©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Vasopressin-stimulated Electrogenic Sodium Transport in A6 Cells Is Linked to a Ca-mobilizing Signal Mechanism (*)

John P. Hayslett (1)(§), Lawrence J. Macala (1), Joan I. Smallwood (1), Leena Kalghatgi (1), Jose Gassala-Herraiz (2), Carlos Isales (2)

From the (1)Department of Internal Medicine, Yale School of Medicine, New Haven, Connecticut 06510 and the (2)Institute for Molecular Medicine, Medical College of Georgia, Augusta, Georgia 30912

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Vasopressin is known to activate two types of cell surface receptors; V coupled to adenylate cyclase, and V linked to a Ca-dependent transduction system. We investigated whether arginine vasopressin (AVP) stimulation of electrogenic sodium transport in A6 cells, derived from Xenopus laevis, is mediated by activation of either one or both types of AVP-specific receptors.

AVP caused a rapid increase in electrogenic sodium transport, reflected by the transepithelial potential difference (V) and equivalent short circuit current (I) measurements. AVP also rapidly increased intracellular Ca (Ca) and total inositol trisphosphate. The increase in I was dependent on the rise in (Ca), because 1,2-bis(2-aminophenoxy)ethane-N,N,N`,N`-tetraacetic acid (BAPTA) dose-dependently inhibited the I response. There was no evidence, however, that activation of adenylate cyclase mediated AVP-stimulated I; transport was not inhibited after AVP-induced activation of adenylate cyclase was abolished by 2`,5`-dideoxyadenosine or when cAMP-dependent protein kinase (PKA) activity was abolished by the specific PKA inhibitor IP. Further studies showed that although both forskolin and 8-(4-chlorophenylthio)-cAMP stimulated I, this occurred by mechanisms independent of PKA activation.

These results indicate that AVP-stimulated Na transport is mediated by a V receptor and a Ca- dependent mechanism.


INTRODUCTION

Vasopressin (AVP)()stimulates hydraulic water transport and electrogenic sodium transport in the collecting duct epithelial cells of mammalian kidney and in some amphibian epithelia(1) . Pioneering studies by Orloff and Handler (2) suggested that cAMP acts as a second messenger for both of these effects. Subsequent studies confirmed that activation of adenylate cyclase, and thus the generation of cAMP, mediates AVP stimulation of water transport, because inhibition of the enzyme with 2`,5`-dideoxyadenosine (DDA) abolishes the response(3) . However, the role of cAMP in AVP stimulation of electrogenic Na transport remains in question, for there are as yet no studies demonstrating a direct dependence of Na transport on cAMP generation.

AVP can bind and activate two types of cell surface receptors: V receptors, present in renal epithelial cells and some amphibian epithelia, are coupled to adenylate cyclase via a cholera toxin-sensitive G protein, whereas V receptors, present mainly in smooth muscle, mesangial cells, hepatocytes, and central nervous system neurons, are linked to a calcium-dependent transduction system(4) . Recent studies, however, have demonstrated V receptors in rabbit cortical collecting tubule cells (5). In addition, we have reported that a calcium-dependent mechanism mediates aldosterone(6) -, adenosine(7) -, and insulin-stimulated (8) electrogenic sodium transport in amphibian renal epithelial (A6) cells. We have therefore investigated whether AVP stimulation of Na transport is similarly linked to a Ca-dependent messenger system via activation of a V or V-like receptor.


MATERIALS AND METHODS

Na Transport

Experiments were performed on a clone of A6 cells derived from the kidney of Xenopus laevis. These cells, provided by Gregory Grillo, Walter Reed Research Institute, were obtained from the American Tissue Type Collection and cloned by limiting dilution (clone A6-S2). The methods employed for cell culture, measurement of net sodium transport, and study of inhibitory agents have been reported recently(7) . In brief, cells were grown to confluence on porous membranes (Millicell-HA cups, Millipore Corp., Bedford, MA) and studied when fully differentiated 10-14 days after subculture. Net Na transport was determined from the equivalent short circuit (I), calculated from the open circuit measurements of V and R using Ohm's law, which represents the net current associated with active ion transport when V = O mV. Data are expressed as the change in I above the initial level observed in control and experimental monolayers at 30 min after addition of agonist or control vehicle. The agonist or vehicle was added to media on the basal surface of monolayers in a volume that was 1% of the media volume. Although basal I varied between different passages, agonist-induced changes were proportional to basal values.

