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Activation of protein kinase in the guinea pig fundic gastric mucosa by histamine
Life Sciences Vol . 17, pp . Printed in the U .S .A .
Pergamon Press
1303-1306
ACTIVATION OF PROTEIN KINASE IN THE GUINFA PIG FUNDIC GASTRIC MUCOSA BY HISTAMINE Armin Wollin, Larry D . Barnes, Yvonne S . Hui and Thomas P . Dousa Mayo Clinic and Foundation, Rochester, Minnesota (Received in final form September 19, 1975) The effect of histamine, 1,4-methylhistamine and ethanol on cyclic AMP levels and protein kinase activation was measured in tissue strips from the fundic region of guinea pig gastric mucosa . Histamine induced a significant elevation of tissue cyclic AMP levels and also in situ activation of the protein kinase . 1,4-methylhistamine, an inactive analog of histamine, and ethanol had no effect on these twn parameters . Results suggest that protein kinase activation is involved in the cyclic AMP-mediated action of histamine on the gastric fundic mucosa . Considerable experimental evidence iadicatea that histamine action on gastric mucosa HCL secretion is mediated by cyclic AMP, at least in some mammalian species . It has been shown that histamine can specifically stim ulate fundic gastric mucosa adenylate cyclase (1-3) and can elevate the tissue (4,5) or gastric juice levels (6) of the cyclic AMP . However, the mechanism by which cyclic AMP generated under the influence of histamine influences the gastric secretioy function is not known . An active cyclic AMPdependent protein kinase has been described in cell-free preparations from fundic gastric mucosa of guinea pig (2) and rabbit (7) . In the present preliminary experiments, we explored whether histamine, through mediation by cyclic AMP, can activate protein kinase in the intact, unbroken cells of the fundic region of guinea pig gastric mucosa . Methods Guinea pigs maintained on ad libitum diet were killed and strips of mucosa were removed from the fundic region of the stomach . Mucosa tissue was kept in ice-cold Kreba-Ringer solution saturated with a mixture of 957 oxygen aad 57. Cda and then preincubated in this solution for 10 min at 37oC . The tissue was then distributed in flasks using a ratio of 0 .3 $m of tissue per 3 ml Krebs-Ringer solution containing also 0 .2 mM 1-methyl-3-isobutylxanthine . Tissue was then incubated with or without addition of histamine or other tested agents for 10 min at 37oC . In preliminary experiment it was determined that histamine increased cyclic AMP level already at 5 min and increased level did not change substantially for at least 20 min . At the end of the incubation period the tissue was removed and homogenized in a medium containing 5 mM potassium phosphate, 2 mM EI7fA, 0 .25 M sucrose and 0 .5 mM 1-methyl-3-isobutylxanthine, pH 7 .0 . An aliquot (0 .5 ml) of homogenate was quickly mixed with an equal volume of ice-cold 10~ trichloroacetic acid (TCA) and stored frozen for cyclic AMP determination . The homogenate was then iumediately centrifuged at OoC (10,000 x g for 10 min) and the protein kinase activity in the fresh supernate was assayed without or with addition of 5 x 10' 6M of cyclic AMP . The method for protein kinase incubation 1303
1304
Activation of Protein Kinase by Histamine
Vol. 17, No .
