Life Sciences Vol . 19, pp . 233-242, 1976 . Printed in the U.S .A .
Pergamon Press
CALCIUM AND CYCLIC NUCLEOTIDE INVOLVEMENT IN EXOCRINE PANCREATIC ENZYME SECRETION STUDIED WITH THE IONOPHORE A-23187 Seymour Heislerl Department of Physiology and Pharmacology C.H .U ., Université de Sherbrooke Sherbrooke, Québec JlH 5N4 (Received in final form June 4, 1976) Summary The ionophore, A-23187, was an effective pancreatic secretagogue . The response to A-23187 was Cat+-dependent ; Mgt+ reduced the secretory response to the ionophore . A-23187stimulated enzyme release was potentiated by dibutyryl cyclic AMP ; in the presence of carbachol, output of pancreatic protein paralleled the response to A-23187 alone. The timecourse for secretion with A-23187 was similar to that observed with carbachol. The ionophore did not affect basal cyclic AMP levels but did stimulate a rapid Cat+-dependent production of pancreatic cyclic GNP which preceded the onset of the secretory response . A-23187 did not significantly alter basal or carbachol-stimulated 45 Ca efflux from isotope preloaded glands ; yet in Cat+-lowered media, it inhibited (reversed) the secretory response to carbachol, an effect which may have been due to an outward transport by the ionophore of cholinergic-mobilized intracellular Cat+ . Like carbachol, A-23187, inhibits the incorporation of amino acid into new protein, the effect being partially dependent on extracellular Cat+ . The data suggest that the pancreatic cholinergic receptor acts as a Ca t+-ionophore and that extracellular Cat+ is utilized in the synthesis of cyclic GMP . In the exocrine pancreas some sequential temporal events associated with cholinergic stimulation are a) a decrease in basal cyclic AMP levels (1) ; bj a rapid increase in cyclic GMP synthesis (2) ; c) an increase in the rate of 4 Ca efflux from isotope-preloaded glands (3-7) ; d) an increase in the rate of secretion of zymogen protein and e) an initial inhibition of 3H-leucine incorporation into newly synthesized protein (8) . Calcium has been implicated as an important mediator in a variety of hormone and neurohormone-mediated events (9, 10) . The antibiotic A-23187 (EliLilly) is an agent capable of bypassing hormone receptors and of introducing extracellular Cat* into a variety of different target tissues (11-14) including the pancreas (15-17) . As a result of this activity, the antibiotic ought to
lFresent address : Department of Pharmacology, Université Laval, Cité Universitaire, Québec, Canada, GlK 7P4 233
234
Ca t+/cGMP in Pancreatic Secretion
Vol . 19, No . 2
duplicate all hormonal actions in tissues in which Cat+ is actively involved as a mediator . To date, only the secretory (15-17) and efflux (17) aspects of the ionophore action have been studied in the pancreas . The current study was designed to test the extent of the Cat"' requirement in pancreatic function by ascertaining if all of the carbachol-provoked events noted above could be mimicked by A-23187 . Methods Female Sprague-Dawley rats (180-225 g) were guillotined ; pancreata were excised amd adherent fat and mesentery removed in cold incubation medium . Pancreata were fragmented and 50-100 mg wet weight/flask used in subsequent experiments . Secretion of 3H-labeled protein (60 min of incubation at 370) from pancreatic fragments was studied in Krebs-Ringer bicarbonate buffer as described by Heisler et al . (18) . In these studies, extracellular Cat+ concen tration was modified, as indicated in Results . Cyclic AMP was measured according to Heisler et al . (19) following 1, 3 or 5 min of incubation of tissue samples in Krebs-Ringer bicarbonate buffer . Results are expressed as pmoles cyclic AMP formed - mg protein-1 . Cyclic GMP formed following 30 sec of incubation of pancreatic fragments, was quantitated by a radioimmunoassay technique modified by Harper and Brooker (20) . Assays were conducted in 50 mM sodium acetate buffer (pH 6 .2) [125 11_2'0-succinyl cyclic GMP tyrosine methyl using cyclic GMP antiserum and eater from a cyclic GMP kit (2) . Results are expressed as fmoles cyclic GMP formed " mg protein-1 " 30 sec-1 . Release of 45 Ca was studied in fragmented glands which were preincubated