Isotype-specific Activation of Cystic Fibrosis Transmembrane Conductance Regulator-Chloride Channels by cGMP-dependent Protein Kinase II

Abstract

Type II cGMP-dependent protein kinase (cGKII) isolated from pig intestinal brush borders and type Iα cGK (cGKI) purified from bovine lung were compared for their ability to activate the cystic fibrosis transmembrane conductance regulator (CFTR)-Cl− channel in excised, inside-out membrane patches from NIH-3T3 fibroblasts and from a rat intestinal cell line (IEC-CF7) stably expressing recombinant CFTR. In both cell models, in the presence of cGMP and ATP, cGKII was found to mimic the effect of the catalytic subunit of cAMP-dependent protein kinase (cAK) on opening CFTR-Cl− channels, albeit with different kinetics (2-3-min lag time, reduced rate of activation). By contrast, cGKI or a monomeric cGKI catalytic fragment was incapable of opening CFTR-Cl− channels and also failed to potentiate cGKII activation of the channels. The cAK activation but not the cGKII activation was blocked by a cAK inhibitor peptide. The slow activation by cGKII could not be ascribed to counteracting protein phosphatases, since neither calyculin A, a potent inhibitor of phosphatase 1 and 2A, nor ATPγS (adenosine 5′-O-(thiotriphosphate)), producing stable thiophosphorylation, was able to enhance the activation kinetics. Channels preactivated by cGKII closed instantaneously upon removal of ATP and kinase but reopened in the presence of ATP alone. Paradoxically, immunoprecipitated CFTR or CF-2, a cloned R domain fragment of CFTR (amino acids 645-835) could be phosphorylated to a similar extent with only minor kinetic differences by both isotypes of cGK. Phosphopeptide maps of CF-2 and CFTR, however, revealed very subtle differences in site-specificity between the cGK isoforms. These results indicate that cGKII, in contrast to cGKIα, is a potential activator of chloride transport in CFTR-expressing cell types. Type II cGMP-dependent protein kinase (cGKII) isolated from pig intestinal brush borders and type Iα cGK (cGKI) purified from bovine lung were compared for their ability to activate the cystic fibrosis transmembrane conductance regulator (CFTR)-Cl− channel in excised, inside-out membrane patches from NIH-3T3 fibroblasts and from a rat intestinal cell line (IEC-CF7) stably expressing recombinant CFTR. In both cell models, in the presence of cGMP and ATP, cGKII was found to mimic the effect of the catalytic subunit of cAMP-dependent protein kinase (cAK) on opening CFTR-Cl− channels, albeit with different kinetics (2-3-min lag time, reduced rate of activation). By contrast, cGKI or a monomeric cGKI catalytic fragment was incapable of opening CFTR-Cl− channels and also failed to potentiate cGKII activation of the channels. The cAK activation but not the cGKII activation was blocked by a cAK inhibitor peptide. The slow activation by cGKII could not be ascribed to counteracting protein phosphatases, since neither calyculin A, a potent inhibitor of phosphatase 1 and 2A, nor ATPγS (adenosine 5′-O-(thiotriphosphate)), producing stable thiophosphorylation, was able to enhance the activation kinetics. Channels preactivated by cGKII closed instantaneously upon removal of ATP and kinase but reopened in the presence of ATP alone. Paradoxically, immunoprecipitated CFTR or CF-2, a cloned R domain fragment of CFTR (amino acids 645-835) could be phosphorylated to a similar extent with only minor kinetic differences by both isotypes of cGK. Phosphopeptide maps of CF-2 and CFTR, however, revealed very subtle differences in site-specificity between the cGK isoforms. These results indicate that cGKII, in contrast to cGKIα, is a potential activator of chloride transport in CFTR-expressing cell types. INTRODUCTIONGuanosine 3′,5′-cyclic monophosphate (cGMP) has been identified as an important intracellular mediator of salt and water secretion in intestinal epithelium(1Field M. Graf L.H. Laird W.J. Smith P.L. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 2800-2804Crossref PubMed Scopus (432) Google Scholar, 2Hughes J.M. Murad F. Chang B. Guerrant R.L. Nature. 1978; 271: 755-756Crossref PubMed Scopus (252) Google Scholar, 3Field M. Rao M.C. Chang E.B. N. Engl. J. Med. 1989; 321 (879-883): 800-806Crossref PubMed Scopus (232) Google Scholar). Secretagogues acting through the cGMP-signaling pathway include the family of heat-stable enterotoxins (STs), 1The abbreviations used are: STheat-stable enterotoxinCFTRcystic fibrosis transmembrane conductance regulatorCFcystic fibrosiscGKcGMP-dependent protein kinasecAKcAMP-dependent protein kinasePKIWalsh inhibitor peptide (PKI(5-24)-amide)ATPγSadenosine 5′-O-(thiotriphosphate)PAGEpolyacrylamide gel electrophoresis. low molecular weight peptides secreted by enteropathogenic bacteria, and guanylin, a recently discovered endogenous ST-like peptide hormone(3Field M. Rao M.C. Chang E.B. N. Engl. J. Med. 1989; 321 (879-883): 800-806Crossref PubMed Scopus (232) Google Scholar, 4Currie M.G. Fok K.F. Kato J. Moore R.J. Hamra F.K. Duffin K.L. Smith C.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 947-951Crossref PubMed Scopus (502) Google Scholar). Binding of ST or guanylin to the receptor domain of an intestine-specific isoform of guanylyl cyclase (GC-C) triggers cyclase activation, cGMP accumulation, and stimulation of net fluid secretion through the activation of apical Cl− channels in parallel with inhibition of coupled NaCl transporters(3Field M. Rao M.C. Chang E.B. N. Engl. J. Med. 1989; 321 (879-883): 800-806Crossref PubMed Scopus (232) Google Scholar, 5Schultz S. Green C.K. Yuen P.S.T. Garbers D.L. Cell. 1990; 63: 941-948Abstract Full Text PDF PubMed Scopus (516) Google Scholar, 6Vaandrager A.B. Schulz S. De Jonge H.R. Garbers D.L. J. Biol. Chem. 1993; 268: 2171-2179Google Scholar). The cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial Cl− channel mutated in CF patients(7Collins F.S. Science. 1992; 256: 774-779Crossref PubMed Scopus (712) Google Scholar, 8Welsh M.J. Smith A.E. Cell. 1993; 73: 1251-1254Abstract Full Text PDF PubMed Scopus (1216) Google Scholar), appears to be involved in the Cl− secretory response to ST and cGMP analogues, as evidenced by the absence of this response in CF intestine(9De Jonge H.R. Bijman J. Sinaasappel M. Pediatr. Pulmonol. Suppl. 1987; 1: 54-57Google Scholar, 10Baxter P.S. Goldhill J. Hardcastle P.T. Taylor C.J. Nature. 1988; 335: 211Crossref PubMed Scopus (39) Google Scholar).Several mechanisms have been proposed to link cGMP to the CFTR-Cl− channels, including (i) cGMP cross-activation of cAMP-dependent protein kinase (11Forte L.R. Thorne P.K. Eber S.L. Krause W.J. Freeman R.H. Francis S. Corbin J.D. Am. J. Physiol. 1992; 263: C607-C615Crossref PubMed Google Scholar, 12Tien X.-Y. Brasitus T.A. Kaetzel M.A. Dedman J.R. Nelson D.J. J. Biol. Chem. 1994; 269: 51-54Abstract Full Text PDF PubMed Google Scholar, 13Chao A.C. de Sauvage F.J. Dong Y.-J. Wagner J.A. Goeddel D.V. Gardner P. EMBO J. 1994; 13: 1065-1072Crossref PubMed Scopus (231) Google Scholar) followed by of J. R.J. M.J. Smith A.E. Cell. Full Text PDF PubMed Scopus Google Scholar), of cGMP with the CFTR R.J. S. Pediatr. Pulmonol. Suppl. 