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Cyclic guanosine-5′-phosphate and protease secretion from rat submandibular gland in vitro
Archs oral Biol. Vol. 25 pp. 75 to 76 Pergamon Press
Ltd 1980.Prmted
in Great Britam
CYCLIC GUANOSINE-5’-PHOSPHATE AND PROTEASE SECRETION FROM RAT SUBMANDIBULAR GLAND IN VITRO T. N. SPEARMANand E. T. PRITCHARD Department of Oral Biology, University of Manitoba, Winnipeg. Manitoba, Canada R3E 0W3
Summary-Secretion of kallikrein and two other proteases from rat submandibular gland slices was induced by a-adrenergic stimulation in the presence of calcium. This process was not mediated by cyclic GMP. Carbamylcholine. which had no effect on protease release, markedly increased the gland content of cyclic GMP.
Most investigators now believe that proteases, such as the kallikreins, are contained within the convoluted granular tubule cells of rat (Matthews, 1974; Orstavik et al., 1975), mouse (Gresik et al., 1978; Simson et al., 1978), cat (Hojima et al., 1977) and guinea-pig (Schachter, Maranda and Moriwaki, 1978) submandibular glands, and their secretion is effected by a-adrenergic stimuli (Abe and Dawes, 1978; Orstavik and Gautvik, 1977; Hosoi, Aoyama and Ueha, 1978). However, Albano et a/. (1976) claimed that kallikrein secretion can be elicited by either a-adrenergic or muscarinic cholinergic stimulation in guinea-pig slice preparations. They also maintained that this effect was mediated by cyclic guanosine-S-phosphates (GMP). Because Spearman (1979) found that three proteases in rat submandibular gland were released only by a-adrenergic agonists, we re-examined this tissue for possible concomitant changes in the levels of cyclic GMP during protease secretion. The experimental protocol was similar to that used previously (Spearman and Pritchard, 1977) concerned Table 1. Influence
with K+-release. Slices (20_30mg/tube) were obtained from submandibular glands of male Lpng Evans rats, 34 months of age, using a McIlwain tissue chopper (The Mickle Co., Gomshall, U.K.). The slices were placed in plastic tubes containing 3 ml Krebs-Ringer bicarbonate buffer, pH 7.4, in a gyrorotary water bath at 37°C. Each tube was individually and continuously gassed with 95 per cent 02-C02. After a lo-15 min pre-incubation, the buffer was removed and replaced with 1 ml fresh, warm buffer containing the appropriate antagonists (EGTA (Ca”), phentolamine (cr), propranolol (/?) and atropine (muscarinic cholinergic). A phosphodiesterase inhibitor, 3-isobutyl-l-methyl xanthine, was present in samples designated for cyclic GMP analysis. After further 15 min incubation, agonists were added and the incubation continued for 3 min (cyclic GMP) or 30 min (proteases). Kallikrein and the pH 8.2 and 9.5 proteases were measured in both medium and tissue of each sample using a-Nbenzoyl+arginine ethyl ester (kallikrein) and a-Nbenzoyl-L-arginine-b-naphthylamide (proteases) as
of a-, fl-adrenergic and muscarinic cholinergic stimulation on protease GMP accumulation in rat submandibular gland slices
Additions None Phenylephrine + 4 mM EGTA Epinephrine + propranolol + phentolamine Carbamycholine + atropine Eledoisin-related peptide Physalaemin Substance P
Per cent of total activity secreted Kallikrein pH 9.5 protease pH 8.2 protease 19.9 *41.5 22.2 *62.1 *48.4 23.1 21.1
* k f + + k k
1.5 (15) 1.6 (15) 4.1 (3) 2.9 (6) 0.9 (3) 1.0 (3) 2.4 (6)
19.3 k 2.7 (6) *40.1 k 1.4 (6) *51.2 *42.7 21.8 21.3
t + k +
2.3 (6) 1.7 (3) 0.8 (3) 3.5 (3)
16.9 *32.2 16.3 *48.3 *39.2 19.9 16.9