for 30 min at 37 0 in 10 ml Krebs-Ringer bicarbonate buffer containing 0 .05 mM CaC12 and 0.5 UCi/ml 45Ca . Following removal of the tissue, and washing in isotope-free buffer, the fragments were subdivided into 25 ml flasks containing 2 ml of 45 Ca-free buffer and were than transferred to fresh media 3 times over the next 70 min. Test agents were added and tissues were transferred at 10 min intervals, for the next 50 min, essentially as described by Case and Clausen (3) . During the efflux period, CaC12 was not added to the incubation medium ; ethyleneglycolbis-(ß-aminoethyl)-N,N'-tetraacetic acid (EGTA), 0 .05 UM, was present . At the end of the last 10 min period, tissues removed, homogenized in 5% trichloroacetic acid, and extracted for 60 min at room temperature prior to centrifugation . The pellets were neutralized and used to measure protein Counts, in the efflux content (21) ; an aliquot of the supernatant was counted . media and extracted tissues were corrected for background and efficiency, and totaled. 45 Ca released is calculated as the amount (x) of isotope remaining in the tissue at the end of each 10 min intervai . Incorporation of 3H-leucine into .trichloroacetic acid precipitable protein was estimated in Krebs-Ringer phosphate buffer as outlined by Irvin and Tenenhouse (8) . All experiments were performed a minimum of 3 times and student's t test for unpaired data was used in the statistical comparison of results obtained . Materiale The ionophore A-23187 suspended in solution according to Prince et al . Carbamylcholine chlo(22), was a gift from Eli-Lilly Co . (Indianapolis, Ind .) .
Vol . 19, No . 2
Cat+ /cGMP in Pancreatic Secretion
23 5
ride (carbachol), diburyryl cyclic AMP, cyclic AMP and cyclic GMP were purchased from [8Sigma Chemical Co . (St . Louis, Mo .) . 3H] -Cyclic AMP (20.8 Ci/mmole) and the cyclic GMP assay kit were obtained from Schwars/Mann (Montreal, Qua) ; L- [4,5-3Hj-leucine (53 Ci/mmole) and 45 Ca (15.6 uCi/ug) were purchased from Amersham/Searle Corp . (Oakville, Ont .) . All other chemicals used were of reagent grade and were acquired locally. Results The effect of the ionophore on the pancreatic secretory respônee was observed in a number of different experiments . In the first of these, pancreatic fragments were exposed to a fixed concentration of the ionophore, while the eztracellular concentration of Cat+ was varied . In a parallel study, Mgt+ was omitted from the incubation medium since it had been reported (23) that the ionophore was also capable of forming a complex with this cation . Results obtained are summarised in Fig . 1.
log ~am ]
mm
FIG. 1 Secretory responses of the pancreas to the ionophore A-23187 (0 .25 ug/ml) in the presence of a varying concentration of Incubation Cat+ was for 1 hr at 37 0. Experiments were performed in the presence or absence of added Mgt+ (1 .2 mM) . Control, no Mgt+ ( 0 ) ; control with Mgt+ ( m ) ; A-23187, Mgt* no ( a ) ; A-23187, with Mgl+ Values are means t S .E .M . of 4 observations . The release of 3H-labeled protein in response to the ionophors with or without Mgt+ present, is dependent on the eztracellular Cat+ concentration ; optimal output was observed between 0 .5 - 0 .75 mM CaC12 . Interestingly enough, the response to the ionophore is reduced by the simultaneous presence of Mg . Mgt* in the incubation medium, indirectly suggesting that both Cat+ and compete in complex formation with the ionophore. The basal response is also reduced by 2+ Mg -
Ca t+ /cGMP in Pancreatic Secretion
236
Vol . 19, No . 2
Fig . 2 illustrates the pancreatic response to A-23187 alone, or in combination with carbachol, dibutyryl cyclic AMP, or carbachol + dibutyryl The ionophore effec cyclic AMP (extracellular CaC12 concentration - 2 .5 mM) . tively stimulated protein secretion at concentrations of 0 .25 fig - 2 .5 ug/ml . At higher concentrations it was twice as effective in stimulating secretion compared to carbachol . Carbachol by itself also significantly stimulated secretion (P 0.5 ug/ml, the output of protein in response to both secretagogues together was identical to that observed with the ionophore alone. Dibutyryl cyclic AMP alone also significantly increased basal enzyme release (P
