphate (IP3) measurements, we found that IMCD suspensions incubated with AVP or DDAVP (10-8 M) displayed no measurable increase in IP3, whereas IMCD suspensions incubated with the muscarinic cholinergic agent carbachol (100 µM) induced a significant increase in IP3 production. Similarly, carbachol, but not AVP or DDAVP, induced a significant increase in membrane-associated protein kinase C (PKC) enzyme activity. To test what specific PKC isoforms are activated by carbachol in IMCD, we first characterized the PKC isoforms in IMCD suspensions by immunoblotting using affinity-purified antibodies against different PKC isoforms. We identified one classic PKC isoform (alpha ), three novel PKC isoforms (delta , epsilon , eta ), and one atypical PKC isoform (zeta ) in the IMCD. Carbachol induced a cytosol-to-membrane translocation of the PKC-eta isoform but did not alter the distribution of any other isoform. In contrast, AVP had no effect on the distribution of any PKC isoform tested. These data, taken together, demonstrate that carbachol is an activator of the phosphoinositide hydrolysis pathway in IMCD but do not demonstrate signaling via this pathway in response to AVP or DDAVP. These results suggest that the previously observed AVP-stimulated Ca2+ mobilization in IMCD may be due to a mechanism other than activation of the phosphoinositide hydrolysis pathway.

inositol 1,4,5-trisphosphate; protein kinase C; vasopressin; muscarinic; phorbol ester

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

THE ANTIDIURETIC HORMONE, arginine vasopressin (AVP), plays a central role in the urine concentrating mechanism of mammalian kidney by regulating transepithelial urea and water transport across the inner medullary collecting duct (IMCD) and through other actions in the kidney. It is generally accepted that these actions of vasopressin are mediated by binding to V2 vasopressin receptors, which activate adenylyl cyclase and increase the intracellular cAMP level. In addition to increasing the cAMP level, AVP also has been shown to increase intracellular free calcium concentration ([Ca2+]i) in both microdissected IMCD segments (3, 7, 17, 23) and in cultured IMCD cells (15). Theoretically, AVP is capable of raising [Ca2+]i by binding to the V1 vasopressin receptors and activating phospholipase C, as has been seen in various cell types. The mechanism of the AVP-induced Ca2+ rise in IMCD, however, remains an unresolved issue, because the predominant vasopressin receptor in this tissue is the V2 type, whose intracellular signaling pathway is linked to activation of adenylyl cyclase. In fact, quantitative reverse transcription-polymerase chain reaction studies have not detected V1 receptor mRNA expression in the IMCD (7, 8).

Previous studies on AVP-stimulated Ca2+ mobilization in IMCD using indo 1 and fura 2 Ca2+ indicator dyes demonstrated that AVP at >= 10 nM induced a rapid (within 1 min after administration of AVP) and transient rise in [Ca2+]i (3, 7, 15, 17, 23). Furthermore, Ca2+ mobilization was also seen in response to the V2-selective vasopressin analog, 1-desamino-8-D-arginine vasopressin (DDAVP), and was blocked by a specific V2-receptor antagonist, [d(CH2)51,D-Ile2,Ile4,Arg8]vasopressin (7), but not by antagonists of the V1 or oxytocin receptor (3), suggesting that AVP-induced Ca2+ mobilization is linked to V2 receptors. The Ca2+ mobilization, however, was not seen in response to cAMP or forskolin (3, 15, 23), suggesting that the effect of AVP on [Ca2+]i is probably proximal to stimulation of the effector enzyme, adenylyl cyclase. In this context, it is noteworthy that several G protein-coupled receptors are coupled to dual signaling pathways and are capable of activating both adenylyl cyclase and phospholipase C (1, 29). Thus the demonstration of V2 receptor-mediated Ca2+ mobilization in IMCD cannot exclude the possibility that AVP may increase [Ca2+]i by activating phospholipase C and therefore stimulating the phosphoinositide hydrolysis.

A direct test to see whether AVP stimulates phosphoinositide hydrolysis is to measure the intracellular inositol 1,4,5-trisphosphate (IP3) production, a second messenger known to induce Ca2+ release from intracellular stores. However, results from two previous studies have yielded different conclusions. Teitelbaum (25), using cultured rat IMCD cells, showed that V2 agonist DDAVP stimulates IP3 production in a dose-dependent fashion (10-11-10-7 M). AVP itself stimulated IP3 production only at a low concentration (10-13 M) but not at higher concentrations. Because the IP3 response was mimicked by oxytocin and can be blocked by an oxytocin antagonist, it was concluded that AVP or V2 agonist stimulated phosphoinositide hydrolysis in IMCD cells via occupancy of oxytocin receptor. On the other hand, using freshly isolated IMCD cells from rabbit kidney, Garg and Kapturczak (9) found no increase in IP3 in response to AVP. They concluded that a vasopressin-activated phosphoinositide hydrolysis pathway is not present in IMCD. It is not clear whether the differences between two studies can be attributable to the different tissues used (freshly isolated cells vs. cultured cells) or to different species of animal (rat vs. rabbit).