& k k k f f f
1.3 (15) 1.8 (15) 2.1 (3) 2.6 (6) 0.8 (3) 0.9 (3) 2.1 (6)
16.3 k 2.7 (3) 19.5 * 2.0 (3) 19.4 & 3.1 (3)
22.0 k 2.7 (3) 24.5 & 3.2 (3) 25.2 k 3.0 (3)
release and cyclic
fmoles cyclic GMP/mg protein 223 k 45 (14) 245 + 35 (6) 131 f 12 (4) 251 _t 64 (6) 218 k 43 (17) 210 k 45 (18) *1168 * 149 (18) 285 + 98 (9) 208 k 26 (9) 209 * 50 (9) 247t_61 (9)
Incubations as described in text with inhibitors (phentolamine, propranolol, atropine at 200 PM and EGTA at 4 mM) being present 15 min before stimulants. After the addition of agonists (phenylephrine, epinephrine, carbamylcholine at 50 PM and peptides at 1 PM), the incubations were continued for 3 min for cyclic GMP assay, and 30 min for proteases. Results are mean + SE with n in parenthesis. * Indicates the result is significantly different from its respective control at p < 0.01. 75
T. N. Spearman and E. T. Pritchard
16
substrates (Orstavik et al., 1977; Reikkinen and Niemi, 1968). Original tissue enzyme activity was 55.4 + 5.5 (kallikrein), 10.0 k 1 @H 8.2) and 14.0 f 1.2 (pH 9.5) as nmoles substrate hydrolysed/min/mg protein. The medium was obtained by aspiration into graduated centrifuge tubes followed by centrifugation at 1OOOg for 1Omin to remove debris. Tissues were homogenized in 2ml of water. Cyclic GMP was estimated in the supernatant fractions prepared from 5 per cent trichloracetic acid homogenates that had been ether-extracted and purified by QAE-column chromatography after succinylation (Frandsen and Krishna, 1976) by the radioimmunoassay procedure of Steiner, Parker and Kipnis (1972). The results (Table 1) showed that only a-adrenergic stimulation effectively released protease from submandibular gland slices. Extracellular Ca* + was essential for this process as the omission of Ca* + (not shown) or the addition of the Ca*+-chelator, EGTA, to the medium abolished secretion of protease. However, r-adrenergic stimulation did not change cyclic GMP levels, in contrast to guinea-pig submandibular gland slices where, according to Albano et al. (1976), both u-adrenergic and choline& agonists cause an increase in cyclic GMP and promote kallikrein release. They also reported that tie acinar cells were depleted during kallikrein secretion. It seems unlikely, however, that the disappearing acinar granules contained kallikrein as Schachter, Maranda and Moriwaki (1978) have clearly demonstrated that all the kallikrein in guinea-pig submandibular glands is within the convoluted granular tubule cells. The tachykinin peptides, physalaemin, eledoisin, substance P (all obtained from Sigma Chemical Co.. St. Louis, MO, U.S.A.), which induce K+ release in rat parotid (Rudich and Butcher, 1976; Putney, 1977) and submandibular (Spearman and Pritchard, 1977) glands, did not cause either protease secretion or cyclic GMP accumulation (Table 1). Our study shows that only muscarinic cholinergic stimulation increases the level of cyclic GMP in rat submandibular slices. It appears that a-adrenergicevoked processes, such as protease release and K+ efflux, do not involve cyclic GMP as a mediator, nor, would it appear, does tachykinin peptide-induced K’ release. Acknowledgements-Miss Joanne E. Cushnie gave technical assistance. This work was supported by a grant (MT-3565) from the Medical Research Council of Canada. REFERENCES
Abe K. and Dawes C. 1978. The effects of electrical and pharmacological stimulation on the types of proteins secreted by rat parotid and submandibular glands. Archs oral Biol. 23, 367-312.