Pergamon Press
CALCIUM AND CYCLIC NUCLEOTIDE INVOLVEMENT IN EXOCRINE PANCREATIC ENZYME SECRETION STUDIED WITH THE IONOPHORE A-23187 Seymour Heislerl Department of Physiology and Pharmacology C.H .U ., Université de Sherbrooke Sherbrooke, Québec JlH 5N4 (Received in final form June 4, 1976) Summary The ionophore, A-23187, was an effective pancreatic secretagogue . The response to A-23187 was Cat+-dependent ; Mgt+ reduced the secretory response to the ionophore . A-23187stimulated enzyme release was potentiated by dibutyryl cyclic AMP ; in the presence of carbachol, output of pancreatic protein paralleled the response to A-23187 alone. The timecourse for secretion with A-23187 was similar to that observed with carbachol. The ionophore did not affect basal cyclic AMP levels but did stimulate a rapid Cat+-dependent production of pancreatic cyclic GNP which preceded the onset of the secretory response . A-23187 did not significantly alter basal or carbachol-stimulated 45 Ca efflux from isotope preloaded glands ; yet in Cat+-lowered media, it inhibited (reversed) the secretory response to carbachol, an effect which may have been due to an outward transport by the ionophore of cholinergic-mobilized intracellular Cat+ . Like carbachol, A-23187, inhibits the incorporation of amino acid into new protein, the effect being partially dependent on extracellular Cat+ . The data suggest that the pancreatic cholinergic receptor acts as a Ca t+-ionophore and that extracellular Cat+ is utilized in the synthesis of cyclic GMP . In the exocrine pancreas some sequential temporal events associated with cholinergic stimulation are a) a decrease in basal cyclic AMP levels (1) ; bj a rapid increase in cyclic GMP synthesis (2) ; c) an increase in the rate of 4 Ca efflux from isotope-preloaded glands (3-7) ; d) an increase in the rate of secretion of zymogen protein and e) an initial inhibition of 3H-leucine incorporation into newly synthesized protein (8) . Calcium has been implicated as an important mediator in a variety of hormone and neurohormone-mediated events (9, 10) . The antibiotic A-23187 (EliLilly) is an agent capable of bypassing hormone receptors and of introducing extracellular Cat* into a variety of different target tissues (11-14) including the pancreas (15-17) . As a result of this activity, the antibiotic ought to
lFresent address : Department of Pharmacology, Université Laval, Cité Universitaire, Québec, Canada, GlK 7P4 233
234
Ca t+/cGMP in Pancreatic Secretion
Vol . 19, No . 2
duplicate all hormonal actions in tissues in which Cat+ is actively involved as a mediator . To date, only the secretory (15-17) and efflux (17) aspects of the ionophore action have been studied in the pancreas . The current study was designed to test the extent of the Cat"' requirement in pancreatic function by ascertaining if all of the carbachol-provoked events noted above could be mimicked by A-23187 . Methods Female Sprague-Dawley rats (180-225 g) were guillotined ; pancreata were excised amd adherent fat and mesentery removed in cold incubation medium . Pancreata were fragmented and 50-100 mg wet weight/flask used in subsequent experiments . Secretion of 3H-labeled protein (60 min of incubation at 370) from pancreatic fragments was studied in Krebs-Ringer bicarbonate buffer as described by Heisler et al . (18) . In these studies, extracellular Cat+ concen tration was modified, as indicated in Results . Cyclic AMP was measured according to Heisler et al . (19) following 1, 3 or 5 min of incubation of tissue samples in Krebs-Ringer bicarbonate buffer . Results are expressed as pmoles cyclic AMP formed - mg protein-1 . Cyclic GMP formed following 30 sec of incubation of pancreatic fragments, was quantitated by a radioimmunoassay technique modified by Harper and Brooker (20) . Assays were conducted in 50 mM sodium acetate buffer (pH 6 .2) [125 11_2'0-succinyl cyclic GMP tyrosine methyl using cyclic GMP antiserum and eater from a cyclic GMP kit (2) . Results are expressed as fmoles cyclic GMP formed " mg protein-1 " 30 sec-1 . Release of 45 Ca was studied in fragmented glands which were preincubated for 30 min at 37 0 in 10 ml