Previously, our laboratory showed in isolated IMCDs that the muscarinic cholinergic agent carbachol also induces a transient increase in [Ca2+]i that has its time course and the magnitude of spike height similar to that induced by vasopressin or DDAVP (7). Carbachol has been clearly demonstrated to stimulate the phosphoinositide hydrolysis in rabbit IMCD through a muscarinic receptor in these cells (18, 19). We therefore examined whether AVP, DDAVP, and carbachol activate the phosphoinositide hydrolysis pathway in rat IMCD. To test this, we have measured the intracellular IP3 level and protein kinase C (PKC) enzyme activity in IMCD suspensions. We also performed immunoblotting experiments to characterize the PKC isoforms in IMCD and tested for agonist-induced translocation of specific PKC isoforms. With all three types of measurements, our results showed that carbachol activated phosphoinositide signaling in IMCD suspensions but did not demonstrate a similar effect of AVP or DDAVP. Therefore, the present study does not support a role for phosphoinositide hydrolysis in AVP-stimulated Ca2+ signaling in the rat IMCD.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

IMCD Suspension Preparation

IMCD suspensions prepared from 250- to 300-g Sprague-Dawley rat (Taconic Farm, Germantown, NY) kidney were used throughout this study. IMCD suspensions were prepared based on the method of Stokes (24). The rats had free access to drinking water and rodent diet (NIH-31; Zeigler Bros., Gardner, PA) prior to the experiment. In brief, after death of the rats by decapitation, the kidneys were quickly removed and placed in ice-chilled bicarbonate-buffer suspension fluid (in mM: 118 NaCl, 25 NaHCO3, 5 KCl, 4 Na2HPO4, 2 CaCl2, 1.2 MgSO4, 5.5 glucose). The inner medullas were dissected, minced to ~1-mm3 cubes, and incubated with digestion solution (2 mg/ml collagenase B and 600 U/ml hyaluronidase in bicarbonate buffer solution) for 60 min and for another 20 min in digestion solution containing 0.001% DNase I. The suspension was gently aspirated through a large-bore Pasteur pipette every 20 min to facilitate the digestion.

At the end of incubation period, the whole inner medulla suspensions were centrifuged at 80 g for 10 s. The supernatant containing the lighter, thin structures was carefully removed. The pellet containing predominantly the IMCD fragments was washed with suspension fluid containing 0.1% BSA. This low-speed centrifugation and wash procedure was repeated twice. The final IMCD suspensions were placed on ice for 10 min for recovery. After removal of the supernatant, the tubule fragments were resuspended with the desired volume of suspension fluid, divided into aliquots in polypropylene tubes (Falcon 2063), and warmed to 37°C before studies. These IMCD suspensions normally contain a mixture of fragments of IMCD tubules and clumps of IMCD cells and a few thin structures (thin limbs and vasa recta).

Intracellular IP3 Measurement

Sample preparation. The aliquots of IMCD suspensions (~0.4 mg protein/100 µl) were preincubated with 10 mM LiCl for 10 min before exposure to different agents (50 µl). After the indicated incubation period with various agonists, the reaction was arrested by adding 150 µl of 15% ice-cold TCA and stored on ice for 10 min to lyse the cells. The samples were subjected to a 2,300-g centrifugation for 10 min (4°C). The supernatant was transferred to a siliconized microcentrifuge tube for IP3 assay; the remaining pellet was used for protein measurement by Bradford method (Bio-Rad protein assay).

IP3 assay. The intracellular IP3 was determined by the competitive radioligand binding assay measuring the displacement of [3H]IP3 from a binding protein prepared from bovine adrenal glands. The assay system was purchased from Amersham as a kit (TRK 1000; Amersham, Arlington Heights, IL). Before the IP3 measurement, TCA was extracted by 1 ml of water-saturated ethyl ether four times. Samples were then titrated to pH 7.5 by adding 1.5 M KOH containing 60 mM HEPES, dried in a Speed Vac, and reconstituted with 100 µl of assay buffer. The reconstituted samples were incubated with [3H]IP3 and binding protein in centrifuge tubes on ice for 15 min. After a centrifugation at 3,000 g for 10 min at 4°C, the supernatant was discarded to remove the unbound isotopic IP3, and the residual fluid was allowed to desiccate. The microcentrifuge tube containing the pellet was cut off with a razor blade into a scintillation vial, dissolved in 1 ml of water, and mixed with scintillation fluid to count its radioactivity. The amount of IP3 in each sample was read from the standard curve of 0-25 pmol IP3.

