©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
G Protein Regulation of the Na/H Antiporter in Xenopus laevis Oocytes
INVOLVEMENT OF PROTEIN KINASES A AND C (*)

(Received for publication, January 18, 1995; and in revised form, May 22, 1995)

Stefan Busch (1) Thomas Wieland (1) Helmut Esche (2) Karl H. Jakobs (1) Winfried Siffert (1)(§)

From the  (1)Institut fr Pharmakologie and (2)Institut fr Molekularbiologie, Universitt GH Essen, Hufelandstrasse 55, D-45122 Essen, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

We have characterized the regulation of the endogenous Na/H exchanger in Xenopus laevis oocytes by G proteins and protein kinases by measuring the ethylisopropylamiloride-sensitive Li uptake. Injection of oocytes with the stable GTP analog GTPS stimulated Li uptake up to almost 4-fold, an effect blocked by coinjection with the GDP analog, guanyl-5`-yl thiophosphate. Injection into oocytes of subunits of the heterotrimeric G protein transducin enhanced Li uptake by about 3-fold. This stimulation was blocked by transducin subunits, which by themselves did not influence Li uptake. Using various activators and inhibitors of protein kinases, it is demonstrated that the X. laevis oocyte Na/H antiporter can be stimulated by activation of both protein kinase A and C. Stimulation of Na/H exchanger activity by GTPS but not that induced by transducin subunits was blocked by the protein kinase A inhibitor H-89. On the other hand, transducin subunit-stimulated activity was prevented by the protein kinase C inhibitor, calphostin C. The non-selective protein kinase inhibitor H-7 blocked both GTPS- and transducin subunit-stimulated Na/H exchanger activity. The results suggest that the Na/H exchanger of X. laevis oocytes can be activated by G proteins and that this activation is not direct but mediated by protein kinase A- and/or protein kinase C-dependent pathways.


INTRODUCTION

Na/H exchangers (NHE)()constitute a family of membrane transporters that mediate the electroneutral exchange of Na ions against H ions and that are activated by various receptors coupled to guanine nucleotide-binding proteins (G proteins) and receptors with tyrosine kinase activity(1) . The NHE-1 isoform is ubiquitously expressed and mainly serves intracellular pH and cell volume regulation(2, 3, 4, 5) . The expression of the other hitherto cloned mammalian isoforms, referred to as NHE-2-NHE-4, appears more restricted to the apical membranes of epithelial cells, and their role could reside in mediating transepithelial Na transport (2, 3, 4, 5) . These isoforms differ with respect to their inhibition by amiloride and its analogs (6) and with regard to their regulation by intracellular second messengers. For example, the activity of the NHE-1 and NHE-2 expressed in fibroblast PS120 cells is increased by protein kinase C (PKC)-activating phorbol esters, whereas the activity of the NHE-3 is reduced by this treatment(7) . Recent findings suggest that the NHE-1 belongs to the family of calmodulin-binding proteins, which is activated by a rise in cytosolic free Ca concentration(8, 9) . Variable effects of cAMP on mammalian NHE activity have been reported. Upon expression of NHE-1, NHE-2, and NHE-3 in PS120 cells, no effect of cAMP on these isoforms was found(7) . On the other hand, when studied in their natural environment, both inhibition and stimulation of NHE activity by cAMP have been reported (10, 11, 12) . This variability may be due to the cell type studied and/or the NHE isoform expressed in these cells. The NHE isoform cloned from trout red blood cells unambiguously differs from the mammalian isoforms as this exchanger is phosphorylated and activated by both PKC and protein kinase A (PKA)(13, 14, 15) .

Few information exists regarding the properties of the Na/H exchanger expressed in Xenopus laevis oocytes(16, 17, 18) . In a previous study, we expressed the human NHE-1 in X. laevis oocytes, which increased the exchanger activity above the endogenous oocyte NHE activity(18) . Interestingly, the activity of the expressed NHE-1 was totally suppressed by low concentrations (100 nM) of the novel isoform-selective inhibitor HOE694(6) , whereas the endogenous NHE activity remained unaffected even in the presence of 100 µM of this compound. This pharmacological difference suggested that the functional characteristics of the X. laevis oocyte Na/H exchanger may also be different from those of the mammalian transporters. In the present study, we investigated the regulation of the Na/H exchanger of X. laevis oocytes by G proteins and protein kinases. The data presented here show that the oocyte antiporter can be activated by G proteins and that this regulation involves both PKA- and PKC-dependent mechanisms.