Krebs-Ringer bicarbonate buffer containing 0 .05 mM CaC12 and 0.5 UCi/ml 45Ca . Following removal of the tissue, and washing in isotope-free buffer, the fragments were subdivided into 25 ml flasks containing 2 ml of 45 Ca-free buffer and were than transferred to fresh media 3 times over the next 70 min. Test agents were added and tissues were transferred at 10 min intervals, for the next 50 min, essentially as described by Case and Clausen (3) . During the efflux period, CaC12 was not added to the incubation medium ; ethyleneglycolbis-(ß-aminoethyl)-N,N'-tetraacetic acid (EGTA), 0 .05 UM, was present . At the end of the last 10 min period, tissues removed, homogenized in 5% trichloroacetic acid, and extracted for 60 min at room temperature prior to centrifugation . The pellets were neutralized and used to measure protein Counts, in the efflux content (21) ; an aliquot of the supernatant was counted . media and extracted tissues were corrected for background and efficiency, and totaled. 45 Ca released is calculated as the amount (x) of isotope remaining in the tissue at the end of each 10 min intervai . Incorporation of 3H-leucine into .trichloroacetic acid precipitable protein was estimated in Krebs-Ringer phosphate buffer as outlined by Irvin and Tenenhouse (8) . All experiments were performed a minimum of 3 times and student's t test for unpaired data was used in the statistical comparison of results obtained . Materiale The ionophore A-23187 suspended in solution according to Prince et al . Carbamylcholine chlo(22), was a gift from Eli-Lilly Co . (Indianapolis, Ind .) .
Vol . 19, No . 2
Cat+ /cGMP in Pancreatic Secretion
23 5
ride (carbachol), diburyryl cyclic AMP, cyclic AMP and cyclic GMP were purchased from [8Sigma Chemical Co . (St . Louis, Mo .) . 3H] -Cyclic AMP (20.8 Ci/mmole) and the cyclic GMP assay kit were obtained from Schwars/Mann (Montreal, Qua) ; L- [4,5-3Hj-leucine (53 Ci/mmole) and 45 Ca (15.6 uCi/ug) were purchased from Amersham/Searle Corp . (Oakville, Ont .) . All other chemicals used were of reagent grade and were acquired locally. Results The effect of the ionophore on the pancreatic secretory respônee was observed in a number of different experiments . In the first of these, pancreatic fragments were exposed to a fixed concentration of the ionophore, while the eztracellular concentration of Cat+ was varied . In a parallel study, Mgt+ was omitted from the incubation medium since it had been reported (23) that the ionophore was also capable of forming a complex with this cation . Results obtained are summarised in Fig . 1.
log ~am ]
mm
FIG. 1 Secretory responses of the pancreas to the ionophore A-23187 (0 .25 ug/ml) in the presence of a varying concentration of Incubation Cat+ was for 1 hr at 37 0. Experiments were performed in the presence or absence of added Mgt+ (1 .2 mM) . Control, no Mgt+ ( 0 ) ; control with Mgt+ ( m ) ; A-23187, Mgt* no ( a ) ; A-23187, with Mgl+ Values are means t S .E .M . of 4 observations . The release of 3H-labeled protein in response to the ionophors with or without Mgt+ present, is dependent on the eztracellular Cat+ concentration ; optimal output was observed between 0 .5 - 0 .75 mM CaC12 . Interestingly enough, the response to the ionophore is reduced by the simultaneous presence of Mg . Mgt* in the incubation medium, indirectly suggesting that both Cat+ and compete in complex formation with the ionophore. The basal response is also reduced by 2+ Mg -
Ca t+ /cGMP in Pancreatic Secretion
236
Vol . 19, No . 2
Fig . 2 illustrates the pancreatic response to A-23187 alone, or in combination with carbachol, dibutyryl cyclic AMP, or carbachol + dibutyryl The ionophore effec cyclic AMP (extracellular CaC12 concentration - 2 .5 mM) . tively stimulated protein secretion at concentrations of 0 .25 fig - 2 .5 ug/ml . At higher concentrations it was twice as effective in stimulating secretion compared to carbachol . Carbachol by itself also significantly stimulated secretion (P 0.5 ug/ml, the output of protein in response to both secretagogues together was identical to that observed with the ionophore alone. Dibutyryl cyclic AMP alone also significantly increased basal enzyme release (P