1994; Scholar), and cGMP activation of an intestine-specific isoform of cGMP-dependent protein kinase II Jonge H.R. Nature. Scopus Google Scholar, Jonge H.R. Google Scholar, Jonge H.R. Rao M.C. M. of Scholar, A.B. De Jonge H.R. 1994; PubMed Scopus Google cGKII was discovered as a in intestinal brush Jonge H.R. Nature. Scopus Google Scholar), with a cGMP receptor protein on and Jonge H.R. Google Scholar, Jonge H.R. Rao M.C. M. of Scholar). The intestinal isoform is from the type Iα and cGK identified in as by differences in subunit and for Jonge H.R. Google Scholar, Jonge H.R. Rao M.C. M. of Scholar, A.B. De Jonge H.R. 1994; PubMed Scopus Google Scholar). molecular of cGKII from J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar) and rat J. De Jonge H.R. U. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar) that cGKII is a different and F. 1989; PubMed Scopus Google Scholar, M. U. 1989; PubMed Scopus Google the for a between cGK was from of the activation of CFTR-Cl− channels in membrane patches of an intestinal cell line J. M. J. A. De Jonge H.R. M. A. B. Am. J. Physiol. 1993; PubMed Google or NIH-3T3 fibroblasts stably expressing recombinant R.J. Smith A.E. M.J. Cell. 1993; Full Text PDF Scopus Google Scholar). In both models, of patches to a of cGMP and ATP failed to Cl− channel the of purified cGKII, but not cGKIα, in activation of CFTR-Cl− activation of the CFTR-Cl− channel by cGKII is the of in cGK of and a for the of cGMP as a regulator of Cl− transport in intestinal in to CFTR-expressing cell in cGKII is or A.B. De Jonge H.R. 1994; PubMed Scopus Google Scholar, J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, J. De Jonge H.R. U. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google was from the inhibitor peptide was from U. CF-2, a cloned R domain peptide of CFTR was in and purified as J.A. P. A.C. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). salt and cGMP were from was from were from and CFTR cell that stably CFTR were and as J. M. J. A. De Jonge H.R. M. A. B. Am. J. Physiol. 1993; PubMed Google Scholar, R.J. S. S. Smith A.E. M.J. Science. PubMed Scopus Google Scholar). were by stable of the rat cell line with a J. M. J. A. De Jonge H.R. M. A. B. Am. J. Physiol. 1993; PubMed Google NIH-3T3 CFTR with a R.J. S. S. Smith A.E. M.J. Science. PubMed Scopus Google of was purified from the of by the of The was from with and in membrane were from the intestinal by a and and as F.S. De Jonge H.R. J. PubMed Scopus Google Scholar). cGKII from the and by on was as Jonge H.R. Google Scholar, Jonge H.R. Rao M.C. M. of Scholar) with a for in the of cGKII from the gel with 1 cGMP was in the presence of cGMP and were by in catalytic subunit of type II cAK and cGKI by to be the Iα A. P. F. J. 1992; PubMed Scopus Google were purified from bovine and bovine as F. A.C. U. P. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, U. P. F. P. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of the purified protein as by the D.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar) were and monomeric cGKI fragment was by as C.E. J. Biol. Chem. Full Text PDF PubMed Google were as by A. B. F.J. Scopus Google Scholar). were to a of and potential to the to the with to the of the was used for and was were on an and on a The were or and on a was as by M. J. De Jonge H.R. Bijman J. 1993; PubMed Scopus Google Scholar). In of the that the of CFTR-Cl− channels in membrane patches of the fibroblasts the of or of channels, channel in patches was in as the of channels. The of the and was 1 and Cl− In a low Cl− was used and Cl− patches were in a 1 as by M. J. A.B. Sinaasappel M. De Jonge H.R. Bijman J. 1992; PubMed Scopus Google Scholar). were of CFTR and CF-2 and Phosphopeptide was immunoprecipitated from CFTR and protein as J.A. P. A.C. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). CFTR to protein of or CFTR was for in of 1 and purified protein subunit of cGKII, The phosphorylated were in and on as J.A. P. A.C. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). for CF-2 were similar to for CFTR. kinetic of with was by (i) the to the CF-2 between and the to and of protein were by the of of and protein were with of in of for and to as J.A. P. A.C. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). CF-2 or CFTR were from the with of and of and In the kinetic the of CF-2 was by of CF-2 and CFTR, 1 of was to the gel and the was for J.A. P. A.C. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). The gel were with of for and the were were on by in the followed by in the were to of by with of Cl− channel activation in excised, inside-out membrane patches from R.J. Smith A.E. M.J. Cell. 1993; Full Text PDF Scopus Google Scholar, R.J. J. M.J. Smith A.E. Nature. 1990; PubMed Scopus Google Scholar, M.J. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar), the of catalytic subunit of cAK to the in the presence of in activation 1 of channels of the CFTR-Cl− channel Cl− in Cl− channel results not similar low conductance a was by cAK in patches from the rat intestinal cell line stably expressing J. M. J. A. De Jonge H.R. M. A. B. Am. J. Physiol. 1993; PubMed Google Scholar). In to the effect of cGK on CFTR-Cl− channel opening was in excised, patches from both cell of and cGMP not activate in patches of the cGKI isoform purified from bovine lung failed to the CFTR-Cl− channel by M.J. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). the of of the cGKII a Cl− a that was of the upon of of cAK to the of the cGKII response was in the presence of cGKII, the for activation was found cGKII not In to CFTR-Cl− activation by cGKII was a slow to activation of 1 for and slow activation was to from the of counteracting protein phosphatases, since neither calyculin a potent inhibitor of phosphatase 1 and in CFTR M.J. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar) and A.C. Sci. 1994; Full Text PDF PubMed Scopus Google nor the presence of ATPγS to stable thiophosphorylation, was able to enhance the CFTR activation kinetics not cGKII but not was of CFTR-Cl− channels also in patches from In this low could be a in Cl− a channel conductance of and a in potential upon of the Cl− in the The of CFTR-Cl− channels the of activation by of cGKII not from the of channels of the channel in excised, inside-out membrane patches from A, of channels. chloride was by a low chloride were the channels of the The channel conductance was Cl− of Cl− in the to by with of the of the channel as CFTR from the that cGKII could not enhance channel in patches from their by cAK and ATP not between CFTR-Cl− channel by cAK and cGKII was the that the to upon the removal of kinase and ATP from the but could be instantaneously by the of ATP the of ATP in CFTR-Cl− channel and the that cGKII activation of CFTR from a of the cGKII with a peptide inhibitor of was to the this CFTR-Cl− channel activation in membrane patches by cAK was channel activation by cGKII was not of the channel by cAK in the removal of from the as compared to in the absence of was to of or CF-2 by cGKII the was that in of to CFTR-Cl− channels by could with the activation of CFTR-Cl− channels by the cGK neither of patches with cGKI results not nor the of cGKI and cGKII effect on the rate or extent of CFTR-Cl− channel activation as compared to cGKII of CFTR and CF-2 by cGK with CFTR and CF-2, the recombinant R domain of CFTR that both in for cAK and J.A. P. A.C. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, M.J. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). between and was from the kinetics of in of CF-2 by cGKI and cGKII CF-2 was a for cGKI for cGKII and the of CFTR was also for cGKI of the cGKII of CFTR and CF-2 by purified protein A, CFTR was immunoprecipitated from and phosphorylated as The also catalytic subunit of cAK 1 and cGKI and and cGKII and and in CFTR was The were by The gel was and to CFTR as a of J. R.J. M.J. Smith A.E. Cell. Full Text PDF PubMed Scopus Google The and of cGKII and Jonge H.R. Google and cGKI and the of CF-2 by of and The to of The the kinetic from the the of Phosphopeptide of CF-2 and CFTR by and to the R domain have been to be involved in the activation of CFTR A.C. Sci. 1994; Full Text PDF PubMed Scopus Google In to the of cGK activation of the CFTR-Cl− channel be to the of that was of CF-2 and CFTR phosphorylated by and cGKII phosphorylated the peptides in CF-2 with very differences in the of of peptides also phosphorylated the CFTR on a of the peptides phosphorylated in CF-2 the of cGKII the R in on peptides and J.A. P. A.C. J. Biol. Chem. 1992; Full Text PDF PubMed Google were phosphorylated to a similar In contrast, J.A. P. A.C. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar) for was phosphorylated by cAK and cGKII, but was phosphorylated to a by cGKI and not maps of CF-2 and CFTR. and maps of CF-2 and CFTR, by cAK and cGKI and and cGKII and CF-2 and immunoprecipitated CFTR were phosphorylated for in the presence of cAK cGKI or cGKII as The were by or and by of CF-2 and and J.A. P. A.C. J. Biol. Chem. 1992; Full Text PDF PubMed Google and CFTR were from and for with were by in the in and by in the from 1 to and to in J.A. P. A.C. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). The results of In of CF-2 an to peptide could be that the peptide peptide or in to this a recently cloned cGK J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, J. De Jonge H.R. U. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar), in the membrane of intestinal epithelial Jonge H.R. Nature. Scopus Google Scholar, Jonge H.R. Google Scholar, Jonge H.R. Rao M.C. M. of Scholar, A.B. De Jonge H.R. 1994; PubMed Scopus Google Scholar, J. De Jonge H.R. U. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar, A.B. S. U. De Jonge H.R. J. PubMed Scopus Google Scholar) and to a extent in and J. De Jonge H.R. U. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar), is identified as a potential regulator of the CFTR-Cl− a of a of and purified cGKII, inside-out membrane patches from intestinal or NIH-3T3 and the could mimic the effect of cAK on CFTR-Cl− channel albeit with a is however, that the in the in is the of the cGKII to the and that this opening not a similar kinetic for cGKII in with CFTR in the A.B. De Jonge H.R. 1994; PubMed Scopus Google Scholar, A.B. S. U. De Jonge H.R. J. PubMed Scopus Google Scholar). The of Cl− channel of that the of was the of similar of M.J. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google In with the maps from CF-2 and CFTR phosphorylated in by cGKII and cAK were and different from the by J.A. P. A.C. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). The of CFTR with cAK and cGKII was also similar the rate of CF-2 by cGKII was a between in and be with cGKIα, cGK isoform in cell F. A.C. U. P. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, U. P. F. P. J. Biol. Chem. Full Text PDF PubMed Google Scholar) failed to activate CFTR-Cl− channels in this and an M.J. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar) or to potentiate cGKII activation of the in of ability to in of the in CFTR molecular for the activation of the CFTR-Cl− channel by cGK isotypes is is that not or in CFTR that for the results from the peptide indicate that the of of CF-2 is similar for cGKI and cGKII and the as that of the of of CFTR by the was also similar with the that peptide was phosphorylated to only a very low by cGKI that the of the R domain is and that is for CFTR-Cl− channel J. R.J. M.J. Smith A.E. Cell. Full Text PDF PubMed Scopus Google Scholar), for a of of in the of the in A.C. Sci. 1994; Full Text PDF PubMed Scopus Google Scholar), and for the of and in the R D.J. F.S. Pediatr. Pulmonol. Suppl. 