Albano J., Bhoola K. D.. Heap P. F. and Lemon M. J. C. 1976. Stimulus-secretion coupling; role of cyclic AMP, cyclic GMP and calcium in mediating enzyme (kallikrein) secretion in the submandibular gland. J. Physiol., Lond. 258, 631-658. Frandsen E. K. and Krishna G. 1976. A simple ultrasensitive method for the assay of cyclic AMP and cyclic GMP in tissues. L.$e Sci. 18, 529-542. Gresik E., Michelakis A.. Barka T. and Ross T. 1978. Immunochemical localization of renin in the submandibular gland of the mouse. J. Histochem. Cytochem. 26, 855-861. Hojima Y., Maranda B., Moriwaki C. and Schachter M. 1977. Direct evidence for the localization of kallikrein in the striated ducts of the cat’s submandibular gland by use of specific antibody. J. Physiol., Land. 268, 793-801. Hosoi K., Aoyama K. and Ueha T. 1978. Regulation of the secretory process of granular components from the convoluted tubular cells of the mouse submandibular gland. J. dent. Rex 57, 87-90. Matthews R. V. 1974. The effects of autonomic stimulation upon the rat submandibular gland. Archs oral Biol. 19. 989-994. Orstavik T. B., Brandtzaeg P., Nustad K. and Halvorsen K. M. 1975. Cellular localization of kallikrein in rat submandibular and sublingual salivary glands. Acta histochem. (Jena) 54, 183-192. Orstavik T. B. and Gautvik K. M. 1977. Regulation of salivary kallikrein secretion in the rat submandibular gland. Acta physiol. stand. 100, 33-44. Orstavik T. B., Nustad K. and Brandtzaeg P. 1977. A biochemical and immunohistochemical study of kallikrein in normal and isoproterenol-stimulated rat salivary glands during postnatal development. Archs oral Biol. 22, 495-502. Putney J. W. 1977. Muscarinic, alpha-adrenergic and peptide receptors regulate the same calcium influx sites in the parotid gland. J. Physiol., Lond. 268, 139-149. Reikkinen P. J. and Niemi M. 1968. Androgen-dependent salivary gland protease in the rat. EndocrinoL 83, 12241231. Rudich L. and Butcher F. R. 1976. Effect of substance P and eledoisin on K+ efflux, amylase release and cyclic nucleotide levels in slices of rat parotid gland. Biochim. hiophys. Acta 444, 704-711. Schachter M., Maranda B. and Moriwaki C. 1978. Localization of kallikrein in the coagulating and submandibular glands of the guinea-pig. J. histochem. cytochem. 26. 318-321. Simson J. A. V., Hazen D., Spicer S. S., Murphy R. A. and Young M. 1978. Secretagogue-mediated discharge of nerve growth factor from granular tubules of male mouse submandibular glands: an immunocytochemical study. Anat. Rec. 192, 375-388. Spearman T. N. 1979. Secretion and the submandibular gland of the rat. Ph.D. Thesis, University of Manitoba. Spearman T. N. and Pritchard E. T. 1977. Potassium release from submandibular salivary gland in vitro. Biochim. biophys. Acta 466, 19X-207. Steiner A. L., Parker C. W. and Kipnis D. M. 1972. Radioimmunoassay for cyclic nucleotides. J. biol. Chem. 247, 1106-1113.
Ltd 1980.Prmted
in Great Britam
CYCLIC GUANOSINE-5’-PHOSPHATE AND PROTEASE SECRETION FROM RAT SUBMANDIBULAR GLAND IN VITRO T. N. SPEARMANand E. T. PRITCHARD Department of Oral Biology, University of Manitoba, Winnipeg. Manitoba, Canada R3E 0W3
Summary-Secretion of kallikrein and two other proteases from rat submandibular gland slices was induced by a-adrenergic stimulation in the presence of calcium. This process was not mediated by cyclic GMP. Carbamylcholine. which had no effect on protease release, markedly increased the gland content of cyclic GMP.
Most investigators now believe that proteases, such as the kallikreins, are contained within the convoluted granular tubule cells of rat (Matthews, 1974; Orstavik et al., 1975), mouse (Gresik et al., 1978; Simson et al., 1978), cat (Hojima et al., 1977) and guinea-pig (Schachter, Maranda and Moriwaki, 1978) submandibular glands, and their secretion is effected by a-adrenergic stimuli (Abe and Dawes, 1978; Orstavik and Gautvik, 1977; Hosoi, Aoyama and Ueha, 1978). However, Albano et a/. (1976) claimed that kallikrein secretion can be elicited by either a-adrenergic or muscarinic cholinergic stimulation in guinea-pig slice preparations. They also maintained that this effect was mediated by cyclic guanosine-S-phosphates (GMP). Because Spearman (1979) found that three proteases in rat submandibular gland were released only by a-adrenergic agonists, we re-examined this tissue for possible concomitant changes in the levels of cyclic GMP during protease secretion. The experimental protocol was similar to that used previously (Spearman and Pritchard, 1977) concerned Table 1. Influence