Protein Kinase C Assay

Sample preparation. To study the ligand-activated PKC enzyme activity, IMCD suspensions were divided into aliquots in polypropylene tubes (0.4 mg protein/200 µl) and incubated with agents (100 µl) at 37°C for 1 min. The reaction was stopped by adding 2 ml of ice-cold suspension fluid followed by a 5,600-g centrifugation at 4°C for 20 s to harvest the cells. IMCD cells were homogenized in 500 µl homogenization buffer (in mM: 50 Tris, 10 EGTA, 5 EDTA, 250 sucrose, and 10% glycerol) containing beta -mercaptoethanol (0.3% wt/vol), 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 mM benzamidine, using a motor-driven homogenizer at 15,000 rpm. This homogenate was centrifuged at 100,000 g for 1 h. The supernatant was collected as the "cytosolic fraction." The pellet was treated with 200 µl homogenization buffer containing 0.1% Triton X-100 for 1 h on ice, followed by another 100,000-g centrifugation for 1 h. This supernatant was collected as the "Triton-soluble membrane fraction."

PKC enzyme activity. The PKC enzyme activity assay is based on the ability of PKC in the sample to catalyze the transfer of the gamma -32P of ATP to the threonine group on a substrate peptide (histone type IIIS). The assay system was purchased from Amersham as a kit (Amersham RPN 77). The Triton-soluble membrane fraction was diluted to 0.03% Triton X-100 before PKC activity assay. In this assay, 25 µl of cytosolic or membrane samples were first mixed with 25 µl of 30°C prewarmed reaction mixture containing 3 mM calcium acetate, phosphatidyl-L-serine 2%, 6 µg/ml phorbol 12-myristate 13-acetate (PMA), 225 µM peptide, and 7.5 mM dithiothreitol. To start the reaction, 25 µl of ice-cold Mg-[32P]ATP were added to the sample and allowed to incubate for 10 min at 30°C. The reaction was stopped by adding 100 µl of stop reagent. To separate the phosphorylated peptide, 25 µl of reaction mixture were pipetted onto a phosphocellulose membrane (Pierce phosphocellulose unit) and centrifuged at 3,000 g for 30 s. The membrane was washed twice with 500 µl of 75 mM phosphoric acid and transferred into scintillation vial for counting its radioactivity. To measure the Ca2+- and phospholipid-independent PKC activity, the cytosolic and membrane samples were mixed with reaction mixture in the absence of calcium acetate, phosphatidyl-L-serine, and PMA.

Electrophoresis and immunoblotting of membranes. To study the translocation of PKC isoforms, the cytosolic and membrane fraction samples were dissolved in Laemmli buffer. Sample proteins were resolved by SDS-PAGE, using 8% Tris glycine gels (Novex, San Diego, CA), and were electrophoretically transferred onto nitrocellulose membranes. The blots were blocked for 1 h with 5% nonfat dry milk in wash buffer (42 mM Na2HPO4, 8 mM NaH2PO4, 150 mM NaCl, 0.05% Tween-20, pH 7.5), washed in wash buffer, and incubated with primary antibody overnight at 4°C. The immune complexes were detected with horseradish peroxidase-conjugated donkey anti-rabbit IgG in 1:5,000 dilution (Pierce, Rockford, IL). Sites of antibody-antigen reaction were visualized using an enhanced chemiluminescence detection kit (Kirkegaard & Perry, Gaithersburg, MD). The optical density was analyzed by scanning densitometry (Molecular Dynamics, Sunnyvale, CA) and normalized by the amount of protein (in µg) loaded in each lane.

Intracellular Calcium Measurement

To measure the intracellular calcium, IMCD suspensions were prepared from kidney inner medullas of two rats as described earlier, except that they were washed with HEPES-buffered suspension fluid (in mM: 118 NaCl, 5 KCl, 4 Na2HPO4, 2 CaCl2, 1.2 MgSO4, 5.5 glucose, 10 HEPES, pH 7.4) after incubation with collagenase and hyaluronidase. To load the suspensions with fura 2, 150 µl of calcium indicator mixture was added to 1,850 µl of IMCD suspension, and the suspension was incubated at room temperature for 1 h. The calcium indicator mixture contains 50 µg fura 2-AM in 7.5 µl DMSO, 7.5 µl Pluronic F-127 (Molecular Probes, Eugene, OR), and 135 µl HEPES-buffered suspension fluid. The final fura 2-AM concentration after addition to the suspensions was 25 µM. After the loading period, the IMCD suspensions were washed with HEPES-buffered suspension fluid three times and resuspended to a final volume of 2 ml. For each measurement, 1 ml of IMCD suspension was transferred into cuvette containing a micro spin bar for continuous mixing. The intracellular calcium measurements were carried out at room temperature in a SPEX spectrofluorometer (Edison, NY). The emission intensity at 506 nm was measured with excitation at 340 and 380 nm. The intracellular calcium signals were presented as fluorescence intensity ratios (340 nm/380 nm) as a function of time.

