Hormonal Control of ADP-ribosyl Cyclase Activity in Pancreatic Acinar Cells from Rats

Abstract

Cyclic ADP-ribose, a metabolite of NAD+ evokes Ca2+ release from intracellular stores in different cells. We have determined the activity of cADPr-producing enzymes (ADP-ribosyl cyclases) in different cellular fractions prepared from isolated pancreatic acinar cells by measuring the conversion of the β-NAD+ analogs 1,N 6-etheno-NAD and nicotinamide guanine dinucleotide to the fluorescent products 1,N 6-etheno-cADPr and cyclic GDP-ribose, respectively. Substrate/product analyses were carried out by reverse-phase high pressure liquid chromatography. In all subcellular fractions examined (cytosol, mitochondria, plasma, and intracellular membranes), ADP-ribosyl cyclase activity was detected except in zymogen granular membranes. Western blot analysis and immunoprecipitation experiments revealed the presence of the ADP-ribosyl cyclase CD38 in both plasma membranes and mitochondria but not in the cytosol. Hormonal stimulation of intact acinar cells for 1 min with acetylcholine (ACh), cholecystokinin (CCK), or a membrane-permeant analog of cGMP increased ADP-ribosyl cyclase activity in the cytosol by 1.8-, 1.6-, and 1.9-fold, respectively, as compared with the control but had no effect in any other fraction. Both ACh and CCK also increased accumulation of cGMP in the cells by about 2-fold. Bombesin had no significant effect on either ADP-ribosyl cyclase activity or cGMP accumulation within this short period of stimulation. We conclude that at least two types of ADP-ribosyl cyclases are present in pancreatic acinar cells: membrane-bound CD38 and a cytosolic enzyme different from CD38. Stimulation of pancreatic acinar cells with CCK or ACh results in exclusive activation of the cytosolic ADP-ribosyl cyclase activity, most likely mediated by cGMP. Cyclic ADP-ribose, a metabolite of NAD+ evokes Ca2+ release from intracellular stores in different cells. We have determined the activity of cADPr-producing enzymes (ADP-ribosyl cyclases) in different cellular fractions prepared from isolated pancreatic acinar cells by measuring the conversion of the β-NAD+ analogs 1,N 6-etheno-NAD and nicotinamide guanine dinucleotide to the fluorescent products 1,N 6-etheno-cADPr and cyclic GDP-ribose, respectively. Substrate/product analyses were carried out by reverse-phase high pressure liquid chromatography. In all subcellular fractions examined (cytosol, mitochondria, plasma, and intracellular membranes), ADP-ribosyl cyclase activity was detected except in zymogen granular membranes. Western blot analysis and immunoprecipitation experiments revealed the presence of the ADP-ribosyl cyclase CD38 in both plasma membranes and mitochondria but not in the cytosol. Hormonal stimulation of intact acinar cells for 1 min with acetylcholine (ACh), cholecystokinin (CCK), or a membrane-permeant analog of cGMP increased ADP-ribosyl cyclase activity in the cytosol by 1.8-, 1.6-, and 1.9-fold, respectively, as compared with the control but had no effect in any other fraction. Both ACh and CCK also increased accumulation of cGMP in the cells by about 2-fold. Bombesin had no significant effect on either ADP-ribosyl cyclase activity or cGMP accumulation within this short period of stimulation. We conclude that at least two types of ADP-ribosyl cyclases are present in pancreatic acinar cells: membrane-bound CD38 and a cytosolic enzyme different from CD38. Stimulation of pancreatic acinar cells with CCK or ACh results in exclusive activation of the cytosolic ADP-ribosyl cyclase activity, most likely mediated by cGMP. Hormonal stimulation of pancreatic acinar cells stimulates production of inositol 1,4,5-trisphosphate (IP3) 1The abbreviations used are: IP3, d-myo-inositol-1,4,5-trisphosphate; ACh, acetylcholine; BSA, bovine serum albumin; CCK, cholecystokinin; cADPr, cyclic ADP-ribose; ϵ-AD, 1,N 6-ethenoadenine; ϵ-AMP, 1,N 6-ethenoadenosine 5′-monophosphate; ϵ-ADP, 1,N 6-ethenoadenosine-5′-diphosphate; ϵ-ADPr, 1,N 6-etheno-ADP-ribose; ϵ-cADPr, 1,N 6-ethenocyclic ADP-ribose; ϵ-NAD+, 1,N 6-etheno-NAD+; HPLC, high performance liquid chromatography; NGD, nicotinamide guanine dinucleotide; MES, 2-(N-morpholino)ethanesulfonic acid. with a consequent release of Ca2+ from intracellular Ca2+ stores (1Streb H. Irvine R.F. Berridge M.J. Schulz I. Nature. 