with K+-release. Slices (20_30mg/tube) were obtained from submandibular glands of male Lpng Evans rats, 34 months of age, using a McIlwain tissue chopper (The Mickle Co., Gomshall, U.K.). The slices were placed in plastic tubes containing 3 ml Krebs-Ringer bicarbonate buffer, pH 7.4, in a gyrorotary water bath at 37°C. Each tube was individually and continuously gassed with 95 per cent 02-C02. After a lo-15 min pre-incubation, the buffer was removed and replaced with 1 ml fresh, warm buffer containing the appropriate antagonists (EGTA (Ca”), phentolamine (cr), propranolol (/?) and atropine (muscarinic cholinergic). A phosphodiesterase inhibitor, 3-isobutyl-l-methyl xanthine, was present in samples designated for cyclic GMP analysis. After further 15 min incubation, agonists were added and the incubation continued for 3 min (cyclic GMP) or 30 min (proteases). Kallikrein and the pH 8.2 and 9.5 proteases were measured in both medium and tissue of each sample using a-Nbenzoyl+arginine ethyl ester (kallikrein) and a-Nbenzoyl-L-arginine-b-naphthylamide (proteases) as
of a-, fl-adrenergic and muscarinic cholinergic stimulation on protease GMP accumulation in rat submandibular gland slices
Additions None Phenylephrine + 4 mM EGTA Epinephrine + propranolol + phentolamine Carbamycholine + atropine Eledoisin-related peptide Physalaemin Substance P
Per cent of total activity secreted Kallikrein pH 9.5 protease pH 8.2 protease 19.9 *41.5 22.2 *62.1 *48.4 23.1 21.1
* k f + + k k
1.5 (15) 1.6 (15) 4.1 (3) 2.9 (6) 0.9 (3) 1.0 (3) 2.4 (6)
19.3 k 2.7 (6) *40.1 k 1.4 (6) *51.2 *42.7 21.8 21.3
t + k +
2.3 (6) 1.7 (3) 0.8 (3) 3.5 (3)
16.9 *32.2 16.3 *48.3 *39.2 19.9 16.9
& k k k f f f
1.3 (15) 1.8 (15) 2.1 (3) 2.6 (6) 0.8 (3) 0.9 (3) 2.1 (6)
16.3 k 2.7 (3) 19.5 * 2.0 (3) 19.4 & 3.1 (3)
22.0 k 2.7 (3) 24.5 & 3.2 (3) 25.2 k 3.0 (3)
release and cyclic
fmoles cyclic GMP/mg protein 223 k 45 (14) 245 + 35 (6) 131 f 12 (4) 251 _t 64 (6) 218 k 43 (17) 210 k 45 (18) *1168 * 149 (18) 285 + 98 (9) 208 k 26 (9) 209 * 50 (9) 247t_61 (9)
Incubations as described in text with inhibitors (phentolamine, propranolol, atropine at 200 PM and EGTA at 4 mM) being present 15 min before stimulants. After the addition of agonists (phenylephrine, epinephrine, carbamylcholine at 50 PM and peptides at 1 PM), the incubations were continued for 3 min for cyclic GMP assay, and 30 min for proteases. Results are mean + SE with n in parenthesis. * Indicates the result is significantly different from its respective control at p < 0.01. 75
T. N. Spearman and E. T. Pritchard
16
substrates (Orstavik et al., 1977; Reikkinen and Niemi, 1968). Original tissue enzyme activity was 55.4 + 5.5 (kallikrein), 10.0 k 1 @H 8.2) and 14.0 f 1.2 (pH 9.5) as nmoles substrate hydrolysed/min/mg protein. The medium was obtained by aspiration into graduated centrifuge tubes followed by centrifugation at 1OOOg for 1Omin to remove debris. Tissues were homogenized in 2ml of water. Cyclic GMP was estimated in the supernatant fractions prepared from 5 per cent trichloracetic acid homogenates that had been ether-extracted and purified by QAE-column chromatography after succinylation (Frandsen and Krishna, 1976) by the radioimmunoassay procedure of Steiner, Parker and Kipnis (1972). The results (Table 1) showed that only a-adrenergic stimulation effectively released protease from submandibular gland slices. Extracellular Ca* + was essential for this process as the omission of Ca* + (not shown) or the addition of the Ca*+-chelator, EGTA, to the medium abolished secretion of protease. However, r-adrenergic stimulation did not change cyclic GMP levels, in contrast to guinea-pig submandibular gland slices where, according to Albano et al. (1976), both u-adrenergic and choline& agonists cause an increase in cyclic GMP and promote kallikrein release. They also reported that tie acinar cells were depleted during kallikrein secretion. It seems unlikely, however, that the disappearing acinar granules contained kallikrein as Schachter, Maranda and Moriwaki (1978) have clearly demonstrated that all the kallikrein in guinea-pig submandibular glands is within the convoluted granular tubule cells. The tachykinin peptides, physalaemin, eledoisin, substance P (all obtained from Sigma Chemical Co.. St. Louis, MO, U.S.A.), which induce K+ release in rat parotid (Rudich and Butcher, 1976; Putney, 1977) and submandibular (Spearman and Pritchard, 1977) glands, did not cause either protease secretion or cyclic GMP accumulation (Table 1). Our study shows that only muscarinic cholinergic stimulation increases the level of cyclic GMP in rat submandibular slices. It appears that a-adrenergicevoked processes, such as protease release and K+ efflux, do not involve cyclic GMP as a mediator, nor, would it appear, does tachykinin peptide-induced K’ release. Acknowledgements-Miss Joanne E. Cushnie gave technical assistance. This work was supported by a grant (MT-3565) from the Medical Research Council of Canada. REFERENCES
Abe K. and Dawes C. 1978. The effects of electrical and pharmacological stimulation on the types of proteins secreted by rat parotid and submandibular glands. Archs oral Biol. 23, 367-312.