The transepithelial potential difference (V) was used to estimate the time of onset of agonist-induced changes in Na transport, because this parameter is more sensitive to rapid changes than measurement of I. V changes in parallel with I. Continuous recordings were made with a strip recorder before and after addition of AVP. To avoid transient disturbances in culture media, culture cups were maintained in an incubator at 27 °C.

Intracellular Ca (Cai)

Cai was measured in suspended cells which had been grown to confluence on porous Falcon membranes with an active surface of 4.5 cm. These larger membranes were used to obtain a yield appropriate for the assay. Previous studies demonstrated that basal and hormone-stimulated changes in I were comparable when cells were grown on Millipore HA culture cups or on Falcon membranes (data not shown). Porous membranes were employed, because cells grown on a nonporous surface, such as glass or culture plastic, did not generate cAMP in response to AVP, suggesting that such growth conditions altered receptor expression and/or availability. However, the transparent porous membranes were found to be unsuitable for measurement of Ca, because autofluorescence prohibited adequate signal to noise ratios. Therefore, suspended cells were used, recovered by gentle scraping in phosphate-buffered saline with 5 mM EGTA (no trypsin).

The method used to measure Ca with Fura-2/AM was recently reported(7) . The use of suspended cells prohibited rapid removal of Fura-2 leaked to the external bathing solution which was detectable after about 5 min. Continuous recordings of Ca, therefore, did not exceed approximately 5 min.

Inositol Trisphosphate (IP)

The methods used to measure total cellular IP were previously reported(7) . Cells were grown to confluence on Falcon membranes. In brief, total IP was measured by high pressure liquid chromatography from cells labeled with myo-[H]inositol (50 µCi/ml) for 48 h in inositol free amphibian Ringer's solution. Preliminary studies demonstrated that under this condition basal and hormone-stimulated I values were comparable with those in normal culture media (data not shown). LiCl was not added to the external bathing solution.

Cyclic AMP

Total cellular cAMP was measured as described (7) with a radioimmunoassay kit obtained from Biomedical Technologies Inc. (Stoughton, MA). Confluent cells grown on Falcon membranes were exposed to the phosphodiesterase inhibitor RO-201724 (100 µM) for 15 min before addition of agonist. Following a 10-min treatment with agonist, the reactions were terminated by addition of cold 6% trichloroacetic acid. After centrifugation to remove precipitated material, trichloroacetic acid was extracted by the Freon/tri-N-octylamine technique(9) . Results are expressed as total cAMP accumulated at 10 min/mg of protein.

cAMP-dependent Protein Kinase (PKA) Measurements

The activity of PKA was determined with a commercial kit (Promega, Inc., Madison, WI) that measures the phosphorylation of a fluorescently labeled substrate peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide)(10) . Subsequent electrophoresis in an 0.8% agarose gel provides clear separation of the substrate species: the phosphorylated species migrates toward the anode, whereas the unphosphorylated remainder migrates toward the cathode. The measured fluorescence of the phosphorylated band, visualized under UV light, is directly proportional to the amount of peptide phosphorylated by active kinase.