was the same as in our previous study (8) except that 100 pg of calf thymus histories (Type II, Sigma Co .) were used as substrate. Ten pl of extr~çt containing about 30 ug protein was added to incubation mixture. The ZP incorporated into protein was determined using the method described previously (9) . In preliminary experiments it was determined that 5 x 10 -~M cyclic AMP stimulated maximally protein kinase in the above described guinea pig gastric mucosa extracts . The average basal protein kinase activity was 31 .2 + 2 .6 pmol Pi incorporated into protein per mg of extract protein per 5 min(+ SEM) . Addition of 5 x 10 -6M cyclic AMP increased the activity an average~ + 35 .2 _+ 6.17. (+ SEM) ; P < 0.005, t-test . The extent of protein kinase activation was assessed by measurement of the ratio of the activity of enzyme assayed in the absence of added cyclic AMP to the activity after addition of 5 x 10 -6M cyclic AMP,(i .e .,-cyclic AMP/+cyclic AMP) the protein kinase activity ratio (10) . The control (-cyclic AMP/+cyclic AMP) protein kinase activity ratio was rather high and stimulation of protein kinase with added cyclic AMP was relatively small . This may be due to the fact, that under present experimental conditions, protein kinase might have been in part activated,that substances interfering with protein kinase activation by cyclic AMP were present in high quantity or that cyclic AMP-dependent protein kinase represents only small portion of protein kinases contained in assayed extracts . TCA-precipitated protein was removed from samples for cyclic AMP determination by centrifugation, TCA was removed by the extraction with water-saturated ether and cyclic AMP was determined by the radioimmunoassay method of Steiner (11) . Proteins were determined by the method of Lowry (12) . Results and Discussion The results outlined in Table I show that incubation with histamine produced a marked increase in tissue levels of cyclic AMP and a significant increase in the (-cyclic AMP/+cyclic AMP) protein kinase activity ratio . Protein kinase activity assayed without addition of exogenous cyclic AMP was higher in extracts from tissues exposed to histamine (39 .8 + 2 .3) than controls (31 .2 _+ 2.6) ; on the other hand, activity assayed in presence of 5 x 10 -6M cyclic AMP was slightly lower in extracts from histamine-exposed slices than in controls . On the other hand, 1,4-methylhistamine, an analog of histamine which is unable to elicit HC1 secretion (13) produced no significant change in the tissue level of cyclic AMP or in the (-cyclic AMP/+cyclic AMP) protein kinase activity ratio . In our previous studies we have shown that histamine produces a dose-dependent stimulation of guinea pig fundic gastric mucosa adenylate cyclase activity (2,3) while 1,4-methylhistamine had no effect on this enzyme (2,3) . Neither histamine nor 1,4-methylhistamine influence the activity of cyclic AMP phosphodiesterase (2,3) . Present results indicate that the differential effect of these two compounds on adenylate cyclase is reflected in their effect on the tissue level of cyclic AMP . Ethanol in lower concentration may evoke by direct action on stomach The present HC1 secretion but the mechanism of its action is unclear (14) . results, which show that 1~ ethanol neither elevated cyclic AMP levels, an observation analogous to findings in rat (15), nor activated protein kinase, suggest that this compound promotes HC1 secretion either indirectly, such as by releasing natural secretagogues in vivo, or by acting directly on HC1-secreting cells in biochemical steps subsequent to cyclic AMP formation and protein kinase activation . Although the presence of protein kinase in cell-free extracts of the fundic gastric mucosa of guinea pig (2) and rabbit (7) was reported, it has not been yet demonstrated that this enzyme can be activated in whole un-
8
Vol . 17, No . 8
Activation of Protein Kinase by Histamine
1305
TABLE 1 Effect of histamine and other agents on cyclic MIP level end (-cyclic AMP/+cyclic AMP) protein kimse activity ratio in strips of fundic gastric mucosa Significantly dLfferent from control alt +P~.005, ++p~0 .05~ (paired t-test) . N=number of observations .