1983; 306: 67-69Crossref PubMed Scopus (1794) Google Scholar). This leads to an increase in cytosolic [Ca2+], which propagates from the luminal to the basolateral cell pole as a Ca2+ wave (2Nathanson M.H. Padfield P.J. O'Sullivan A.J. Burgstahler A.D. 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At present, only limited information is available about hormonal regulation of the enzymes, which can produce cADPr in pancreatic acinar cells. The ectoenzyme CD38 is a candidate (18Fukushi Y. Kato I. Takasawa S. Sasaki T. Ong B.H. Sato M. Ohsaga A. Sato K. Shirato K. Okamoto H. Maruyama Y. J. Biol. Chem. 2001; 276: 649-655Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), and indeed it was shown that following muscarinic stimulation of isolated acini, the cADPr concentration was lower in the homogenate of pancreatic acinar cells from CD38 knockout mice as compared with wild type mice (18Fukushi Y. Kato I. Takasawa S. Sasaki T. Ong B.H. Sato M. Ohsaga A. Sato K. Shirato K. Okamoto H. Maruyama Y. J. Biol. Chem. 2001; 276: 649-655Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Despite a model consisting of NAD export via connexin 43, extracellular synthesis of cADPr by ecto-CD38, and reimport of cADPR via CD38 or via nucleoside transporters (20Graeff R.M. Franco L. De Flora A. Lee H.C. J. Biol. Chem. 1998; 273: 118-125Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), it remains mainly unclear how an ecto-ADP-ribosyl cyclase such as CD38 can efficiently and rapidly produce an intracellular second messenger. A soluble ADP-ribosyl cyclase was purified from Aplysia californica ovotestis (19Hellmich M.R. Strumwasser F. Cell Regul. 1991; 2: 193-202Crossref PubMed Scopus (144) Google Scholar), and soluble ADP-ribosyl cyclase activities have also been detected in sea-urchin eggs (20Graeff R.M. Franco L. De Flora A. Lee H.C. J. Biol. Chem. 1998; 273: 118-125Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), brain homogenates (21Matsumura N. Tanuma S. Biochem. Biophys. Res. Commun. 1998; 253: 246-252Crossref PubMed Scopus (23) Google Scholar), and human T cells (22Guse A.H. da Silva C.P. Berg I. Skapenko A.L. Weber K. Heyer P. Hohenegger M. Ashamu G.A. Schulze-Koops H. Potter B.V. Mayr G.W. Nature. 1999; 398: 70-73Crossref PubMed Scopus (270) Google Scholar). In the present study, we show that in a cytosolic fraction from pancreatic acinar cells, which is of an increase in both ADP-ribosyl cyclase activity and cGMP accumulation was detected following hormonal stimulation of the cells with acetylcholine or cholecystokinin (CCK), had no The effect of hormonal activation of ADP-ribosyl cyclase by the cGMP indicating of cGMP and a in this and of from were to and in of 1 and with bovine serum with was carried out in two with and of for min at of were with A and were in A with cells were by in a with for 1 of cellular were carried out cells, zymogen and were by at for the was at for and the was in A The of this was for In to Schulz I. T. W. Am. J. Physiol. 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Google Scholar) a of enzymes in cellular fractions as compared with the in a of cytosolic ADP-ribosyl cyclase it was to the enzyme activities rapidly the the at for the was The cell membranes and mitochondria was at for The was as and the as enzymes for other were in the of this to the shown in I. of from the granular were by in of to with with 1 and and were on for The was at for and the was in A F. J. Schulz I. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). for ADP-ribosyl cyclase activity was in subcellular fractions Biochem. 