EXPERIMENTAL PROCEDURES

Chemicals

All nucleotides were purchased from Boehringer Mannheim. Gentamycin, streptomycin, penicillin, calphostin C, calf intestine alkaline phosphatase, forskolin, PKA inhibitor (type III from bovine heart), and phorbol 12-myristate 13-acetate (PMA) were from Sigma. The protein kinase inhibitors, H-7 and H-89, and ethylisopropylamiloride (EIPA) were from Calbiochem. S-adenosine 3`,5`-cyclic monophosphorothioate ((S)-cAMPS) was from Bio-Log (Bremen, Germany). Transducin and subunits were purified from illuminated bovine rod outer segments with GTP as activating nucleotide as previously described(19) .

Reverse Transcription, PCR Analysis, and DNA Sequencing

First strand cDNA synthesis from oligo(dT)-selected poly(A) RNA was carried out with 25 pmol of random primers (Life Technologies, Inc.). The single strand cDNA was then subjected to 30 cycles of PCR amplification (1 min, 94 °C; 1 min, 48 °C; 1.5 min, 72 °C), using 10 pmol of two NHE-1- and NHE-specific oligonucleotide primers encompassing the region between the putative transmembrane domains Va and VIII. The sequences of the primers were: sense 5`-GAATTCTCGGCCGTGGACCCCGTGGC-3` (NHE bp 667-686, NHE-1 bp 1110-1129) and antisense 5`-GAATTCCGCTCACGCTGCTCCACATC-3` (NHE bp 1135-1116, NHE-1 bp 1578-1559). Using the EcoRI endonuclease restriction sites of the 5`-ends of each primer, the amplification products were subcloned into the complementary restricted pBluescript SK plasmid (Stratagene) for sequence analysis. DNA sequencing was carried out using fluorescent nucleotides in an automatic sequencer (Pharmacia Biotech Inc.).

Oocyte Injection

Cytoplasmic injections into defolliculated X. laevis oocytes were performed as previously detailed(18) . 50 nl of nucleotides or transducin subunits were injected to yield the indicated final intracellular concentrations, assuming a mean oocyte volume of 1 µl. After resealing of the plasma membrane, the oocytes were transferred to Li-containing medium as described below. Some oocytes were incubated for 30 min in OR-2 medium containing 1 µCi of [H]sorbitol (DuPont NEN), for which no endogenous transporter in X. laevis oocytes has been described. Oocytes were considered tight in the absence of any radioactivity above background after extensive washing.

Determination of Na/H Exchange Activity

Na/H antiporter activity was determined by measuring EIPA-sensitive Li uptake, i.e. Li/H-exchange, as described in detail previously(18) . This method avoids the use of radioactive Na. In brief, oocytes were incubated for various time periods within the linear range of Li uptake (18) in a medium containing 65 mM LiCl, 2.5 mM lithium citrate, 8 mM MgCl, and 5 mM Mops, pH 7.4. Thereafter, the cells were removed from the medium, washed five times in ice-cold 0.1% (w/v) CsCl solution, and transferred to plastic tubes containing 1 ml of 0.1% (w/v) CsCl solution. After oocyte homogenization and removal of yolk proteins, lithium concentration was determined in the clear supernatant by analytical flame photometry using a Pye Unicam spectrophotometer SP9. Assuming a mean oocyte volume of 1 µl and after correcting for dilution, intracellular Li concentrations were calculated. Initial velocities of Na/H exchanger activity are presented here in mmol of Li/liter of oocyte and h. All data represent means ± S.D. performed on 10 oocytes from at least two different animals. Data were analyzed by Student's t-test, and differences were regarded significant at p < 0.05.