Nt Cyclic AMP (pmol/mg protein) = SFH (_cyclic AMP/+cyclic AMp) protein kinase activity ratio ! SEM
(8)
Control (no edditione)
14 .71 11 .70
(10) 0.739 39 .036
N;
(9)
(10)
Histamine (10 -4M)
64 .64+ =11 .46
0 .897 ~ .OiO
N:
1,4-methylhistemlne (10-41I)
Nt
Ethanol (Lx v/v)
(7)
16 .57 =2 .60
(4)
16 .52 11 .34
(9)
0 .703 *-0 .025
(6)
0.736 39 .024
broken cells by cyclic AMP formed endogenously under the influence of histamine. Soderling, Corbin and Park (10) demonstrated that the degree of protein kinase activation in intact tissues exposed to hormones or other agents can be at least semiquantitatively assessed by measuring the (-cyclic AMP/+cyclic AMP) protein kinase activity ratio (10) . Using this principle, the present results demonstrate that histamine-induced elevation of tissue cyclic AMP levels is accompanied by significant _in _situ activation of the protein kinase in fundic gastric mucosa . The specificity of this response is stressed by the fact that an inactive histamine analog under the same conditions failed to elicit such an effect . Although the role of protein kinase in the cellular action of histamine on the fundic gastric mucosa is not known, the present results show that histamine-induced cyclic AMP synthesis leads to activation of protein kinase and suggests that the increase in protein kinase activity and cyclic AMP-dependent protein phosphorylations may represent an important step in the cellular mechanism of action of histamine on fundic gastric mucosa, subsequent to the histamine-elicited cyclic AMP generation . Acknowledgements These investigations were supported by United States Public Health Service Research Grant AM-16105 and Grant AM-17328 to CURE from the National Institutes of Health, by a Research Grant-in-Aid from the American Heart Association, and by the Mayo Foundation . Dr . Armin Wollin is a fellow from the Canadian Medical Research Council, Dr . Larry Barnes is a Senior Mayo Research Fellow and Dr . Thomas Dousa is an Established Investigator of the American Heart Association. Expert technical assistance of D, Heublein and C . Wilson as well as the secretarial assistance of Mrs . Joyce Wellik is greatly acknowledged . Reprint requests should be addressed to Dr . T. P . Dousa . References 1. 2. 3. 4. 5.
C. P . Sung, B, C . Jenkins, L . Racey Burnes, V. Hackney, J . G . Spenney, G. Sachs and V . D . Wiebelhaus, Am . J. Physiol . 225, 1359-1363 (1973) . T. P . Dousa and C . F. Code, International Sympo~lan on Histamine HaReceptor Antagonist, p 319-328, Smith, Kline S French Laboratories, Ltd . Welwyn Garden City, Deltakos (UK) Ltd . London W1 (1973) . T. P . Dousa and C . F . Code, J. Clin . Invest . 53, 334-337 (1974) . H. 0 . Karppanen, P. J . Neuvonen, P. R. Bieck ând E . Westermann, NaunynSchmiedeberR's Arch . Pharmacol. 284, 15-23 (1974) . T . H . McNeill and S . C . Verma, Br :J . Pharmacol . 52, 104-106 (1974) . s
1306 6. 7. 8. 9. 10 .
11 . 12 . 13 . 14 . 15 .
Activation of Protein Kinase by Histamine
Vol . 17, No . 8
P . R. Bieck, J. A. Ostes, G . A . Robison and R. B, Adkins, Aur. J. Physiol. 224, 158-164 (1973) . Wit . Reimami and N . G. Rapino, Biochim, Biophys . Acts 350, 201-214 (1974) . T . P . Douse, H . Sands, 0. Hechter, Endocrinolo 91, 75%?63 (1972) . m T . P . Douse, Endocrinology 96, 1359-1366 1974 . T. R . Soderling, J. D . Corby and C . R, Park, Techniques for Study of Protein Kinase Activation in Intact Cells in Methods in Enzymolopy Vol . XXXVIII Part C, eds S, P . Colowick and N . 0. Kaplan, pA 358-367 Âca am c ess, ew York (1974) . A. L . Steiner, Assay of Cyclic Nucleotides by Radioimmunoassay Methods in Methods in Enzymology , Vol . XXRVIII Part C, eds S . P. Colowick and N. 0 . Kaplan, pp . 96-105, ca em c ress, ew York (1974) . 0 . H . Lowry, N . R, Rosebrough, A, L, Farr and R . L . Randall, J. Biol . Chem . 193, 265-275 (1951) . M. I . Grossuran, C . Robertson and C . E, Rosiere, J . Pharmacol . Exp . Therap . 104, 275-277 (1952) . T . M. Ritchie, The Aliphatic Alcohols in The PharmacoloSical Basis of Therapeutics , IV Edition , eds L. S . Goodman and A. Gilman, pp . 138, The Macmillan Co ., New York (1970) . L . L. Taque and L . L . Shanbour, Life Sci . 14, 1065-1073 (1974) .