Albano J., Bhoola K. D.. Heap P. F. and Lemon M. J. C. 1976. Stimulus-secretion coupling; role of cyclic AMP, cyclic GMP and calcium in mediating enzyme (kallikrein) secretion in the submandibular gland. J. Physiol., Lond. 258, 631-658. Frandsen E. K. and Krishna G. 1976. A simple ultrasensitive method for the assay of cyclic AMP and cyclic GMP in tissues. L.$e Sci. 18, 529-542. Gresik E., Michelakis A.. Barka T. and Ross T. 1978. Immunochemical localization of renin in the submandibular gland of the mouse. J. Histochem. Cytochem. 26, 855-861. Hojima Y., Maranda B., Moriwaki C. and Schachter M. 1977. Direct evidence for the localization of kallikrein in the striated ducts of the cat’s submandibular gland by use of specific antibody. J. Physiol., Land. 268, 793-801. Hosoi K., Aoyama K. and Ueha T. 1978. Regulation of the secretory process of granular components from the convoluted tubular cells of the mouse submandibular gland. J. dent. Rex 57, 87-90. Matthews R. V. 1974. The effects of autonomic stimulation upon the rat submandibular gland. Archs oral Biol. 19. 989-994. Orstavik T. B., Brandtzaeg P., Nustad K. and Halvorsen K. M. 1975. Cellular localization of kallikrein in rat submandibular and sublingual salivary glands. Acta histochem. (Jena) 54, 183-192. Orstavik T. B. and Gautvik K. M. 1977. Regulation of salivary kallikrein secretion in the rat submandibular gland. Acta physiol. stand. 100, 33-44. Orstavik T. B., Nustad K. and Brandtzaeg P. 1977. A biochemical and immunohistochemical study of kallikrein in normal and isoproterenol-stimulated rat salivary glands during postnatal development. Archs oral Biol. 22, 495-502. Putney J. W. 1977. Muscarinic, alpha-adrenergic and peptide receptors regulate the same calcium influx sites in the parotid gland. J. Physiol., Lond. 268, 139-149. Reikkinen P. J. and Niemi M. 1968. Androgen-dependent salivary gland protease in the rat. EndocrinoL 83, 12241231. Rudich L. and Butcher F. R. 1976. Effect of substance P and eledoisin on K+ efflux, amylase release and cyclic nucleotide levels in slices of rat parotid gland. Biochim. hiophys. Acta 444, 704-711. Schachter M., Maranda B. and Moriwaki C. 1978. Localization of kallikrein in the coagulating and submandibular glands of the guinea-pig. J. histochem. cytochem. 26. 318-321. Simson J. A. V., Hazen D., Spicer S. S., Murphy R. A. and Young M. 1978. Secretagogue-mediated discharge of nerve growth factor from granular tubules of male mouse submandibular glands: an immunocytochemical study. Anat. Rec. 192, 375-388. Spearman T. N. 1979. Secretion and the submandibular gland of the rat. Ph.D. Thesis, University of Manitoba. Spearman T. N. and Pritchard E. T. 1977. Potassium release from submandibular salivary gland in vitro. Biochim. biophys. Acta 466, 19X-207. Steiner A. L., Parker C. W. and Kipnis D. M. 1972. Radioimmunoassay for cyclic nucleotides. J. biol. Chem. 247, 1106-1113.