RESULTS

Effects of Guanine Nucleotides and G Protein Subunits on Li Uptake

To study whether the X. laevis oocyte Na/H exchanger is regulated by G proteins, we first examined the effects of injected nucleotides (5 µM, final intracellular concentration) on Li uptake. Injection of the stable GTP analog GTPS stimulated EIPA-sensitive Li uptake by almost 4-fold (Table 1). In contrast, GTP, the ATP analog ATPS, and the GDP analog GDPS had no or only slight effects on Li uptake. When GTPS and GDPS were injected simultaneously at a molar ratio of 1:100, the GTPS-induced stimulation of Li uptake was completely suppressed. Thus, these data suggested that activated G proteins can enhance the X. laevis oocyte Na/H exchanger activity.



Possible G protein regulation of Na/H exchanger activity was additionally studied by injecting and subunits (1 µM, final concentration) of the retinal G protein transducin into oocytes. Whereas injected subunits had no effect on Li uptake, injection of subunits strongly increased EIPA-sensitive Li uptake by about 3-fold (Table 2). When and subunits were coinjected, no effect on Li uptake was observed. Thus, in addition to GTPS, free subunits of transducin can stimulate the X. laevis oocyte Na/H exchanger.



Effects of PKC and PKA on Li Uptake

To determine whether the stimulatory effects of GTPS and transducin subunits are due to a direct G protein activation of the X. laevis oocyte Na/H antiporter or due to the formation of second messengers and consequently of second messenger-activated protein kinases, we first studied the possible regulation of Li uptake by directly activated PKC and PKA. Pretreatment of oocytes with the phorbol ester, PMA (10 nM), increased EIPA-sensitive Li uptake by almost 4-fold (Table 3). This stimulation was completely suppressed by the specific PKC inhibitor calphostin C (1 µM) (20) but only slightly reduced by H-89 (100 µM), a rather selective PKA inhibitor(21) . These findings suggest that the Na/H exchanger expressed in X. laevis oocytes can be activated by a PKC-dependent mechanism.



To study whether the X. laevis oocyte Na/H antiporter is also regulated by PKA, oocytes were treated with the direct PKA activator, (S)-cAMPS(22) , and the direct adenylyl cyclase activator, forskolin. Both agents strongly stimulated Li uptake. (S)-cAMPS increased Li uptake to 370, 450, and 470% of controls at 1, 10, and 100 µM, respectively (data not shown). Forskolin (100 µM) increased EIPA-sensitive Li uptake by 5-6-fold (Table 4). The stimulatory effect of forskolin was completely abolished in oocytes pretreated with the PKA inhibitor, H-89 (100 µM), or injected with the bovine heart PKA inhibitor (0.1 µM). In contrast, the PKC inhibitor, calphostin C (1 µM), had no effect on forskolin-stimulated Li uptake. Thus, the Na/H exchanger expressed in X. laevis oocytes is apparently under the control of both PKC and PKA.



The stimulation of X. laevis oocyte Na/H exchanger by both PKA and PKC could be due to the expression of a NHE isoform resembling the NHE, which is stimulated by both PKA and PKC, or to the expression of two distinct isoforms being differentially controlled by PKA and PKC. PCR analysis of cDNA synthesized from oocyte RNA with oligonucleotide primers homologous to both the human NHE-1 and the NHE yielded one major reaction product of 417 bp. After subcloning of this PCR product, 10 different clones were analyzed, which all exhibited an identical nucleotide sequence, suggesting the expression of only one NHE transcript in these cells. Sequence comparison with corresponding sequences of the human NHE-1 and the NHE indicated a 79 and 73% homology, respectively, on the nucleotide level, which was increased to 89 and 80%, respectively, on the amino acid level.

Effects of Protein Kinase Inhibitors on G Protein-mediated Li Uptake

Finally, we investigated whether protein kinases, specifically PKA and PKC, are involved in stimulation of the X. laevis Na/H antiporter by GTPS and transducin subunits. Preincubation of oocytes with H-7 (100 µM), a non-selective protein kinase inhibitor(21) , completely suppressed Li uptake stimulated by either injected GTPS or transducin subunits (Fig. 1), suggesting the involvement of a protein kinase-dependent mechanism(s) in stimulation of Na/H antiporter by either agent. On the other hand, the selective PKA inhibitor, H-89 (100 µM), strongly reduced Li uptake stimulated by GTPS, whereas transducin subunit-stimulated Li uptake was not affected. Vice versa, pretreatment of oocytes with calphostin C (1 µM) completely inhibited Li uptake stimulated by transducin subunits but was without effect on stimulation by GTPS.