1994; Scholar). the presence of a phosphorylated by but not by cGKII or cAK since CFTR-Cl− channel activation by cGKII or cAK was not by cGKI is that the CFTR protein in membrane is to the cGK and that this is or in CF-2 peptide differences in or between the monomeric cGKII protein Jonge H.R. Google Scholar, Jonge H.R. Rao M.C. M. of Scholar, A.B. De Jonge H.R. 1994; PubMed Scopus Google Scholar) and the cGKI not a of a monomeric fragment failed to CFTR-Cl− channel the of has been that the for the of the type II cGK with in the J. De Jonge H.R. U. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar). in intestinal brush borders have also to a of a fragment in cGKII to the Jonge H.R. Google Scholar, Jonge H.R. Rao M.C. M. of Scholar). In the presence of a for in cGKII J. De Jonge H.R. U. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar) have is in Smith PubMed Scopus Google Scholar), in to activate CFTR. a cGK activation of the CFTR-Cl− channel on the of a protein in of epithelial (IEC-CF7) and have been recently in the of a different of Cl− channels J.R. C.J. L.R. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, B. F. M. J. Nature. 1993; PubMed Scopus Google Scholar). the opening of CFTR-Cl− channels in by cAK not an protein C.E. N. R.J. M. J.R. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar). with the cGK isotypes to between a and of CFTR of cGKII of CFTR-Cl− channels in membrane patches with the of cGKII and absence of cGKI in intestinal Jonge H.R. Google Scholar, Jonge H.R. Rao M.C. M. of Scholar, A.B. De Jonge H.R. 1994; PubMed Scopus Google Scholar, A.B. S. U. De Jonge H.R. J. PubMed Scopus Google Scholar) a cGKII as the of the of cGMP and in this and transport A.B. S. U. De Jonge H.R. J. PubMed Scopus Google Scholar) also a between cGKII and but not Cl− secretion in rat intestinal and cell M. A.C. Am. J. Physiol. 1992; PubMed Google Scholar), the cell not of cGKII A.B. S. U. De Jonge H.R. J. PubMed Scopus Google Scholar) but able to activate CFTR in response to as a of cGMP followed by cross-activation of L.R. Thorne P.K. Eber S.L. Krause W.J. Freeman R.H. Francis S. Corbin J.D. Am. J. Physiol. 1992; 263: C607-C615Crossref PubMed Google Scholar, 12Tien X.-Y. Brasitus T.A. Kaetzel M.A. Dedman J.R. Nelson D.J. J. Biol. Chem. 1994; 269: 51-54Abstract Full Text PDF PubMed Google Scholar, 13Chao A.C. de Sauvage F.J. Dong Y.-J. Wagner J.A. Goeddel D.V. Gardner P. EMBO J. 1994; 13: 1065-1072Crossref PubMed Scopus (231) Google Scholar). in a low of cGMP to in the brush membrane could be of rat to ST in F.S. De Jonge H.R. PubMed Scopus Google Scholar). in contrast to ST not enhance Cl− secretion in rat intestinal and in the in the presence of cGMP A.B. S. U. De Jonge H.R. J. PubMed Scopus Google Scholar). The for of cGKII and CFTR also cGMP activation of Cl− secretion is not in CFTR-expressing cell including M.J. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). INTRODUCTIONGuanosine 3′,5′-cyclic monophosphate (cGMP) has been identified as an important intracellular mediator of salt and water secretion in intestinal epithelium(1Field M. Graf L.H. Laird W.J. Smith P.L. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 2800-2804Crossref PubMed Scopus (432) Google Scholar, 2Hughes J.M. Murad F. Chang B. Guerrant R.L. Nature. 