Monolayers of A6 cells, grown to confluence on Falcon membranes, were treated for 30 min with AVP and were then immediately placed on ice and washed with ice-cold phosphate-buffered saline (pH 7.2). The phosphate-buffered saline was aspirated from the apical side and replaced with 250 µl of 20 mM Tris-HCl buffer (pH 7.4), containing 10 µM leupeptin, 25 µg/ml aprotinin, 1 mM disodium pyrophosphate, 1 mM 1,4-dithiothreitol, and 1 mM EGTA. Cells were scraped from the membrane with a rubber policeman and homogenized by probe sonication for 15 s (Kontes, model KT-50 Micro Ultrasonic Cell Disrupter). Samples were aliquoted and stored at -70 °C until assayed.

The PKA assay reaction was run with 6-9 µg of homogenate protein, 1 mM ATP, 10 mM MgCl, 20 mM Tris-HCl (pH 7.4), and 60 µM fluorescently tagged Kemptide (PepTag A1 peptide) in a final volume of 25 µl. Samples were incubated at 30 °C for 15 min. Each cell extract was assayed in duplicate in both the absence and presence of 1 µM cAMP to determine the fraction of total enzyme (+cAMP) that was endogenously activated (-cAMP). In parallel samples, the inclusion of the PKA-specific inhibitor IP (5 µM) (11) enabled calculation of true PKA activity: non-inhibitable (non-PKA) phosphorylation was subtracted from total measured phosphorylation.

After agarose electrophoresis of all samples, the relevant gel pieces were excised, brought to a volume of 0.5 ml with deionized water, and heated in boiling water until the gel was liquified. Fluorescence was then determined (Perkin-Elmer LS-5 spectrofluorometer), using an excitation wavelength of 568 nm and an emission wavelength of 592 nm. Calculated PKA-specific enzyme activities are expressed in fluorescence units generated per 10 µg of cellular protein/15 min.

In Situ Inhibition of PKA

Experiments were also performed to directly inhibit PKA in intact A6 cells with the highly selective antagonist IP. To increase cell membrane permeability, and thus increase cellular uptake of the applied polypeptide, monolayers of A cells were exposed to 5 µM IP while chilled, but not frozen, at -20 °C for 10 min (suggested by Dr. Adrian Katz). Subsequently, cells were allowed to recover in the same bathing solution for 90 min in a 27 °C incubator before experiments were performed. The effects of chilling on the basal properties of the monolayers and on hormone responsiveness were determined by comparison with cells from the same seeding that were not subject to the chilling procedure.

Reagents

AVP, 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP), trifluoperazine, Freon, and tri-N-octylamine were obtained from Sigma. Additional chemicals were obtained from the following vendors: chelerythrine, Alomone Laboratories (Jerusalem, Israel); forskolin and 1,9-dideoxyforskolin, Calbiochem; DDA, Pharmacia Biotech Inc.; RO-201724, Biomol Research Laboratory, Inc. (Plymouth Meeting, PA); Fura-2/AM and 5,5`-dimethyl BAPTA/AM, Molecular Probes (Eugene, OR); amiloride was a gift from Merck Sharp and Dohme; and 7,8-dihydroxychloropromazine was provided by Research Biochemical, Inc., as part of the Chemical Synthesis Program of the National Institute of Mental Health, Contract 278-90-007(BS).

Statistical Analysis

Statistical comparisons were made using the unpaired Student's t test. The Dunnett test was used when multiple experimental groups were compared with a single control group. A p < 0.05 was regarded as denoting statistical significance.


RESULTS

Effect of AVP on Electrogenic Sodium Transport

Addition of AVP to medium bathing the basal surface of A6 monolayers stimulated I at a minimal concentration of 1 nM (Fig. 1A). Higher concentrations progressively increased I up to 10-fold at 1 µM. AVP was found to increase V within 6 s (Fig. 1B). The response persisted for about 3.5 h, but decayed progressively after the first hour. To our knowledge, this is the first demonstration of an AVP-induced increase in I in less than 1 min. The stimulation of I was abolished by 100 µM amiloride in the apical solution, indicating that I reflected electrogenic sodium transport: control, 1.6 ± 0.1 µAcm; AVP, 3.1 ± 0.1; AVP + amiloride, 0.4 ± 0.2 (mean ± S.E.).