1996; PubMed Scopus Google Scholar). The fractions were to a concentration of 1 in and 1,N was at at of to ϵ-cADPr, ϵ-ADPr, and other was by an increase and was at and at an of a analysis of products of was carried out by reverse-phase of was by the rapidly to and at for min a The of the enzyme was to to the ADP-ribosyl cyclase activities present in fraction homogenate and fractions cytosolic fraction not the of the cytosolic fraction for of NAD+ not have any significant on the ADP-ribosyl cyclase activity and was not applied In the was used of The were the as for the by the ADP-ribosyl cyclase is cyclic GDP-ribose, which was and by of and of different that can by Biochem. 1996; PubMed Scopus Google Scholar), of the products was This was by K. Mayr G.W. A.H. Biochem. 2001; PubMed Scopus Google Scholar) to that used to cADPr Silva C.P. Potter B.V. Mayr G.W. A.H. J. 1998; PubMed Scopus Google Scholar). were by or at for of ϵ-cADPr, of the was a reverse-phase A from to was used at a of 1 K. Mayr G.W. A.H. Biochem. 2001; PubMed Scopus Google Scholar, Silva C.P. Potter B.V. Mayr G.W. A.H. J. 1998; PubMed Scopus Google Scholar). in the were at at at at and at 1,N adenosine were detected with a and and of the were by the and of with of The ϵ-NAD+, ϵ-ADP, ϵ-AMP, and were was by with Aplysia californica ADP-ribosyl cyclase for was from by to for min K. Mayr G.W. A.H. Biochem. 2001; PubMed Scopus Google Scholar, Silva C.P. Potter B.V. Mayr G.W. A.H. J. 1998; PubMed Scopus Google Scholar). The of the products in as a were for and for Western and of CD38 Western blot were by in and membranes. were for 1 with and with a CD38 and were the immunoprecipitation of of cell to 1 with 1 was with a of a CD38 for min at the addition of of was for were with and with of the were in of and and for min at of ADP-ribosyl cyclase activity, the was in of and the activity was as of cGMP in were determined a available enzyme prepared pancreatic acinar cells of were in of A and for 1 min in the presence of the or of at and of the intracellular cGMP were determined to the ADP-ribosyl cyclase activities in different cellular fractions and we used as of the nicotinamide by of to such as ϵ-AD, ϵ-ADP, ϵ-AMP, ϵ-cADPr, and in an increase in Biochem. 1996; PubMed Scopus Google Scholar). available ADP-ribosyl cyclase all within the different subcellular fractions purified from pancreatic acinar cells enzyme activities not ADP-ribosyl cyclase activities in intact pancreatic acinar cells were detected when was present in the extracellular not activity was detected of not In to the ADP-ribosyl cyclase activity out of the it was to the by a of which were by activity in different pancreatic acinar cell of is fractions were with the of In all the was to different indicating that for NAD+ are present in pancreatic acinar cells. In all except the zymogen cADPr was at and the that the different as compared with the was by the has been used as an ADP-ribosyl cyclase in R.M. Walseth T.F. K. Lee H.C. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, H. A. F. Biol. 1997; PubMed Scopus Google Scholar, Biochem. Biophys. Res. Commun. 1997; PubMed Scopus Google Scholar). The of the of both in cytosolic and fractions were not different from not with The ADP-ribosyl cyclase activity was determined in fraction from the of by and the In different it and was in mitochondria by the plasma and the cytosol The zymogen had ADP-ribosyl cyclase of zymogen granular membranes from the zymogen it that no ADP-ribosyl cyclase activity was present in this fraction as compared with the cell homogenate cyclase activities in cellular fractions from pancreatic acinar cyclase ADP-ribosyl cyclase of granular in a The ADP-ribosyl cyclase activity in pancreatic homogenate from in to of cADPr ADP-ribosyl cyclase activity in the cytosol is with that in other fractions activity in the zymogen and membranes can This that a significant of activity is present in the cytosol to for and cADPr production following hormonal stimulation. is that pancreatic acinar cells the ectoenzyme which ADP-ribosyl and activities (18Fukushi Y. Kato I. Takasawa S. Sasaki T. Ong B.H. Sato M. Ohsaga A. Sato K. Shirato K. Okamoto H. Maruyama Y. J. Biol. Chem. 2001; 276: 649-655Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). shown in the for human CD38 a with the of CD38 in the plasma the and a