Statistics

Data are presented as means ± SE. Comparisons between groups were made by paired or unpaired Student's t-test, as appropriate.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Effect of Carbachol, AVP, and DDAVP on Intracellular IP3 Production

As an initial test of whether AVP and carbachol activate the phosphoinositide hydrolysis pathway, we first compared the effects of 100 µM carbachol, 10-8 M AVP, and 10-8 M DDAVP on intracellular IP3 level in IMCD suspensions. Previous studies demonstrated that these concentrations of each agent elicited a similar increase in [Ca2+]i in microdissected IMCD segments as described in the introduction. The results from five experiments (Fig. 1) showed that only carbachol produced a statistically significant increase in IP3 production compared with vehicle alone (37 ± 9%, P < 0.05, n = 5). IMCD suspensions incubated with AVP or DDAVP displayed no measurable increase in intracellular IP3 (Fig. 1). Thus, despite the fact that all three agents increased the intracellular Ca2+ in IMCD, only carbachol also raised the intracellular IP3 level.


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  		Fig. 1.   Effects of carbachol, arginine vasopressin (AVP), and 1-desamino-8-D-arginine vasopressin (DDAVP) on inositol 1,4,5-trisphosphate (IP3) production of inner medullary collecting duct (IMCD) suspensions. IMCD suspensions in aliquots were incubated with vehicle alone, 100 µM carbachol, 10-8 M AVP, or 10-8 M DDAVP at 37°C for 0.5-1 min. Mean intracellular IP3 production (pmol/mg protein): control, 11.8 ± 1.2; carbachol, 16.1 ± 2.1; AVP, 12.1 ± 1.1; and DDAVP, 11.6 ± 1.5. Only IMCD incubated with carbachol exhibited an increase in IP3 level above the control group; n, no. of experiments. * P < 0.05.

Effect of Carbachol, AVP, and DDAVP on PKC Activity

We went on to test the effect of the same agents on PKC enzyme activity. The PKC enzyme activity in the membrane fraction of IMCD suspensions was measured in the presence (total activity) and in the absence (background activity) of Ca2+ and phosphatidylserine. The results from five experiments, illustrated in Fig. 2, show that IMCD suspensions incubated with carbachol exhibited a significant increase (P < 0.01, n = 5) in total PKC activity in the membrane samples without having a significant change in the background activity. The calculated difference between two measurements (Fig. 2, right) indicates a 53 ± 10% increase (P < 0.01, n = 5) in membrane Ca2+/phosphatidylserine-dependent PKC activity in response to carbachol incubation. In these experiments, a small decrease by 13 ± 3% in cytosol PKC activity was also observed in carbachol-treated samples and was statistically significant (P < 0.05, data not shown). In contrast, neither AVP nor DDAVP had a significant effect on PKC activity in the cytosolic or the membrane samples.


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  		Fig. 2.   Effect of carbachol, AVP, and DDAVP on membrane-associated protein kinase C (PKC) activity of IMCD suspensions. IMCD suspensions in aliquots were incubated with vehicle alone (open bars), 100 µM carbachol (solid bars), 10-8 M AVP (hatched bars), or 10-8 M DDAVP (stippled bars) at 37°C for 1 min. Membrane fraction of IMCD suspensions was prepared and subjected to PKC activity measurement. PKC enzyme activity of each sample was measured in the presence (+) and in the absence (-) of both Ca2+ and phosphatidylserine. Calculated difference between two measurements represents PKC activity that is sensitive to the presence of Ca2+ or phosphatidylserine or to both the Ca2+ and phosphatidylserine in the assay system. * P < 0.01.