Figure 1: Effects of vasopressin (AVP) on I in A6 cells. A, dose-dependent stimulation of electrogenic Na transport (I) by AVP added to the basal solution (5 culture cups per group). Values are mean ± S.E. B, time course analysis of stimulation; transepithelial potential difference (V) was measured over 3 h following exposure of cells to 1 µM AVP. Results of this experiment are representative of five experiments. The initial effect is shown in the inset.



Role of Intracellular Calcium in the Action of AVP

Addition of AVP to A6 cells also resulted in an immediate dose-dependent rise in Ca, as indicated by Fura-2 fluorescence (Fig. 2). The time and dose dependencies of the Ca response were similar to those of the I response (Fig. 1, A and B). AVP appeared not to stimulate Ca influx during the period of observation, because addition of 100 µM MnCl did not quench intracellular Fura-2 fluorescence during stimulation; Mn enters cells via activated Ca channels and displaces Ca from Fura-2(12) .


Figure 2: Effect of AVP on Ca. AVP induced a dose-dependent increase in Ca, reflected by an increase in the fluorescence ratio (expressed in arbitrary units) of Fura-2 at an emission of 510 nm, during dual excitation at 340 and 380 nm (A-C). These data obtained in a single experiment are representative of 10 separate experiments.



To determine whether AVP-stimulated sodium transport was dependent on this increase in Ca monolayers were preloaded (2.5 h) with the calcium chelator 5,5`-dimethyl BAPTA. Fig. 3shows that BAPTA dose-dependently inhibited subsequent stimulation of I by 1 µM AVP, with an apparent Kof approximately 10 µM. These results therefore imply that AVP stimulation of I is mediated by an increase in Ca.


Figure 3: Effect of Ca chelation by BAPTA on AVP-induced I. Dose-dependent inhibition of I in cells preloaded with 5,5` dimethyl BAPTA/AM, at indicated concentrations. There were 5 culture cups in each group. Values are means ± S.E.



Since the above experiments suggested that elevated Ca was entirely due to release from intracellular Ca stores, we tested whether AVP increased phosphoinositide turnover. Initially, total IP was measured before and after addition of 1 µM AVP. As shown in Fig. 4A, an increase in IP was observed at 10 s, reached a maximum at 30 s, and persisted for at least 300 s. Subsequently, a dose-response analysis was performed by measuring IP levels at 30 s after addition of various AVP concentrations. Fig. 4B shows that AVP increased IP in the range of 10 nM to 1 µM. The lack of a progressive rise in IP with high concentrations of AVP may have resulted from increased rates of degradation (uninhibited because LiCl was not used).


Figure 4: AVP stimulation of total inositol trisphosphate (IP). A, time-dependent effect of 1 µM AVP. B, dose-dependent stimulation of IP by AVP. Data are derived from two separate experiments, each with two samples for each time interval or concentration. Values are mean ± S.E.



These studies together provide strong evidence that the action of AVP to stimulate electrogenic Na transport depends on an increase in Ca via turnover of membrane bound phospholipids and IP-induced release of Ca from non-mitochondrial intracellular stores.

The role of Calcium-dependent Effectors in AVP-stimulated I

The demonstration that AVP causes a rise in IP implies that diacylglycerol, an endogenous activator of protein kinase C (PKC), is also produced. Studies were performed to determine whether PKC appears to play a role in Na transport stimulation by AVP. In these experiments the effect of three PKC antagonists on AVP-stimulated I were examined.