Figure 1: Effects of different protein kinase inhibitors on GTPS- and transducin subunit-stimulated Li uptake. Shown are the effects of pretreatment of oocytes for 6 h without (control, C) and with H-7 (100 µM), H-89 (100 µM), or calphostin C (Cal-C, 1 µM) as indicated before injection of GTPS (5 µM) (leftpanel) or transducin subunits (1 µM) (rightpanel) on Li uptake. Each column reflects means ± S.D. of at least 10 oocytes from at least two different animals.




DISCUSSION

It is generally accepted that the phospholipase C-PKC pathway constitutes one route for NHE activation, with the notable exception of the NHE-3, which is inhibited by phorbol ester-activated PKC(7) . On the other hand, data on regulation of mammalian Na/H exchangers by PKA-dependent phosphorylation are rather variable, with no effects, inhibition, and stimulation being reported(7, 10, 11, 12) . The Na/H antiporter cloned from trout red blood cells, NHE, is activated by agents causing an increase in intracellular cAMP concentration and by direct PKA activators(13, 14, 15) . The data presented herein indicate that the Na/H exchanger expressed in X. laevis oocytes is stimulated by both PKC- and PKA-dependent mechanisms, thus resembling the regulation of the NHE of trout red cells. In oocytes injected with calf intestine alkaline phosphatase (0.01 units/oocyte), not only PMA- and forskolin-stimulated but also unstimulated Li uptake was reduced to about 50% of untreated controls (data not shown), suggesting that even basal transport activity is maintained by phosphorylation.

A dual regulation of the X. laevis oocyte Na/H exchanger was also observed by activating oocyte endogenous G proteins with GTPS and by injecting G protein subunits. Recently, evidence has been accumulated that subunits of heterotrimeric G proteins can directly activate phospholipase C, preferentially the 2 and 3 isoforms of this enzyme(23, 24) , and a phospholipase C with homologies to the mammalian phospholipase C-3 enzyme has been cloned from X. laevis oocytes(25) . Transducin subunits injected into X. laevis oocytes stimulated NHE activity, and this stimulation was prevented by coinjected subunits, suggesting the stimulation being due to free subunits. Similar data were reported for phospholipase C activation by transducin subunits(23) . However, due to the suppression by the protein kinase inhibitors, H-7 and calphostin C, the stimulatory effect of subunits is unlikely due to a direct interaction with the oocyte NHE. We rather propose that upon injection of subunits the oocyte phospholipase C is activated, resulting in the hydrolysis of phosphoinositides and the subsequent activation of PKC, then activating directly or via additional kinases the oocyte Na/H exchanger. Such a signal transduction cascade could be initiated in vivo via muscarinic or angiotensin II receptors, which, in oocytes, are coupled to phospholipase C via pertussis toxin-sensitive G proteins(26, 27, 28) .

Activation of the oocyte endogenous G proteins by injected GTPS also evoked a strong stimulation of the Na/H exchanger, which was nucleotide-specific and antagonized by the GDP analog, GDPS. Interestingly, the stimulatory effect of GTPS was almost completely inhibited by the non-selective protein kinase inhibitor, H-7, and, most notably, by the PKA inhibitor, H-89, but not by the PKC inhibitor, calphostin C. These findings suggest that activation of the oocyte Na/H exchanger by GTPS-activated G proteins is not direct but is mediated by a PKA-dependent mechanism most likely due to activation of adenylyl cyclase by GTPS-activated G proteins. GTPS is a nonspecific activator of G proteins, and the cellular response, therefore, depends on the isoform pattern and relative amounts as well as the availability of the different G proteins and their effectors expressed in a given cell type. Although circumstantial, the finding that the PKC inhibitor calphostin C was without effect on GTPS-injected oocytes, in contrast to its strong inhibitory effect on subunit-injected cells, suggests that GTPS did not release sufficient subunits to activate the endogenous phospholipase-PKC pathway.

