Conditions that alter intracellular cAMP levels affect expression of the cAMP phosphodiesterase gene in Dictyostelium.

Abstract

We examined expression of the Dictyostelium cAMP phosphodiesterase (PDE) gene under conditions that alter intracellular cAMP levels during in vitro differentiation of wild-type strain V12M2 and a sporogenous derivative, HB200. In control cultures, cellular PDE activity peaked at 6 hr and declined by 8 hr, while secreted PDE activity continued to increase through 8 hr. Lowering intracellular cAMP levels with caffeine or progesterone increased cellular and secreted PDE activities 2-fold, increased stalk cell differentiation, and inhibited spore differentiation. In contrast, exposure to 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP; a membrane-permeable cAMP analog) or ammonia (which promotes intracellular cAMP accumulation in V12M2 and HB200 cells) lowered PDE activities by as much as 45%, decreased stalk cell differentiation, and increased spore differentiation. Simultaneous exposure to 8-Br-cAMP and caffeine gave intermediate PDE activities as would be expected if 8-Br-cAMP entered the cell and bypassed the caffeine-mediated block to adenylate cyclase activation. In all cases, we observed commensurate changes in developmental PDE transcript levels. The developmental time course of expression was not significantly altered by these treatments. These results suggest that the magnitude of PDE gene expression is negatively regulated by intracellular cAMP levels and provide evidence for one of the earliest changes in gene expression that is consistent with cell-type specificity. These results are discussed in terms of a bistable switch employing intracellular cAMP as a regulator of cell fate. feine and progesterone, which inhibit intracellular cAMP accumulation by different mechanisms (12,13), inhibit spore and promote stalk cell differentiation in concentrationdependent manners (7). Conversely, conditions that raise intracellular cAMP levels by hyper-inducing adenylate cyclase activity increase spore and decrease stalk cell differentiation (14). Ammonia, which promotes intracellular cAMP accumulation by inhibiting its secretion (15), and 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), a membrane-permeable cAMP analog with low affinity for the cell surface cAMP receptor (16)

Role of sarcoplasmic reticulum in control of membrane potential and nitrergic response in opossum lower esophageal sphincter

“…However, the concomitant application of caffeine (10 m M ) still abolished MPfs and the sIJP, and hyperpolarized MP by 8.2±1.2 mV ( n =5), a degree of hyperpolarization that was not significantly different from that induced by caffeine alone. Caffeine has also been reported to be a potent inhibitor of cAMP phosphodiesterase in different tissues (Rivedal & Sanner, 1985; Riley & Barclay, 1990; Tesarik et al , 1992 ; Pozo et al , 2002 ), resulting in intracellular cAMP accumulation, which activates protein kinase C‐dependent signal cascade. A membrane‐permeable cAMP analog, 8‐bromo‐cAMP (8‐Br‐cAMP), was used to exclude this possibility.…”

“…These effects are unlikely to be due to opening of BK (Ca) channels, as they were not affected by preapplication of the BK (Ca) channel blocker TEA (2 m M ) (Figure 2). Caffeine has been reported to be a potent cAMP phosphodiesterase inhibitor, resulting in intracellular cAMP accumulation (Rivedal & Sanner, 1985; Riley & Barclay, 1990; Tesarik et al , 1992 ; Sinha et al , 1993 ). However, our finding that 8‐Br‐cAMP, a membrane‐permeable cAMP analog, had no effect on MP or MPfs precludes this mechanism as playing a role in the observed findings.…”

The ABC transporter, AbcB3, mediates cAMP export in D. discoideum development

“…We developed D. discoideum cells for 0-, 3-, and 6-hours and treated them with the membrane permeable cAMP analog, 8-Br-cAMP, for 2 hours to mimic intracellular cAMP cargo overload (24). We then measured the transcript abundance of each of the 68 ABC transporters by quantitative RT-PCR (qRT-PCR) and compared them between treated and untreated cells.…”

Functional complementation of a yeast knockout strain by Schistosoma mansoni Rho1 GTPase in the presence of caffeine, an agent that affects mutants defective in the protein kinase C signal transduction pathway

“…At first, it was thought to inhibit phosphodiesterase activity in the cAMP/Ras pathway of glucose signaling (Riley & Barclay 1990). However, double deletions in the genes encoding the two isoenzymes of phosphodiesterase in yeast (Dpde1, Dpde2) showed no modification of the Mpk1p phosphorilation status, a protein which is situated downstream of PKC1 on its signaling pathway (Martin et al 2000).…”