Dihydroxychloropromazine, highly specific for PKC with a K of 8 µM for PKC (K values for other kinases > 50 µM)(13) , dose-dependently inhibited AVP stimulation of I (Fig. 5A), with an apparent Kof approximately 12 µM. The second selective antagonist, chelerythrine, is a benzophenanthridine alkaloid that exhibits an apparent K for PKC of 0.7 µM in rat brain tissue under in vitro conditions (K values for other kinases > 100 µM)(14) . Chelerythrine half-maximally inhibited AVP-stimulated I in intact cells at approximately 3 µM (Fig. 5B). In addition, trifluoperazine, which inhibits calmodulin kinase as well as PKC, reduced the action of AVP by 50% at a K of approximately 25 µM (Fig. 5C), a value that corresponds to the K for PKC inhibition in reports for other types of intact cells(15, 16) . Taken together, these results suggest that the onset of AVP stimulation of I is dependent on PKC activation.


Figure 5: Effects of specific kinase inhibitors on AVP-stimulated I. A, dose-dependent effect of the PKC inhibitor dihydroxychloropromazine (DHCP). B, inhibitory profile of the PKC inhibitor, chelerythrine (Chel), on AVP-stimulated I. C, effect of trifluoperazine (TFP), a PKC and calmodulin inhibitor, on I. Values are mean ± S.E. There were 5 culture cups in each control and experimental group (A-C).



As reported previously (7) these antagonists did not affect either basal transport or R in the concentrations employed in these experiments, suggesting that they were not toxic to the cultured cells.

Role of Adenylate Cyclase and cAMP in the Action of AVP

Addition of AVP to the basal medium also resulted in a dose-dependent increase in cAMP generation (Fig. 6A) that paralleled the stimulation of I. The cAMP level, in the presence of the phosphodiesterase inhibitor RO-201724, increased 8-fold from the control value of 19 ± 1.6 pmol/mg protein to 138 ± 12 at 1 µM AVP. However, after preloading cells with the adenylate cyclase inhibitor DDA, which binds to the ``P'' site on the catalytic subunit of the enzyme(17) , AVP failed to induce cAMP generation at any concentration that was previously shown to stimulate I (1 nM to 1 µM, Fig. 1A).


Figure 6: The effect of DDA on the AVP-induced accumulation of cAMP and I. A, AVP-induced accumulation of cAMP in the absence and presence of DDA (100 µM), a specific inhibitor of adenylate cyclase. These data obtained in a single experiment are representative of three separate experiments. B, the action of DDA on AVP-stimulated I, with 5 culture cups in each group. Values are mean ± S.E.



The corresponding effect of DDA on AVP stimulation of I is shown in Fig. 6B. Despite its abolition of cAMP generation, DDA did not significantly reduce the ability of AVP to stimulate I, indicating that AVP stimulation of Na transport was independent of cAMP generation.

Role of Protein Kinase A

PKA is the major intracellular target for cAMP. Activation of this enzyme is therefore expected to occur as a result of AVP-induced cAMP generation. However, one would predict that DDA would abolish such PKA activation if it were completely effective in inhibiting adenylate cyclase. Further experiments were, therefore, performed to test these predictions.

The percent of PKA activation (-cAMP/+cAMP) in control monolayers was 44 ± 7%, as shown in Fig. 7. Addition of 1 µM AVP increased the percent activation of PKA to 62 ± 4% (p < 0.05) an increase of 41% above control. This change is consistent with other reports regarding PKA stimulation by high concentrations of other cAMP-elevating agonists; isoproterenol and prostaglandin E, for example, increased fractional activation of true PKA by 50% in tracheal smooth muscle(18) . As predicted, preloading A6 cells with the adenylate cyclase inhibitor DDA abolished AVP stimulation of PKA; the fractional PKA activation was then only 46 ± 5% (p = not significant, compared with control).


Figure 7: The effect of AVP on PKA activity in the absence or presence of DDA (100 µM). Shown is PKA-specific kinase activity in A6 cells, expressed as a percent of maximal activity. Data were derived from five separate experiments performed in duplicate. Results are mean ± S.E. The asterisk indicates p < 0.05, compared with control.