Characterization of PKC Isoforms in IMCD

PKC is a family of at least eleven isoforms divided into three distinct classes (the classic, the novel, and the atypical), depending on their sensitivity to Ca2+, phospholipid, and diacylglycerol (20). To further explore whether AVP or carbachol has any effect on the specific isoforms, we first determined what PKC isoforms are expressed in IMCD. To do this, the inner medullary suspensions were subjected either to a 1,000-g centrifugation for 5 min to pellet all cell types, yielding a whole inner medulla (whole IM) sample, or to three low-speed centrifugations (80 g, 10 s) to separate most of the thin structures in the inner medulla in supernatant (non-IMCD) from the pellet enriched with the IMCD fragments (IMCD). Samples from all three components were then homogenized and dissolved in Laemmli buffer before loading for immunoblotting. The enrichment of IMCD cells in IMCD suspensions was compared with aquaporin-2 (AQP-2), an IMCD marker, and aquaporin-1 (AQP-1), a thin limb marker, shown in Fig. 3B. Previous immunoelectron microscopic studies have demonstrated that AQP-1 and AQP-2 are the predominant water channel proteins in inner medullary thin descending limb and collecting duct, respectively.


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  		Fig. 3.   Characterization of PKC isoforms in rat kidney inner medulla. A: PKC isoforms distributed in two different components of the renal inner medulla were compared. Samples were prepared as follows. Inner medulla was dissected, minced, and enzymatically digested into suspensions as described in METHODS. One-third of the inner medulla suspension was collected without fractionation at 1,000-g centrifugation as whole inner medulla (whole IM). Remaining two-thirds of the inner medulla suspension was subjected to three low-speed centrifugations (80 g, 10 s) to separate the IMCD-enriched fragments in the pellets (IMCD) from the lighter non-IMCD structures in the supernatants (non-IMCD). Samples were homogenized and dissolved in Laemmli buffer before loading for SDS-PAGE. Antibodies for PKC-alpha , -beta , -gamma , -delta , -epsilon , and -zeta were purchased from Life Technologies (Gaithersburg, MD) and used at a concentration of 1.5 µg/ml. Antibodies for PKC-eta and -theta were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and used at a concentration of 0.2 µg/ml. B: enrichment of IMCD in suspension preparation as tested by the presence of water channel proteins, aquaporins AQP-1 and AQP-2. AQP-1 was present predominantly in the non-IMCD, whereas AQP-2 was present predominantly in the IMCD suspensions. Distributions are consistent with the known localization of these proteins in the thin descending limb and the collecting duct, respectively.

Eight commercially available affinity-purified polyclonal antibodies to different PKC isoforms (alpha , beta , gamma , delta , epsilon , zeta , eta , theta ) were tested. The results, shown in Fig. 3A, illustrate the presence of one classic PKC isoform (alpha ), four novel PKC isoforms (delta , epsilon , eta , theta ), and one atypical PKC isoforms (zeta ) in inner medulla. Neither PKC-beta nor -gamma was detected. Among the six isoforms detected in the inner medulla, only PKC-zeta was enriched in the IMCD suspensions relative to the whole inner medulla homogenates. PKC-epsilon was more abundant in IMCD than in non-IMCD fractions. PKC-delta and -eta were nearly equally distributed between the IMCD and non-IMCD components of the inner medulla. PKC-alpha was most abundant in non-IMCD structures but was also present in IMCD cells. PKC-theta was enriched in the non-IMCD component. The small amount of PKC-theta detected in IMCD component might be attributable to contamination of thin limbs in the IMCD component based on a comparison with the AQP-1 immunoblot (Fig. 3B). The calculated enrichment factor of each PKC isoform, as defined by the ratio of amount of specific protein in IMCD component to amount of specific protein in whole inner medulla, was displayed in Table 1. Figure 4 shows the preadsorption controls, using the immunizing peptides confirming the specificity of the appropriate bands.

                              
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  		Table 1.   Enrichment of PKC isoforms in IMCD suspensions


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  		Fig. 4.   Immunospecificity of PKC antibodies (Ab). Preadsorption control using the immunizing peptides confirmed the specificity of the appropriate bands in IMCD homogenates. Each PKC antibody was preincubated with the corresponding immunizing peptide at 4°C for 5 h before use.

Carbachol- and Vasopressin-Mediated Translocation of PKC Isoforms

To test what PKC isoform can be activated by carbachol or vasopressin, we performed translocation studies. The amount of PKC protein in the cytosolic and the membrane fractions derived from IMCD suspensions treated with different agents were compared by immunoblotting (Fig. 5) with analysis by densitometry (Fig. 6).The calculated membrane-to-cytosol PKC ratio, used as index of translocation of specific isoform, is summarized in Table 2. In this series of experiments, we have included the effect of phorbol ester (PMA), a potent activator of PKC, as a positive control for the translocation event. As shown in Fig. 5, PMA strongly decreased the amount of PKC-alpha , -delta , -epsilon , and -eta in the cytosol but had no effect on PKC-zeta . PMA significantly increased the membrane PKC-delta and -eta , although it appeared to reduce the membrane PKC-alpha and -epsilon . Moreover, in all PKC isoforms tested, except for PKC-zeta , PMA markedly increased the membrane-to-cytosol PKC ratio by severalfold (Table 2), demonstrating PMA activated the PKC isoforms of the classic (alpha ) and the novel (delta , epsilon , eta ) classes but not PKC of the atypical class (zeta ).