There exists some recent evidence for an activation of the Na/H exchanger by specific G protein subunits. Expression of mutationally activated subunits of the G proteins G, G, G, and G in human embryonic kidney cells resulted in increased NHE activity by activated and subunits, whereas the other activated subunits left the antiporter unaffected(29) . Whereas activation by was assumed to be mediated via the stimulated phospholipase C pathway, stimulation of Na/H exchanger by was independent of the inositol phosphate and the cAMP pathways. Expression of mutationally activated , , , , and subunits in COS-1 cells enhanced NHE activity by , , and (30) . While expression of was without effect, even inhibited NHE activity. The stimulatory effect of and was apparently mediated by the PKC pathway, whereas the activation by was preserved in cells in which PKC had been down-regulated(30) . The signaling cascade involved in this protein kinase C-independent NHE activation by activated remains to be elucidated. Although our experiments have yet to yield any evidence for a direct G protein regulation of the Na/H exchanger of X. laevis oocytes injected with GTPS or G protein subunits, final statements on this issue require the determination of potential effects evoked by injection of different G protein subunits.

In conclusion, the data presented here suggest that the Na/H exchanger of X. laevis oocytes can be activated by G proteins and that this activation is most likely due to increased formation of second messengers resulting in activation of either PKC and/or PKA. Whether this specific Na/H exchanger actually contains consensus sequences for PKA- and PKC-mediated phosphorylation has to await the full-length cloning of this transporter, which is currently being performed in our laboratory.


FOOTNOTES

*
This work was supported by the Deutsche Forschungsgemeinschaft and the Ministerium fr Wissenschaft und Forschung Nordrhein/Westfalen. 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. Tel.: 49-201-7233470; Fax: 49-201-7235968.

The abbreviations used are: NHE, Na/H exchanger; PMA, phorbol 12-myristate 13-acetate; EIPA, ethylisopropylamiloride; GDPS, guanyl-5`-yl thiophosphate; GTPS, guanosine 5`-O-(thiotriphosphate); PKA, protein kinase A; PKC, protein kinase C; (S)-cAMPS, S-adenosine 3`,5`-cyclic monophosphorothioate; bp, base pair(s); Mops, 4-morpholinepropanesulfonic acid; PCR polymerase chain reaction; ATPS, adenosine 5`-O-(thiotriphosphate).