Pergamon Press
1303-1306
ACTIVATION OF PROTEIN KINASE IN THE GUINFA PIG FUNDIC GASTRIC MUCOSA BY HISTAMINE Armin Wollin, Larry D . Barnes, Yvonne S . Hui and Thomas P . Dousa Mayo Clinic and Foundation, Rochester, Minnesota (Received in final form September 19, 1975) The effect of histamine, 1,4-methylhistamine and ethanol on cyclic AMP levels and protein kinase activation was measured in tissue strips from the fundic region of guinea pig gastric mucosa . Histamine induced a significant elevation of tissue cyclic AMP levels and also in situ activation of the protein kinase . 1,4-methylhistamine, an inactive analog of histamine, and ethanol had no effect on these twn parameters . Results suggest that protein kinase activation is involved in the cyclic AMP-mediated action of histamine on the gastric fundic mucosa . Considerable experimental evidence iadicatea that histamine action on gastric mucosa HCL secretion is mediated by cyclic AMP, at least in some mammalian species . It has been shown that histamine can specifically stim ulate fundic gastric mucosa adenylate cyclase (1-3) and can elevate the tissue (4,5) or gastric juice levels (6) of the cyclic AMP . However, the mechanism by which cyclic AMP generated under the influence of histamine influences the gastric secretioy function is not known . An active cyclic AMPdependent protein kinase has been described in cell-free preparations from fundic gastric mucosa of guinea pig (2) and rabbit (7) . In the present preliminary experiments, we explored whether histamine, through mediation by cyclic AMP, can activate protein kinase in the intact, unbroken cells of the fundic region of guinea pig gastric mucosa . Methods Guinea pigs maintained on ad libitum diet were killed and strips of mucosa were removed from the fundic region of the stomach . Mucosa tissue was kept in ice-cold Kreba-Ringer solution saturated with a mixture of 957 oxygen aad 57. Cda and then preincubated in this solution for 10 min at 37oC . The tissue was then distributed in flasks using a ratio of 0 .3 $m of tissue per 3 ml Krebs-Ringer solution containing also 0 .2 mM 1-methyl-3-isobutylxanthine . Tissue was then incubated with or without addition of histamine or other tested agents for 10 min at 37oC . In preliminary experiment it was determined that histamine increased cyclic AMP level already at 5 min and increased level did not change substantially for at least 20 min . At the end of the incubation period the tissue was removed and homogenized in a medium containing 5 mM potassium phosphate, 2 mM EI7fA, 0 .25 M sucrose and 0 .5 mM 1-methyl-3-isobutylxanthine, pH 7 .0 . An aliquot (0 .5 ml) of homogenate was quickly mixed with an equal volume of ice-cold 10~ trichloroacetic acid (TCA) and stored frozen for cyclic AMP determination . The homogenate was then iumediately centrifuged at OoC (10,000 x g for 10 min) and the protein kinase activity in the fresh supernate was assayed without or with addition of 5 x 10' 6M of cyclic AMP . The method for protein kinase incubation 1303
1304
Activation of Protein Kinase by Histamine
Vol. 17, No .