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  		Fig. 5.   Immunoblot detection of translocation of PKC isoforms in IMCD suspensions. IMCD suspensions were incubated with vehicle, 100 µM carbachol, 10-8 M AVP, or 4 µM phorbol 12-myristate 13-acetate (PMA) at 37°C for 1 min. Cytosolic and the Triton-soluble membrane fractions of each sample were prepared as described in METHODS. Immunoblots show typical response of each individual PKC isoform induced by tested agents in 4 experiments. Display in each column is same exposure of same blot. PMA is a known potentiator of the PKC isoforms of the classic and the novel classes but not of the atypical class, as was demonstrated here.


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  		Fig. 6.   Result of densitometric analysis. Densitometric analysis of experiments shown in Fig. 5. Percentage change from control value of PKC amount in the cytosolic fraction (Cytos.), in the membrane fraction (Membr.) of IMCD suspensions, and the calculated membrane-to-cytosol PKC ratio (Membr./Cytos.) was presented. Open bars, vehicle-treated IMCD; solid bars, carbachol-treated IMCD; hatched bars, AVP-treated IMCD. Cytosolic PKC-delta and -epsilon tended to be increased slightly, but not statistically significantly, by AVP. Because increased cytosolic PKC and reduced membrane-to-cytosol PKC ratios were in opposite direction to those expected for PKC activation as demonstrated by PMA, significance of these changes is unknown.

                              
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  		Table 2.   Calculated membrane-to-cytosol ratio of PKC isoforms in IMCD

As also demonstrated in Fig. 5, carbachol stimulated translocation of PKC-eta as seen with PMA but with less potency. IMCD suspensions incubated with carbachol exhibited a reduced cytosol PKC-eta by 51 ± 7%, which was associated with an increased membrane PKC-eta by 40 ± 19% (Fig. 6). A calculated 228% increase in the membrane-to-cytosol PKC-eta ratio demonstrates the translocation of this isoform in response to carbachol (Fig. 6, Table 2). In contrast, AVP has no effect on redistribution of PKC-eta among subcellular fractions.

Neither carbachol nor AVP caused a statistically significant change in amount of other PKC isoforms tested (alpha , delta , epsilon , and zeta ) in the membrane and cytosolic fractions of the IMCD cells (Fig. 6), and there is no significant difference in the calculated membrane-to-cytosol ratios of these PKC isoforms compared with the value of the control group (Fig. 6, Table 2).

Effects of Carbachol, AVP, and DDAVP on Intracellular Calcium

To confirm that the IMCD cells, as prepared for these experiments, exhibit increases in intracellular calcium in response to agonists, as reported previously in isolated tubules (7), we carried out intracellular calcium measurements in IMCD suspensions using fura 2 indicator dye. Figure 7 depicts typical intracellular calcium responses induced by each agent. As can be seen, carbachol (100 µM), AVP (10-8 M), and DDAVP (10-8 M) induced increases in intracellular calcium in IMCD suspensions (n = 4). Whereas AVP and DDAVP produce transient intracellular calcium increases, carbachol produces a more sustained increase in intracellular calcium.


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  		Fig. 7.   Intracellular calcium measurements in IMCD suspensions. Change in the intracellular calcium signal, expressed as the emission ratio at 340 and 380 nm, in response to DDAVP (10-8 M), AVP (10-8 M), or carbachol (100 µM) was measured in IMCD suspensions at room temperature. After an initial 2 min basal period, 10 µl of vehicle (A) or DDAVP (B), AVP (C), or carbachol (D), each at 100 times final concentration, were injected into the IMCD suspension through PE-50 tubing.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

In this study, we have demonstrated that carbachol, at a concentration associated with an increase in [Ca2+]i in IMCD segments, significantly increased the intracellular IP3 level, increased the PKC enzyme activity, and caused the translocation of PKC-eta in IMCD suspensions. These results suggest that the previously observed carbachol-induced Ca2+ rise in IMCD may be attributable to activation of the phosphoinositide pathway. However, vasopressin at the concentration previously used to induce calcium mobilization in IMCD did not have a significant effect on IP3 production or PKC activity and did not cause redistribution of any PKC isoform in IMCD membrane fractions. This suggests that the intracellular Ca2+ rise induced by AVP may be caused by mechanism other than hydrolysis of the phosphoinositide pathway.

