Biochemical SocietyTransactions ( 1 992) 20
Activation of intracellular signalling pathways induces secretion of mucin by isolated rat gastric mucosal cells. ANDREW C. KEATES, JULIE M. WILLIAMS and PETER J. HANSON Pharmaceutical Sciences Institute, Aston University, Aston Triangle, Birmingham B4 7ET, U.K. Gastric mucous cells can release mucus by a slow and continuous exocytosis o r by apical expulsion which involves a massive and rapid degranulation [l]. The aims of this work were to determine whether activation of intracellular signalling pathways could induce the release of mucin from a suspension of cells isolated from the rat fundic mucosa, and to examine whether release was transient or sustained. Cells were prepared from everted sacs of fundus by pronase-digestion and intermittent calcium chelation [2]. The cells were incubated in Eagle’s minimum essential medium containing 20 mM-N-2-hydroxyethylpiperazineN’-2-ethanesulphonic acid, 1 mg bovine serum albumidml and 50 pg gentamicidml as described previously [3]. Mucin was assayed by an indirect competitive binding enzyme-linked immunoassay (e.1.i.s.a.). The preparation of the antibody and the execution of the e.1.i.s.a. have been described previously 141. Use of indirect immunoperoxidase techniques showed that the antibody recognized cells only on the surface and foveolar regions of sections of the stomach. Protein blotting demonstrated t h a t the antibody recognised only material that was retained within the 4% stacking gel. Results obtained with serially-diluted samples of incubation medium in the e.1.i.s.a. could be superimposed on the standard CUNC.
Under both control conditions and in the presence of 50 pM-forskolin release of mucin into the medium was essentially linearly related to time over the period 15-60 min (Fig. 1). At all of the times investigated forskolin significantly stimulated the release of mucin. The cellular content of immunologically detectable mucin was 124 f 20 l g / 107 cells (Mean f S. E. M. from five cellbatches). Incubation for l h in the absence or presence of forskolin released 2.6 f 0.5% and 4.7 f 1.2% of the cellular material. The percentage of cells excluding trypan blue was 90% a t the start and at the end of the incubation period. The half-maximally effective concentration of forskolin for the stimulation of mucin release was 673 nM, with 50 pM-forskolin producing a 62 f 9% (n=3) stimulation of release. The phorbol ester 12-0-tetradecanoylphorbl 13acetate (TPA) also stimulated mucin release. The halfmaximally effective concentration was 25 nM and a lpM concentration produced a stimulation of 203 f 23% (n=3). A synergistic stimulation (effect of two agents together greater that the sum ofthe effects of the agents alone ) of the release of mucin into the medium was found with 10 nM-TPA in combination with either 5 pM-forskolin or 1 pM-A23187. The 4 a-phorbol derivative of TPA had no effect on mucin release. Forskolin activates adenylate cyclase and increases the intracellular content of cyclic AMP. Raising the cyclic AMP content of mucous cells did not produce a rapid burst of secretory activity, but rather modulated the process of slow continuous release. The secretory machinery inside mucous cells was also activated by A23187, one action of which is to raise intracellular Ca2+, and by TPA which activates protein kinase C. Evidence for a positive interaction between the pathway involving protein kinase C and those involving Ca2+ and cyclic AMP was obtained. The increases in m u c h release that occurred with activation of secretory pathways were quite small. This was probably not a consequence of damage to the cells during isolation because similar results have been obtained with a more intact rabbit explant system [5]. It is possible that some of the mucin that was released remained attached to the cells. However, previous experiments [3] which examined the effect of dithiothreitol on the release of radiolabelled mucin did not suggest that retention of secreted mucin was a major problem. In conclusion, activation of secretory pathways in rat gastric mucous cells does not produce a massive and rapid degranulation but rather seems to modulate a process of slow continuous release. 1.