1978; 271: 755-756Crossref PubMed Scopus (252) Google Scholar, 3Field M. Rao M.C. Chang E.B. N. Engl. J. Med. 1989; 321 (879-883): 800-806Crossref PubMed Scopus (232) Google Scholar). Secretagogues acting through the cGMP-signaling pathway include the family of heat-stable enterotoxins (STs), 1The abbreviations used are: STheat-stable enterotoxinCFTRcystic fibrosis transmembrane conductance regulatorCFcystic fibrosiscGKcGMP-dependent protein kinasecAKcAMP-dependent protein kinasePKIWalsh inhibitor peptide (PKI(5-24)-amide)ATPγSadenosine 5′-O-(thiotriphosphate)PAGEpolyacrylamide gel electrophoresis. low molecular weight peptides secreted by enteropathogenic bacteria, and guanylin, a recently discovered endogenous ST-like peptide hormone(3Field M. Rao M.C. Chang E.B. N. Engl. J. Med. 1989; 321 (879-883): 800-806Crossref PubMed Scopus (232) Google Scholar, 4Currie M.G. Fok K.F. Kato J. Moore R.J. Hamra F.K. Duffin K.L. Smith C.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 947-951Crossref PubMed Scopus (502) Google Scholar). Binding of ST or guanylin to the receptor domain of an intestine-specific isoform of guanylyl cyclase (GC-C) triggers cyclase activation, cGMP accumulation, and stimulation of net fluid secretion through the activation of apical Cl− channels in parallel with inhibition of coupled NaCl transporters(3Field M. Rao M.C. Chang E.B. N. Engl. J. Med. 1989; 321 (879-883): 800-806Crossref PubMed Scopus (232) Google Scholar, 5Schultz S. Green C.K. Yuen P.S.T. Garbers D.L. Cell. 1990; 63: 941-948Abstract Full Text PDF PubMed Scopus (516) Google Scholar, 6Vaandrager A.B. Schulz S. De Jonge H.R. Garbers D.L. J. Biol. Chem. 1993; 268: 2171-2179Google Scholar). The cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial Cl− channel mutated in CF patients(7Collins F.S. Science. 1992; 256: 774-779Crossref PubMed Scopus (712) Google Scholar, 8Welsh M.J. Smith A.E. Cell. 1993; 73: 1251-1254Abstract Full Text PDF PubMed Scopus (1216) Google Scholar), appears to be involved in the Cl− secretory response to ST and cGMP analogues, as evidenced by the absence of this response in CF intestine(9De Jonge H.R. Bijman J. Sinaasappel M. Pediatr. Pulmonol. Suppl. 1987; 1: 54-57Google Scholar, 10Baxter P.S. Goldhill J. Hardcastle P.T. Taylor C.J. Nature. 1988; 335: 211Crossref PubMed Scopus (39) Google Scholar).Several mechanisms have been proposed to link cGMP to the CFTR-Cl− channels, including (i) cGMP cross-activation of cAMP-dependent protein kinase (11Forte L.R. Thorne P.K. Eber S.L. Krause W.J. Freeman R.H. Francis S. Corbin J.D. Am. J. Physiol. 1992; 263: C607-C615Crossref PubMed Google Scholar, 12Tien X.-Y. Brasitus T.A. Kaetzel M.A. Dedman J.R. Nelson D.J. J. Biol. Chem. 1994; 269: 51-54Abstract Full Text PDF PubMed Google Scholar, 13Chao A.C. de Sauvage F.J. Dong Y.-J. Wagner J.A. Goeddel D.V. Gardner P. EMBO J. 1994; 13: 1065-1072Crossref PubMed Scopus (231) Google Scholar) followed by of J. R.J. M.J. Smith A.E. Cell. Full Text PDF PubMed Scopus Google Scholar), of cGMP with the CFTR R.J. S. Pediatr. Pulmonol. Suppl. 1994; Scholar), and cGMP activation of an intestine-specific isoform of cGMP-dependent protein kinase II Jonge H.R. Nature. Scopus Google Scholar, Jonge H.R. Google Scholar, Jonge H.R. Rao M.C. M. of Scholar, A.B. De Jonge H.R. 1994; PubMed Scopus Google cGKII was discovered as a in intestinal brush Jonge H.R. Nature. Scopus Google Scholar), with a cGMP receptor protein on and Jonge H.R. Google Scholar, Jonge H.R. Rao M.C. M. of Scholar). The intestinal isoform is from the type Iα and cGK identified in as by differences in subunit and for Jonge H.R. Google Scholar, Jonge H.R. Rao M.C. M. of Scholar, A.B. De Jonge H.R. 1994; PubMed Scopus Google Scholar). molecular of cGKII from J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar) and rat J. De Jonge H.R. U. 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