The muscarinic cholinergic agent, carbachol, was chosen as the positive control for activating hydrolysis of phosphoinositide pathway for several reasons. First, a cholinergic-responsive phosphoinositide hydrolysis has been reported in IMCD cells isolated from rabbit kidney (18). Moreover, using a receptor binding technique, the same group had demonstrated the presence of specific high-affinity cholinergic receptor in isolated rabbit IMCD cells (19). Second, using the isolated tubule microperfusion technique, Han et al. (12) showed that carbachol had an inhibitory effect on osmotic water permeability of IMCD tubules, which can be blocked by a specific PKC inhibitor, calphostin C (12). Carbachol itself, however, has no effect on cAMP level. These data suggest that carbachol is indeed an activator of PKC in rat IMCD segments. Third, results from previous studies of our laboratory had shown that the intracellular Ca2+ increases in response to AVP and DDAVP are similar in magnitude to those in response to carbachol (7). Thus, if AVP-induced calcium mobilization is mediated via the same mechanism as the one that carbachol induces, i.e, the phophoinositide hydrolysis pathway, one would expect to see a change in IP3 production or PKC activity similar to those induced by carbachol.

In the present study, carbachol increased IP3 production in IMCD suspensions, but AVP and DDAVP had no effect. The IP3 measurement in the present study was a direct mass measurement of IP3. In the previous study, using more sensitive radiolabeling measurements, AVP even at higher concentrations did not change IP3 production in rabbit IMCD cells (9). Thus our result agrees with the conclusion drawn previously by Garg and Kapturczak (9) that vasopressin-induced phosphoinositide hydrolysis was not present in IMCD cells.

Activation of phosphoinositide hydrolysis is generally associated with activation of PKC activity. After an increase in [Ca2+]i, PKC translocates from cytosol to plasma membrane of the cell, where it interacts with phosphatidylserine. The increase in IP3 production in response to agonist stimulation prompted us to examine the effect of tested agents on PKC activity. In parallel to the IP3 results, we found that only IMCD incubated with carbachol displayed a significant increased PKC activity in membrane fractions. On the other hand, AVP and DDAVP, which produced no measurable effect on IP3 production, also had no significant effect on PKC activity. Thus measurements of IP3 and PKC activity indicate that carbachol is an activator of phosphoinositide pathway in IMCD cells but do not provide evidence for a similar role of AVP or DDAVP.

To further explore the relative roles of carbachol and AVP, we carried out immunoblotting studies. PKC is a family of at least eleven closely related isoforms, classified into three groups (20): the classic Ca2+-phospholipid-diacylglycerol dependent (alpha , beta I, beta II, gamma ), the novel (or new) Ca2+ independent but phospholipid and diacylglycerol dependent (delta , epsilon , eta , theta , µ), and the atypical Ca2+-diacylglycerol independent but phospholipid dependent (zeta , lambda ). Previously, the distribution of the various PKC isoforms have been characterized in the whole kidney (6, 21), in the inner medulla (6, 21), in various renal sites, such as glomeruli (14), proximal tubule (16), and cortical collecting duct (5, 28), but not in IMCD. In the present study, we have identified five PKC isoforms (alpha , delta , epsilon , eta , and zeta ) in rat IMCD. Among these, PKC-delta , -epsilon , eta , and zeta  were present in relatively large amounts in IMCD suspensions. Therefore, the predominant PKC isoforms in IMCD are the Ca2+-independent PKCs. Previous immunoblotting studies also identified several PKC isoforms in immunodissected cortical collecting duct cells from rabbit kidney (5, 28). DeCoy et al. (5) had identified two PKC isoforms (epsilon  and zeta ), whereas Wilborn and Schafer (28) had identified five PKC isoforms (alpha , epsilon , eta , theta , and zeta ).1

Furthermore, using the cytosolic and membrane samples prepared from IMCD suspensions, we also demonstrated by immunoblotting that carbachol induced specifically the translocation of PKC-eta , but not other isoforms, from the cytosolic to the membrane fraction of the IMCD cells. This result suggests that PKC-eta (Ca2+ independent but phospholipid dependent) is the isoform largely responsible for the increased PKC enzyme activity induced by carbachol. AVP, however, did not induce translocation of any PKC isoform. In contrast, PMA induced the translocation of classical (alpha ) and novel PKC isoforms (delta , epsilon , eta ) but not atypical PKC-zeta , a result consistent with previous observations that PKC-zeta is insensitive to phorbol esters (4, 27).