Fig. 1. W e c t of time on the release of muci n into the n medium, Results are means f S.E.M. from six batches of cells. ; 50 pM-forskolin . The effect of forskolin control at each time was assessed by a paired t-test. * P
Activation of intracellular signalling pathways induces secretion of mucin by isolated rat gastric mucosal cells. ANDREW C. KEATES, JULIE M. WILLIAMS and PETER J. HANSON Pharmaceutical Sciences Institute, Aston University, Aston Triangle, Birmingham B4 7ET, U.K. Gastric mucous cells can release mucus by a slow and continuous exocytosis o r by apical expulsion which involves a massive and rapid degranulation [l]. The aims of this work were to determine whether activation of intracellular signalling pathways could induce the release of mucin from a suspension of cells isolated from the rat fundic mucosa, and to examine whether release was transient or sustained. Cells were prepared from everted sacs of fundus by pronase-digestion and intermittent calcium chelation [2]. The cells were incubated in Eagle’s minimum essential medium containing 20 mM-N-2-hydroxyethylpiperazineN’-2-ethanesulphonic acid, 1 mg bovine serum albumidml and 50 pg gentamicidml as described previously [3]. Mucin was assayed by an indirect competitive binding enzyme-linked immunoassay (e.1.i.s.a.). The preparation of the antibody and the execution of the e.1.i.s.a. have been described previously 141. Use of indirect immunoperoxidase techniques showed that the antibody recognized cells only on the surface and foveolar regions of sections of the stomach. Protein blotting demonstrated t h a t the antibody recognised only material that was retained within the 4% stacking gel. Results obtained with serially-diluted samples of incubation medium in the e.1.i.s.a. could be superimposed on the standard CUNC.
Under both control conditions and in the presence of 50 pM-forskolin release of mucin into the medium was essentially linearly related to time over the period 15-60 min (Fig. 1). At all of the times investigated forskolin significantly stimulated the release of mucin. The cellular content of immunologically detectable mucin was 124 f 20 l g / 107 cells (Mean f S. E. M. from five cellbatches). Incubation for l h in the absence or presence of forskolin released 2.6 f 0.5% and 4.7 f 1.2% of the cellular material. The percentage of cells excluding trypan blue was 90% a t the start and at the end of the incubation period. The half-maximally effective concentration of forskolin for the stimulation of mucin release was 673 nM, with 50 pM-forskolin producing a 62 f 9% (n=3) stimulation of release. The phorbol ester 12-0-tetradecanoylphorbl 13acetate (TPA) also stimulated mucin release. The halfmaximally effective concentration was 25 nM and a lpM concentration produced a stimulation of 203 f 23% (n=3). A synergistic stimulation (effect of two agents together greater that the sum ofthe effects of the agents alone ) of the release of mucin into the medium was found with 10 nM-TPA in combination with either 5 pM-forskolin or 1 pM-A23187. The 4 a-phorbol derivative of TPA had no effect on mucin release. Forskolin activates adenylate cyclase and increases the intracellular content of cyclic AMP. Raising the cyclic AMP content of mucous cells did not produce a rapid burst of secretory activity, but rather modulated the process of slow continuous release. The secretory machinery inside mucous cells was also activated by A23187, one action of which is to raise intracellular Ca2+, and by TPA which activates protein kinase C. Evidence for a positive interaction between the pathway involving protein kinase C and those involving Ca2+ and cyclic AMP was obtained. The increases in m u c h release that occurred with activation of secretory pathways were quite small. This was probably not a consequence of damage to the cells during isolation because similar results have been obtained with a more intact rabbit explant system [5]. It is possible that some of the mucin that was released remained attached to the cells. However, previous experiments [3] which examined the effect of dithiothreitol on the release of radiolabelled mucin did not suggest that retention of secreted mucin was a major problem. In conclusion, activation of secretory pathways in rat gastric mucous cells does not produce a massive and rapid degranulation but rather seems to modulate a process of slow continuous release. 1.
Fig. 1. W e c t of time on the release of muci n into the n medium, Results are means f S.E.M. from six batches of cells. ; 50 pM-forskolin . The effect of forskolin control at each time was assessed by a paired t-test. * P