REFERENCES

  1. Grinstein, S., Rotin, D., and Mason, M.(1989)Biochim. Biophys. Acta 988, 73-97 [Medline] [Order article via Infotrieve]
  2. Counillon, L., and Pouyssgur, J.(1992)Cell. Physiol. Biochem. 2, 138-149
  3. Wakabayashi, S., Sardet, C., Fafournoux, P., Counillon, L., Meloche, S., Pags, G., and Pouyssgur, J.(1992)Rev. Physiol. Biochem. Pharmacol. 119,157-186 [Medline] [Order article via Infotrieve]
  4. Counillon, L., and Pouyssgur, J. (1993) in Molecular Biology and Function of Carrier Proteins (Reuss, L., Russell, J. M., and Jennings, M. L., eds) pp. 169-185, The Rockefeller University Press, New York
  5. Tse, M., Levine, S., Yun, C., Brant, S., Pouyssgur, J., and Donowitz, M. (1994)J. Am. Soc. Nephrol. 4, 969-975 [Abstract]
  6. Counillon, L., Scholz, W., Lang, H. J., and Pouyssgur, J.(1993) Mol. Pharmacol. 44, 1041-1045 [Abstract]
  7. Levine, S. A., Montrose, M. H., Tse, C. M., and Donowitz, M.(1993)J. Biol. Chem. 268, 25527-25535 [Abstract/Free Full Text]
  8. Bertrand, B., Wakabayashi, S., Ikeda, T., Pouyssgur, J., and Shigekawa, M.(1994)J. Biol. Chem. 269, 13703-13709 [Abstract/Free Full Text]
  9. Wakabayashi, S., Bertrand, B., Ikeda, T., Pouyssgur, J., and Shigekawa, M.(1994)J. Biol. Chem. 269, 13710-13715 [Abstract/Free Full Text]
  10. Helmle-Kolb, C., Montrose, M. H., Stange, G., and Murer, H.(1990) Pflgers Arch. 415, 461-470
  11. Green, J., and Kleeman, C. R.(1992)Am. J. Physiol.262,C111-C121
  12. Gupta, A., Schwiening, C. J., and Boron, W. F.(1994)Am. J. Physiol. 266,C1083-C1092
  13. Borgese, F., Sardet, C., Cappadoro, M., Pouyssgur, J., and Motais, R. (1992)Proc. Natl. Acad. Sci. U. S. A. 89, 6765-6769 [Abstract]
  14. Guizouarn, H., Borgese, F., Pellissier, B., Garcia-Romeu, F., and Motais, R.(1993) J. Biol. Chem. 268, 8632-8639 [Abstract/Free Full Text]
  15. Borgese, F., Malapert, M., Fievet, B., Pouyssgur, J., and Motais, R. (1994)Proc. Natl. Acad. Sci. U. S. A. 91, 5431-5435 [Abstract]
  16. Towle, D. W., Baksinski, A., Richard, N. E., and Kordylewski, M.(1991)J. Exp. Biol. 159, 359-369 [Abstract]
  17. Burckhardt, B.-C., Kroll, B., and Frmter, E.(1992) Pflgers Arch. 420, 78-82
  18. Busch, S., Burckhardt, B.-C., and Siffert, W.(1995) Pflgers Arch. 429, 859-869
  19. Wieland, T., Ulibarri, I., Gierschik, P., and Jakobs, K. H.(1991)Eur. J. Biochem. 196, 707-716 [Abstract]
  20. Kobayashi, E., Nakano, H., Morimoto, M., and Tamaoki, T.(1989)Biochem. Biophys. Res. Commun. 159, 548-553 [Medline] [Order article via Infotrieve]
  21. Hidaka, H., and Kobayashi, R.(1992)Annu. Rev. Pharmacol. Toxicol. 32, 377-397 [CrossRef][Medline] [Order article via Infotrieve]
  22. Rothermel, J. D., and Parker Bothelho, L. H.(1988)Biochem. J. 251, 757-762 [Medline] [Order article via Infotrieve]
  23. Camps, M., Hou, C., Sidiropoulos, D., Stock, J. B., Jakobs, K. H., and Gierschik, P. (1992)Eur. J. Biochem. 206, 821-831 [Abstract]
  24. Park, D., Jhon, D.-Y., Lee, C.-W., Lee, K.-H., and Rhee, S. G.(1993)J. Biol. Chem. 268, 4573-4576 [Abstract/Free Full Text]
  25. Ma, H.-W., Blitzer, R. D., Healy, E. C., Premont, R. T., Landau, E. M., and Iyengar, R. (1993)J. Biol. Chem. 268, 19915-19918 [Abstract/Free Full Text]
  26. Van Wezenbeek, L. A. C. M., Tonnaer, J. A. D. M., and Ruigt, G. S. F.(1988) Eur. J. Pharmacol. 151, 497-500 [Medline] [Order article via Infotrieve]
  27. Sakuta, H., Sekiguchi, M., Okamoto, K., and Sakai Y.(1991)Eur. J. Pharmacol. 208, 31-39 [Medline] [Order article via Infotrieve]
  28. Blitzer, R. D., Omri, G., De Vivo, M., Carty, D. J., Premont, R. T., Codina, J., Birnbaumer, L., Cotecchia, S., Caron, M. G., Lefkowitz, R. J., Landau, E. M., and Iyengar, R.(1993)J. Biol. Chem. 268, 7532-7537 [Abstract/Free Full Text]
  29. Voyno-Yasenetskaya, T., Conklin, B. R., Gilbert, R. L., Hooley, R., Bourne, H. R., and Barber, D. L.(1994)J. Biol. Chem. 269, 4721-4724 [Abstract/Free Full Text]
  30. Dhanasekaran, N., Vara Prasad, M. V. V. S., Wadsworth, S. J., Dermott, J. M., and Van Rossum, G.(1994)J. Biol. Chem.269,11802-11806 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.