Cyclic AMP-dependent Cl Secretion Is Regulated by Multiple Phosphodiesterase Subtypes in Human Colonic Epithelial Cells

Abstract

The role of phosphodiesterase (PDE) isoforms in regulation of transepithelial Cl secretion was investigated using cultured monolayers of T84 cells grown on membrane filters. Identification of the major PDE isoforms present in these cells was determined using ion exchange chromatography in combination with biochemical assays for cGMP and cAMP hydrolysis. The most abundant PDE isoform in these cells was PDE4 accounting for 70-80% of the total cAMP hydrolysis within the cytosolic and membrane fractions from these cells. The PDE3 isoform was also identified in both cytosolic and membrane fractions accounting for 20% of the total cAMP hydrolysis in the cytosolic fraction and 15-30% of the total cAMP hydrolysis observed in the membrane fraction. A large portion of the total cGMP hydrolysis detected in cytosolic and membrane fractions of T84 cells was mediated by PDE5 (50-75%). Treatment of confluent monolayers of T84 cells with various PDE inhibitors produced significant increases in short-circuit current (Isc). The PDE3-selective inhibitors terqinsin, milrinone and cilostamide produced increases in Isc with EC50 values of 0.6 nM, 8.0 nM and 0.5 microM respectively. These values were in close agreement with the IC50 values for cAMP hydrolysis. The effects of the PDE1-(8-MM-IBMX) and PDE4-(RP-73401) selective inhibitors on Isc were significantly less potent than PDE3 inhibitors with EC50 values of >7 microM and >50 microM respectively. However, the effects of 8-MM-IBMX and terqinsin on Cl secretion were additive, suggesting that inhibition of PDE1 also increases Cl secretion. The effect of PDE inhibitors on Isc were significantly blocked by apical treatment with glibenclamide (an inhibitor of the CFTR Cl channel) and by basolateral bumetanide, an inhibitor of Na-K-2Cl cotransport activity. These results indicate that inhibition of PDE activity in T84 cells stimulates transepithelial Cl secretion and that PDE1 and PDE3 are involved in regulating the rate of secretion.

The role of cyclic nucleotide phosphodiesterases in the regulation of adipocyte lipolysis

“…Thus, the effect of cilostamide in the cell-based assay was probably attributable to inhibition of PDE3 rather than other PDE families. Published EC 50 values for cilostamide in cellbased assays, including inhibition of vascular smooth muscle cell proliferation (39) and stimulation of chloride secretion in colonic epithelial cells (40), are similar to the values we have obtained with cultured adipocytes. This suggests that the low relative potency of cilostamide in such assays may be to the result of poor penetration into cells.…”

Calcium-sensing receptor abrogates secretagogue- induced increases in intestinal net fluid secretion by enhancing cyclic nucleotide destruction

“…There are 11 families of cyclic nucleotide PDEs, each exhibiting specific affinities for degrading cAMP and͞or cGMP (40,41). Although little information is available on the specific PDE genes expressed in intestinal epithelial cells, PDE1-5 isoforms have been identified in human colon cancer cells (42,43) and each of these PDEs is sensitive to IBMX. Because inhibition of PLC signaling abolishes CaSR effects on cAMP and cGMP accumulation, activation of Ca 2ϩ -calmodulin-dependent PDE1 family members (40) appear to be involved in receptor-mediated increases in cyclic nucleotide destruction.…”

Phosphodiesterase 4D Forms a cAMP Diffusion Barrier at the Apical Membrane of the Airway Epithelium

“…Although data exist concerning a role for phosphodiesterases in the compartmentation of cAMP in nonpolarized cells (17)(18)(19), little is known about the distribution and actions of cAMP in microdomains such as the apical regions of polarized epithelia. Several PDEs are ex-pressed in both human airway and intestinal epithelia based on molecular (20,21) and pharmacologic approaches (22,23). At least one PDE, PDE3, can augment CFTR responses ex vivo (24), in some animal models (25), but not in humans (26).…”