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Regulation of Anion Secretion by Prostaglandin E2: RESULTS(3)

RESULTS(3)Involvement of cAMP in the PGE2-lnduced lsc Responses

The involvement of cAMP in mediating the PGE2 responses was further tested using agents affecting intracellular с AMP level (Fig. 6). Additions of forskolin (10 |xM, n = 10), an adenylate cyclase activator, or IBMX (0.1 mM, n = 4), a cAMP-phosphodiesterase inhibitor, produced a sustained increase in Isc similar to that induced by PGE2. buy levaquin online

On the other hand, the PGE2-stimulated Isc, both apical and basolateral, could be completely abolished by the pretreatment with MDL (20 (xM, n = 3), an adenylate cyclase inhibitor (Fig. 7). The signaling pathway involved in mediating the PGE2 responses was also investigated by examining whether the effect of PGE2 was additive to the effect induced by agents known to activate specific intracellular signaling pathways. Pretreatment with a Ca2+-mo-bilizing agent, ATP (10 ц,М, apical, n = 4), did not affect the magnitude of subsequent PGE2-stimulated Isc responses (Fig. 8A), indicating that the two effects were independent of one another. However, over 80% of the PGE2-induced responses, both apical and basolateral, were abolished by pretreatment with forskolin (100 |xM, n = 4, Fig. 8B), suggesting large overlap of the two effects. Taken together, the results strongly indicated involvement of cAMP in mediating the PGE2-stimulated responses.
Fig6Regulation of anion secretion
FIG. 6. Forskolin and IBMX mimic the PGE2-stimulated lsc. Isc recordings of the forskolin-activated (A), IBMX-induced (B), and apical PGE2-stimulated lsc (C) with arrows marking the time at which the agonists were added. For tracing A: lb: 5.8 |j,Acrrr2; RT: 1800 flcm2. For tracing B: lb: 6.0 ixAcrrr2; RT: 900 flcm2. For tracing C: lb: 7.5 |j.Acm-2; RT: 450 flcm2.

Fig7Regulation of anion secretion
FIG. 7. Effect of adenylate cyclase inhibitor (MDL) on the lsc. Isc recording (representative of 4 experiments) with arrow marking the time at which basolateral MDL and PGE2 were added. Similar results were obtained for apical addition of PGE2. Ib: 7.1 (xAcrrr2; RT: 450 «cm2.

Fig8Regulation of anion secretion
FIG. 8. Demonstration of the involvement of cAMP-dependent signal transduction pathway in PGE2 response. A) Independent lsc responses stimulated by a Ca2+-mobilizing agent, ATP (10 (xM, apical), and basolateral PGE2. Ib: 5.9 ц,Асггг2; RT: 630 flcm2. B) Reduced basolateral PGE2-stimulated lsc after pretreatment with a cAMP-mobilizing agent, forskolin (100 Ц.М; lb: 5.7 цАспгг2; RT: 900 flcm2). Similar results were obtained for apical addition of PGE2.

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