was the same as in our previous study (8) except that 100 pg of calf thymus histories (Type II, Sigma Co .) were used as substrate. Ten pl of extr~çt containing about 30 ug protein was added to incubation mixture. The ZP incorporated into protein was determined using the method described previously (9) . In preliminary experiments it was determined that 5 x 10 -~M cyclic AMP stimulated maximally protein kinase in the above described guinea pig gastric mucosa extracts . The average basal protein kinase activity was 31 .2 + 2 .6 pmol Pi incorporated into protein per mg of extract protein per 5 min(+ SEM) . Addition of 5 x 10 -6M cyclic AMP increased the activity an average~ + 35 .2 _+ 6.17. (+ SEM) ; P < 0.005, t-test . The extent of protein kinase activation was assessed by measurement of the ratio of the activity of enzyme assayed in the absence of added cyclic AMP to the activity after addition of 5 x 10 -6M cyclic AMP,(i .e .,-cyclic AMP/+cyclic AMP) the protein kinase activity ratio (10) . The control (-cyclic AMP/+cyclic AMP) protein kinase activity ratio was rather high and stimulation of protein kinase with added cyclic AMP was relatively small . This may be due to the fact, that under present experimental conditions, protein kinase might have been in part activated,that substances interfering with protein kinase activation by cyclic AMP were present in high quantity or that cyclic AMP-dependent protein kinase represents only small portion of protein kinases contained in assayed extracts . TCA-precipitated protein was removed from samples for cyclic AMP determination by centrifugation, TCA was removed by the extraction with water-saturated ether and cyclic AMP was determined by the radioimmunoassay method of Steiner (11) . Proteins were determined by the method of Lowry (12) . Results and Discussion The results outlined in Table I show that incubation with histamine produced a marked increase in tissue levels of cyclic AMP and a significant increase in the (-cyclic AMP/+cyclic AMP) protein kinase activity ratio . Protein kinase activity assayed without addition of exogenous cyclic AMP was higher in extracts from tissues exposed to histamine (39 .8 + 2 .3) than controls (31 .2 _+ 2.6) ; on the other hand, activity assayed in presence of 5 x 10 -6M cyclic AMP was slightly lower in extracts from histamine-exposed slices than in controls . On the other hand, 1,4-methylhistamine, an analog of histamine which is unable to elicit HC1 secretion (13) produced no significant change in the tissue level of cyclic AMP or in the (-cyclic AMP/+cyclic AMP) protein kinase activity ratio . In our previous studies we have shown that histamine produces a dose-dependent stimulation of guinea pig fundic gastric mucosa adenylate cyclase activity (2,3) while 1,4-methylhistamine had no effect on this enzyme (2,3) . Neither histamine nor 1,4-methylhistamine influence the activity of cyclic AMP phosphodiesterase (2,3) . Present results indicate that the differential effect of these two compounds on adenylate cyclase is reflected in their effect on the tissue level of cyclic AMP . Ethanol in lower concentration may evoke by direct action on stomach The present HC1 secretion but the mechanism of its action is unclear (14) . results, which show that 1~ ethanol neither elevated cyclic AMP levels, an observation analogous to findings in rat (15), nor activated protein kinase, suggest that this compound promotes HC1 secretion either indirectly, such as by releasing natural secretagogues in vivo, or by acting directly on HC1-secreting cells in biochemical steps subsequent to cyclic AMP formation and protein kinase activation . Although the presence of protein kinase in cell-free extracts of the fundic gastric mucosa of guinea pig (2) and rabbit (7) was reported, it has not been yet demonstrated that this enzyme can be activated in whole un-
8
Vol . 17, No . 8
Activation of Protein Kinase by Histamine
1305
TABLE 1 Effect of histamine and other agents on cyclic MIP level end (-cyclic AMP/+cyclic AMP) protein kimse activity ratio in strips of fundic gastric mucosa Significantly dLfferent from control alt +P~.005, ++p~0 .05~ (paired t-test) . N=number of observations .