In Situ Inhibition of PKA

Taken together, our findings indicated that DDA completely blocked AVP stimulation of the cAMP pathway, but did not affect stimulation of I. Nevertheless, an additional study was performed to rule out PKA in mediating the action of AVP. To directly suppress the enzyme in situ intact cells were loaded with the specific PKA inhibitor IP, added to the external bathing solution at a concentration of 5 µM. This experiment was prompted by reports that sufficient cellular loading of the polypeptide could be achieved in renal proximal tubules to inhibit dopamine-induced, cAMP-mediated inhibition of Na-K-ATPase (19) or the apically located Na:H antiporter(20) . Monolayers were chilled briefly at -20 °C for 10 min to enhance the cellular uptake of the polypeptide by increasing cell membrane permeability. After recovery at a normal temperature, cells were exposed to either control vehicle or 1 µM AVP for 30 min and, then, allocated either for assay for PKA activity or measurement of electrogenic sodium transport.

In control cells (vehicle alone) the percent of PKA activation was 16 ± 0.5%, whereas in the AVP-treated group the activated fraction was 24 ± 2%, an increase of 50% (since data are the mean of two separate experiments, the variance is shown as error bars). In cells preloaded with IP before treatment with AVP the percent activation was 18 ± 0.1%, indicating that intracellular IP completely blocked the action of AVP to stimulate PKA. Transport studies verified that cold exposure did not result in injury to the cell monolayer. Ninety minutes after the chilling process, neither basal resistance nor basal I of monolayers were reduced compared with control values, shown in . In addition, indicates that abolition of PKA activation by IP did not inhibit AVP-stimulated I, compared with control.

Mechanism of Forskolin- and 8-CPT-cAMP-mediated NaTransport

The observation that forskolin and/or analogues of cAMP induce a biological response is often used as evidence that the effect in question is mediated by cAMP. Since these reagents have recently been shown to stimulate various transport processes by cAMP-independent mechanisms, studies were performed to determine their action mechanisms in stimulating electrogenic Na transport. Forskolin is a naturally occurring diterpene that activates adenylate cyclase after binding to the catalytic subunit, or G-catalytic subunit complex (21). Application of forskolin to A6 cells caused an increase in cAMP accumulation at 100 nM and above (Fig. 8A). Preloading cells with DDA abolished cAMP generation at 100 nM forskolin and reduced it by approximately 60% at 1 µM. Despite complete or partial inhibition of cAMP production, DDA did not significantly reduce forskolin stimulation of I (Fig. 8B).


Figure 8: The effect of DDA on forskolin-induced cAMP generation and I. A, forskolin-induced accumulation of cAMP in the absence or presence of DDA (100 µM). B, the effect of DDA on forskolin-stimulated I. C, the effect of 1,9-dideoxyforskolin on I. There were 5 culture cups in each control and experimental group (A-C). Values are mean ± S.E.



In further support of a cAMP-independent mechanism, the forskolin analogue 1,9-dideoxyforskolin, was found to also stimulate Na transport, at concentrations of 10 µM and above (Fig. 8C). This compound does not bind to the catalytic subunit of adenylate cyclase, even in concentrations as high as 100 µM(22) .

To examine the ability of cAMP analogues to stimulate Na transport, we employed the highly permeant and phosphodiesterase-resistant analogue 8-CPT-cAMP. Since synthetic derivatives of cAMP exhibit different binding affinities for the regulatory subunits of PKA(23) , the concentrations needed for half-maximal and maximal activation of PKA by cAMP and 8-CPT cAMP were first compared in homogenates of A6 cells. The dose-response curves (Fig. 9A) indicate similar potencies for both reagents: half-maximum values of approximately 100 nM and maximum, approximately 400 nM. This in vitro experiment suggests that, intracellularly, 8-CPT-cAMP should have a potency that is similar to that of cAMP. Our data are consistent with other reports that cAMP activates PKA at half-maximum and maximum values of approximately 50 nM and 200-400 nM, respectively(23) . By this in vitro data, we can then predict that an externally applied equipotent cAMP analogue, to be similarly biologically effective, should yield intracellular concentrations of about 50-100 nM. Fig. 9B shows the effect of 8-CPT-cAMP applied externally to intact cells on PKA activity. In fact PKA activation increased approximately 50 and 100% at external 8-CPT-cAMP concentrations of 1 and 10 µM, respectively. Thus, the intracellular concentrations of 8-CPT-cAMP are probably about 10% of the external concentration.