was present in zymogen in zymogen granular membranes as as in the cytosolic CD38 was not the of applied to the was increased not the of CD38 from the we and purified CD38 by immunoprecipitation and Western blot shown in the a only from the membranes and not from the cytosolic fraction. of activities Western blot we the ADP-ribosyl cyclase activity in both the and the of the The from the membranes an ADP-ribosyl cyclase activity of of activity in the the in the and no ADP-ribosyl cyclase activity was detected in the This the Western blot and that the ADP-ribosyl cyclase activity in the cytosol is different from CD38. the ADP-ribosyl cyclase activity in the membranes but to the cytosolic ADP-ribosyl cyclase activity in a of ADP-ribosyl cyclase activity indicating that cADPr was indeed by we that CD38 is the ADP-ribosyl cyclase activity in plasma membranes and mitochondria, the cytosolic ADP-ribosyl cyclase activity is an enzyme different from CD38. ADP-ribosyl in was to intact cells, and conversion to was following the addition of ACh CCK or of activity by a from cells We conclude that activity is not in cADPr Hormonal of ADP-ribosyl Stimulation of intact acinar cells for min with ACh or CCK and increased ADP-ribosyl cyclase activity in the cytosolic fraction. stimulation was detected 1 and In to ACh and CCK, had no effect on the ADP-ribosyl cyclase activity in any fraction of CCK not increase ADP-ribosyl cyclase activity as compared with cells, enzyme activity was by CCK of 1 to of to and of to The effect of ACh was at 1 ACh or lower to a activity of ADP-ribosyl cyclase in the cytosol In to in the activation of ADP-ribosyl ADP-ribosyl cyclase activities in membranes and the cytosol following stimulation of intact cells with membrane-permeant analogs of cyclic were of intact cells with 1 to a stimulation of cytosolic ADP-ribosyl cyclase activity compared with control cells, the activity in the fraction stimulation of intact cells with 1 not ADP-ribosyl cyclase activity, of can cGMP to subcellular fractions of pancreatic acinar cells to the ADP-ribosyl cyclase activity, indicating that are for ADP-ribosyl cyclase cGMP is in pancreatic acinar cells following hormonal stimulation R.J. 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This of and Ca2+ in with the the of a Ca2+ wave from the to the basolateral pole when different were used to Ca2+ release (3Pfeiffer F. Sternfeld L. Schmid A. Schulz I. Am. J. Physiol. 1998; 274: C663-C672Crossref PubMed Google Scholar, 7Xu X. Zeng W. Diaz J. Muallem S. J. Biol. Chem. 1996; 271: 24684-24690Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 8Gonzalez A. Schmid A. Sternfeld L. Krause E. Salido G.M. Schulz I. Biochem. Biophys. Res. Commun. 1999; 261: 726-733Crossref PubMed Scopus (39) Google Scholar, J.F. P. Physiol. 2000; PubMed Scopus Google Scholar). stimulation with either ACh or CCK, but not with to activation of cytosolic ADP-ribosyl cyclase of the present the of Ca2+ with CCK and ACh with (3Pfeiffer F. Sternfeld L. Schmid A. Schulz I. Am. J. Physiol. 1998; 274: C663-C672Crossref PubMed Google Scholar, 8Gonzalez A. Schmid A. Sternfeld L. Krause E. Salido G.M. Schulz I. Biochem. Biophys. Res. Commun. 1999; 261: 726-733Crossref PubMed Scopus (39) Google Scholar) also to or production of cADPr and of Ca2+-induced Ca2+ in the present hormonal ADP-ribosyl cyclase activity was for the in the cytosol of acinar cells, the remains of how of this enzyme to the is that a of the enzyme from the cytosol to the plasma hormonal in activities were not in plasma membranes. for a and cytosolic ADP-ribosyl cyclase of a cytosolic such as by an K. S. Biochem. J. 2001; PubMed Scopus Google Scholar). of cyclase by and a in cytosolic had also been in smooth muscle cells Zhang Am. J. Physiol. 2000; Scholar). that stimulates ADP-ribosyl cyclase activity and that that ADP-ribosyl cyclase activity also increase cGMP production and that cGMP a for the by which the cytosolic ADP-ribosyl In that a cytosolic ADP-ribosyl cyclase from CD38 is by CCK or ACh to of cADPr with a consequent Ca2+ release from pancreatic acinar cells.