Nt Cyclic AMP (pmol/mg protein) = SFH (_cyclic AMP/+cyclic AMp) protein kinase activity ratio ! SEM
(8)
Control (no edditione)
14 .71 11 .70
(10) 0.739 39 .036
N;
(9)
(10)
Histamine (10 -4M)
64 .64+ =11 .46
0 .897 ~ .OiO
N:
1,4-methylhistemlne (10-41I)
Nt
Ethanol (Lx v/v)
(7)
16 .57 =2 .60
(4)
16 .52 11 .34
(9)
0 .703 *-0 .025
(6)
0.736 39 .024
broken cells by cyclic AMP formed endogenously under the influence of histamine. Soderling, Corbin and Park (10) demonstrated that the degree of protein kinase activation in intact tissues exposed to hormones or other agents can be at least semiquantitatively assessed by measuring the (-cyclic AMP/+cyclic AMP) protein kinase activity ratio (10) . Using this principle, the present results demonstrate that histamine-induced elevation of tissue cyclic AMP levels is accompanied by significant _in _situ activation of the protein kinase in fundic gastric mucosa . The specificity of this response is stressed by the fact that an inactive histamine analog under the same conditions failed to elicit such an effect . Although the role of protein kinase in the cellular action of histamine on the fundic gastric mucosa is not known, the present results show that histamine-induced cyclic AMP synthesis leads to activation of protein kinase and suggests that the increase in protein kinase activity and cyclic AMP-dependent protein phosphorylations may represent an important step in the cellular mechanism of action of histamine on fundic gastric mucosa, subsequent to the histamine-elicited cyclic AMP generation . Acknowledgements These investigations were supported by United States Public Health Service Research Grant AM-16105 and Grant AM-17328 to CURE from the National Institutes of Health, by a Research Grant-in-Aid from the American Heart Association, and by the Mayo Foundation . Dr . Armin Wollin is a fellow from the Canadian Medical Research Council, Dr . Larry Barnes is a Senior Mayo Research Fellow and Dr . Thomas Dousa is an Established Investigator of the American Heart Association. Expert technical assistance of D, Heublein and C . Wilson as well as the secretarial assistance of Mrs . Joyce Wellik is greatly acknowledged . Reprint requests should be addressed to Dr . T. P . Dousa . References 1. 2. 3. 4. 5.
C. P . Sung, B, C . Jenkins, L . Racey Burnes, V. Hackney, J . G . Spenney, G. Sachs and V . D . Wiebelhaus, Am . J. Physiol . 225, 1359-1363 (1973) . T. P . Dousa and C . F. Code, International Sympo~lan on Histamine HaReceptor Antagonist, p 319-328, Smith, Kline S French Laboratories, Ltd . Welwyn Garden City, Deltakos (UK) Ltd . London W1 (1973) . T. P . Dousa and C . F . Code, J. Clin . Invest . 53, 334-337 (1974) . H. 0 . Karppanen, P. J . Neuvonen, P. R. Bieck ând E . Westermann, NaunynSchmiedeberR's Arch . Pharmacol. 284, 15-23 (1974) . T . H . McNeill and S . C . Verma, Br :J . Pharmacol . 52, 104-106 (1974) . s
1306 6. 7. 8. 9. 10 .
11 . 12 . 13 . 14 . 15 .
Activation of Protein Kinase by Histamine
Vol . 17, No . 8
P . R. Bieck, J. A. Ostes, G . A . Robison and R. B, Adkins, Aur. J. Physiol. 224, 158-164 (1973) . Wit . Reimami and N . G. Rapino, Biochim, Biophys . Acts 350, 201-214 (1974) . T . P . Douse, H . Sands, 0. Hechter, Endocrinolo 91, 75%?63 (1972) . m T . P . Douse, Endocrinology 96, 1359-1366 1974 . T. R . Soderling, J. D . Corby and C . R, Park, Techniques for Study of Protein Kinase Activation in Intact Cells in Methods in Enzymolopy Vol . XXXVIII Part C, eds S, P . Colowick and N . 0. Kaplan, pA 358-367 Âca am c ess, ew York (1974) . A. L . Steiner, Assay of Cyclic Nucleotides by Radioimmunoassay Methods in Methods in Enzymology , Vol . XXRVIII Part C, eds S . P. Colowick and N. 0 . Kaplan, pp . 96-105, ca em c ress, ew York (1974) . 0 . H . Lowry, N . R, Rosebrough, A, L, Farr and R . L . Randall, J. Biol . Chem . 193, 265-275 (1951) . M. I . Grossuran, C . Robertson and C . E, Rosiere, J . Pharmacol . Exp . Therap . 104, 275-277 (1952) . T . M. Ritchie, The Aliphatic Alcohols in The PharmacoloSical Basis of Therapeutics , IV Edition , eds L. S . Goodman and A. Gilman, pp . 138, The Macmillan Co ., New York (1970) . L . L. Taque and L . L . Shanbour, Life Sci . 14, 1065-1073 (1974) .