Figure 9: Effects of 8-CPT-cAMP on PKA activity and I. A, comparison of the dose dependencies of cAMP and 8-CPT-cAMP in A6 cell homogenates. B, comparison of the dose-dependent effect of 8-CPT-cAMP on intact cells to increase the percent activation of PKA (light bars) and to stimulate I (dark bars). Data on PKA activation are representative of two separate experiments. There were 4 culture cups in each group in the transport study. The asterisk indicates that the experimental group exceeds control by the Dunnett Test, with a confidence limit of 95%.



These data further indicate that if the stimulation of Na transport were dependent upon PKA activation by 8-CPT-cAMP, it would occur at external concentrations of less than 10 µM. However, Fig. 9B reveals that 8-CPT-cAMP stimulation of I required concentrations between 1 and 3 orders of magnitude higher than those necessary to activate PKA. Therefore, as found with forskolin, 8-CPT-cAMP stimulation of I is mediated by a PKA-independent mechanism. Furthermore, it is seen that an increase in PKA activity per se does not stimulate Na transport.


DISCUSSION

Although it is known that AVP regulates various biological responses by at least two classes of second messenger systems, one involving the cyclic nucleotide cAMP and the other Ca, it is generally accepted that AVP stimulation of electrogenic Na transport is mediated by cAMP. Evidence for this proposition is based largely on the demonstration that AVP induces the generation of cAMP at concentrations that stimulate sodium transport (2) and the belief, until recently, that AVP target cells with the capability for electrogenic Na transport possess only V receptors coupled to adenylate cyclase(24) . Recent reports have shown, however, that AVP acts to increase Cai, as well as cAMP, in cultured cells from rabbit cortical collecting tubule (5, 25) and toad bladder(26) . That observation, together with reports that activation of receptors which are not linked to adenylate cyclase, including receptors for aldosterone (6) and insulin(8) , and the A adenosine receptor(7) , cause a calcium-dependent stimulation of Na transport, prompted this examination of the action of AVP.

The present study in cultured A6 cells demonstrates that AVP stimulates an immediate increase in I, Ca and IP at concentrations of 10 nM and above. The dependence of AVP-stimulated I on increases in Ca was shown in experiments in which chelation of Ca with the EGTA derivative BAPTA dose-dependently blocked I. Previous studies have indicated that intracellular BAPTA inhibits agonist-stimulated increases in Ca, although basal levels are unaffected(6) . These results therefore show that AVP stimulates electrogenic Na transport by a calcium-mobilizing second messenger system, due primarily to release of Ca from intracellular stores.

We have also begun to explore the possible role of calcium-dependent effectors in the calcium mobilizing transduction system mediating Na transport. The findings in the present study provide evidence that PKC is at least one such effector. First, the demonstration of AVP-stimulated IP implies a rise in diacylglycerol production, the natural PKC activator. Second, specific antagonists of PKC dose-dependently inhibited AVP-stimulated I at concentrations near to those that inhibit the purified enzyme in vitro. Further studies, using more direct experimental approaches, will be required to confirm an effector role for PKC in Na transport.

The demonstration of Ca dependence, however, did not exclude the possibility of an additional cAMP-mediated transduction mechanism acting in a redundant or additive manner. Further experiments were therefore performed to examine the potential role of cAMP in mediating Na transport. First, we sought to determine whether AVP stimulation of Na was dependent upon cAMP production, that is whether Na transport was abolished or reduced when adenylate cyclase was inhibited. Second, we examined the effect of inhibiting PKA on electrogenic Na transport, since this enzyme is thought to be the sole, or at least major, substrate for cAMP. And, finally, we probed the effect of experimentally induced increases in cAMP on Na transport, by loading intact cells with either forskolin or the cAMP analogue 8-CPT-cAMP.