Activation of PKC has been implicated in regulation of epithelial salt and water transport in a variety of tissues and cell lines. However, it was not until recently that the role of specific PKC isoforms was identified. For example, using antisense DNA to downregulate PKC-epsilon in the cortical collecting duct cells, DeCoy et al. (5) demonstrated that AVP induced a sustained increase in Na+ reabsorption that would normally appear only transiently in the untreated cells. This study therefore suggests an important role for PKC-epsilon in the regulation of vasopressin-stimulated salt transport in cortical collecting duct principal cells. Similarly, previous studies from our laboratory and others have shown that agents known to activate phosphoinositide hydrolysis, such as PMA, prostaglandin, and carbachol, had inhibitory effects on transepithelial water transport of collecting duct, which can be blocked by PKC inhibitors (12, 13). These studies suggest that activation of PKC plays an important role in regulating transepithelial water transport of collecting duct. In the present study, we have demonstrated that carbachol activates PKC activity and causes the translocation of PKC-eta . These findings suggest a potential role for PKC-eta in the regulation of water transport in IMCD, although the present study contains no direct evidence to prove it.

The demonstration of activation of PKC by carbachol in the present study and the demonstration of specific muscarinic cholinergic receptors in IMCD cells in the previous studies (19), together with known effects of carbachol on modulation of transepithelial water permeability (12) and Na-K-ATPase activity (10), suggest a physiologically significant role for cholinergic agents in regulating salt and water excretion. Indeed, acetylcholine is known to produce diuresis in mammalian kidney when infused via the renal artery. However, it is not known whether cholinergic agonists like acetylcholine are present in the inner medulla. Previous studies using histochemical labeling approaches have not been able to demonstrate direct parasympathetic innervation in the kidney (2, 11). The only evidence supporting cholinergic innervation in the kidney has been from the work of Pirola et al. (22), who demonstrated in the dog renal cortex the existence of high-affinity choline uptake and the presence of choline acetyltransferase, the enzyme that converts choline to acetylcholine.

It is evident from previous studies that AVP induces Ca2+ mobilization in both cultured and freshly isolated IMCD cells. None of the three different measurements of phosphoinositide signaling carried out in the present study provided evidence for the idea that AVP activates phosphoinositide hydrolysis, leaving the AVP-stimulated Ca2+ mobilization in IMCD a mystery. Presumably, any rise in [Ca2+]i can be attributable to calcium entry from extracellular fluid (either via activating the voltage-operated Ca2+ channel or the receptor-operated Ca2+ channel) or to calcium release from the intracellular stores. Possible calcium entry from extracellular fluid is questionable, because previous studies demonstrated a AVP-induced [Ca2+]i rise in the Ca2+-free medium (15). With regard to Ca2+ release from the intracellular stores, two distinct classes of intracellular calcium channels have been identified. One class is the IP3-sensitive calcium channel (IP3 receptor), which releases intracellular Ca2+ on binding of IP3. Our results do not support a role for AVP to increase intracellular IP3 and hence increase [Ca2+]i via this pathway. A second class of agonist-activated intracellular calcium channels is the ryanodine receptors. Intracellular Ca2+ released from this receptor type has been demonstrated pharmacologically, using agents such as caffeine and ryanodine. This receptor is known to be unresponsive to IP3. To date, three different ryanodine receptor isoforms have been identified, mainly in the excitable cells. Type 1 is expressed predominantly in skeletal muscle cells, type 2 is expressed predominantly in cardiac muscle cells, and type 3 is expressed predominantly in brain. Recently, Tunwell and Lai (26) have demonstrated the presence of type 2 ryanodine receptor protein in rabbit kidney. Whether a ryanodine receptor is expressed in IMCD cells and whether there is a role for the ryanodine receptor in AVP-stimulated Ca2+ increase remains to be tested.

    NOTE ADDED IN PROOF

After this study was prepared, Aristimuño and Good reported the presence of PKC isoforms alpha , beta II, delta , epsilon , and zeta  in rat medullary thick ascending limbs [Am. J. Physiol. 272 (Renal Physiol. 41):F624-F631, 1997].

    ACKNOWLEDGEMENTS

We thank Dr. Maurice Burg for critical reading of this manuscript.

    FOOTNOTES

1 See NOTE ADDED IN PROOF.

Address for reprint requests: C.-L. Chou, LKEM/NHLBI, Bldg. 10, Rm. 6N260, 10 Center Dr., MSC 1603, National Institutes of Health, Bethesda, MD 20892-1603.

Received 9 May 1997; accepted in final form 11 December 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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AJP Renal Physiol 274(3):F564-F572
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