We found that the specific antagonists DDA and IP, when loaded into intact cells, were effective in abolishing hormone stimulation of cAMP production and PKA activation, respectively, but did not alter AVP-stimulated Na transport. This dissociation between cAMP generation and Na transport was confirmed by experiments in which the cAMP analogue was applied externally; full stimulation of PKA did not result in increased Na transport. Together, our results provide strong evidence that electrogenic sodium transport is not mediated by a cAMP-dependent system. It should be noted that this finding is not unprecedented, because there are now numerous other examples of biological responses, initially thought to involve cAMP as a second messenger, which have been shown to involve transduction mechanisms that are unrelated to adenylate cyclase activation(27, 28, 29, 30) .

We also examined the cellular action of forskolin and externally applied 8-CPT-cAMP, since their actions to stimulate Na transport have been regarded as evidence for a cAMP-mediated messenger system. Both reagents were found to stimulate Na transport by mechanisms that were independent of cAMP production or activation of PKA, a finding that invalidates their use as markers for a cAMP-mediated mechanism without further direct confirmation. This observation extends other reports that both forskolin and externally applied cAMP can modulate transport proteins by diverse mechanisms other than activation of the cAMP second messenger system. Both forskolin and 1,9-dideoxyforskolin can directly modulate a number of transport proteins, including the glucose transporter(31) , the nicotinic acetylcholine receptor(32, 33) , and several types of voltage-dependent Na and K channels (see Ref. 34 for review). Externally applied cAMP or analogues of cAMP can modify voltage-dependent ion channels by a PKA-independent mechanism in cardiac pacemaker cells(28) , cardiac myocytes(30) , and olfactory receptor cells(29) .

Since our studies indicate that AVP stimulation of Na transport depends on mobilization of Ca, mention should be made of previous reports suggesting that experimentally induced increases in Ca actually inhibit Na transport(35, 36) . Interpretation of such evidence is confounded, we believe, by the nature of the agents utilized. The Ca-elevating agonists clearly also activate several signaling pathways other than Ca which may exhibit inhibitory effects. Increases in Ca obtained with calcium ionophores are difficult to control and probably cannot mimic the highly organized temporal and spatial patterns of incremental Ca which are induced by physiological agonists, as reviewed elsewhere(37) .

In summary, this study indicates that AVP stimulation of electrogenic Na transport is mediated by a Ca-mobilizing signal transduction system. Contrary to the prevailing dogma and despite extensive probing no evidence was obtained that cAMP acts as a second messenger for the stimulation of Na transport. Along with previous reports showing that the actions of aldosterone, insulin, and adenosine to stimulate Na transport are mediated by the second messenger Ca(6, 7, 8) , these findings imply that multiple agonists that stimulate electrogenic Na transport all utilize a single primary transduction system.

  
Table: Demonstration of the effects of cold exposure and IP loading on transepithelial resistance (R) and AVP-stimulated I

Experiments were performed 90 min after chilling cells at -20 °C to load IP, while controls were not chilled. There were 5 cups in each group and values are mean ± SEM.



FOOTNOTES

*
This study was supported by National Institutes of Health Grants DK 18061 and DK 19813 and by the Fondo de Investigacion sanitaria da Seguridad Social of Spain. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Internal Medicine, Yale School of Medicine, 333 Cedar St., LMP 2076, New Haven, CT 06510. Tel.: 203-785-4185; Fax: 203-785-7068.

The abbreviations used are: AVP, arginine vasopressin; DDA, 2`,5`-dideoxyadenosine; PKA, protein kinase A; PKC, protein kinase C; 8-CPT-cAMP, 8-(4-chlorophenylthio)-cAMP; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N`,N`-tetraacetic acid; IP, inositol trisphosphate.


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