Role of Phosphodiesterases in the Regulation of Gonadotropin- Releasing Hormone Secretion in GT1 Cells
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Increases in the level of cAMP stimulate the secretion of GnRH from GT1 GnRH neuronal cells. We hypothesized that cyclic nucleotide phosphodiesterases (PDEs), the enzymes that hydrolyze cAMP, may constitute a negative feedback signaling mechanism for GnRH regulation by decreasing the level of cAMP. GT1 cells were shown to express three PDEs by RT-PCR analysis: the cAMP-specific PDE4B and PDE4D and the calmodulin-dependent PDE1B. A splice variant of PDE4D, PDE4D3, which is activated when phosphorylated by cAMP-dependent protein kinase (PKA), was identified in GT1 cells by Western analysis. Consistent with PDEs negatively regulating GnRH secretion, treatment with the nonselective PDE inhibitor, IBMX, stimulated GnRH secretion 137% in 30-min static cultures. Furthermore, treatment with the PDE4-specific inhibitors Rolipram and RS-25344 increased GnRH secretion 48 and 125%, while treatment with the PDE1-specific inhibitor 8-MeoM-IBMX only caused a modest increase of 28%. In perifusion studies a rapid multi-fold stimulation of GnRH secretion was observed following treatment with IBMX, Rolipram or RS-25344. In conclusion, the level of PDE activity appears to be an important negative feedback signal for GnRH secretion. We hypothesize that activation of PDE4D3 by PKA may constitute a negative feedback signaling pathway which participates in the regulation of cAMP levels.Keywords:
IBMX
Rolipram
PDE10A
Phosphodiesterase inhibitor
Phosphodiesterase 3
Intact rat peritoneal macrophages (rPM) treated with 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterases (PDEs), accumulated more cGMP than untreated cells. A PDE activity toward [(3)H]cGMP was detected in the soluble and particulate fractions of rPM. The hydrolysis of cGMP was Ca(2+)/calmodulin-independent but increased in the presence of cGMP excess. Similar results were obtained when [(3)H]cAMP was used as a substrate. The hydrolytic activity towards both nucleotides was inhibited in the presence of IBMX. Therefore, the PDEs of families 2, 5, 10 and 11 are potential candidates for cGMP hydrolysis in the rPM. They may not only regulate the cGMP level in a feedback-controlled way but also link cGMP-dependent pathways with those regulated by cAMP.
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Increases in the level of cAMP stimulate the secretion of GnRH from GT1 GnRH neuronal cells. We hypothesized that cyclic nucleotide phosphodiesterases (PDEs), the enzymes that hydrolyze cAMP, may constitute a negative feedback signaling mechanism for GnRH regulation by decreasing the level of cAMP. GT1 cells were shown to express three PDEs by RT-PCR analysis: the cAMP-specific PDE4B and PDE4D and the calmodulin-dependent PDE1B. A splice variant of PDE4D, PDE4D3, which is activated when phosphorylated by cAMP-dependent protein kinase (PKA), was identified in GT1 cells by Western analysis. Consistent with PDEs negatively regulating GnRH secretion, treatment with the nonselective PDE inhibitor, IBMX, stimulated GnRH secretion 137% in 30-min static cultures. Furthermore, treatment with the PDE4-specific inhibitors Rolipram and RS-25344 increased GnRH secretion 48 and 125%, while treatment with the PDE1-specific inhibitor 8-MeoM-IBMX only caused a modest increase of 28%. In perifusion studies a rapid multi-fold stimulation of GnRH secretion was observed following treatment with IBMX, Rolipram or RS-25344. In conclusion, the level of PDE activity appears to be an important negative feedback signal for GnRH secretion. We hypothesize that activation of PDE4D3 by PKA may constitute a negative feedback signaling pathway which participates in the regulation of cAMP levels.
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Rolipram
PDE10A
Phosphodiesterase inhibitor
Phosphodiesterase 3
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Zaprinast
IBMX
Phosphodiesterase 3
Rolipram
PDE10A
Amrinone
Milrinone
Cyclic nucleotide phosphodiesterase
Aminophylline
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The role of cyclic nucleotide phosphodiesterase (PDE) isoforms in the beta2-adrenergic stimulation of the L-type Ca2+ current (ICa,L) was investigated in frog ventricular myocytes using double patch-clamp and double-barrelled microperfusion techniques. Isoprenaline (ISO, 1 nM to 10 microM) was applied on one half of the cell, either alone or in the presence of PDE inhibitors, and the local and distant responses of ICa,L were used to determine the gradient of local vs. distant cAMP concentration (alpha). IBMX (100 microM), a non-selective PDE inhibitor, reduced alpha from 40 to 4.4 indicating a 9-fold reduction in intracellular cAMP compartmentation when all PDE activity was blocked. While PDE1 and PDE2 inhibition had no effect, PDE3 inhibition by milrinone (3 microM) or PDE4 inhibition by Ro 20-1724 (3 microM) reduced alpha by 6- and 4-fold, respectively. A simultaneous application of milrinone and Ro 20-1724 produced a similar effect to IBMX, showing that PDE3 and PDE4 were the major PDEs accounting for cAMP compartmentation. Okadaic acid (3 microM), a non-selective phosphatase inhibitor, or H89 (1 microM), an inhibitor of cAMP-dependent protein kinase (PKA), had no effect on the distant response of ICa,L to ISO indicating that PDE activation by PKA played a minor role in cAMP compartmentation. Our results demonstrate that PDE activity determines the degree of cAMP compartmentation in frog ventricular cells upon beta2-adrenergic stimulation. PDE3 and PDE4 subtypes play a major role in this process, and contribute equally to ensure a functional coupling of beta2-adrenergic receptors with nearby Ca2+ channels via local elevations of cAMP.
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Cyclic nucleotide phosphodiesterase (PDE) enzymes are felt to play a role in the regulation of inflammatory responses through their effects on cAMP. In this study, we investigated the effects of nonselective and isozyme selective PDE inhibitors on the proliferative responses of peripheral blood mononuclear cells (PBMCs) to ragweed (RW, a Th2 stimulus), tetanus toxoid (TT, a Th1 stimulus), and phytohemagglutinin in ragweed-allergic patients. The nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) produced nearly identical concentration-dependent inhibition for PBMCs cultured with either Ag (RW and TT) or mitogen (phytohemagglutinin). Neither the type V inhibitor, zaprinast, nor the type III inhibitor, siguazodan, was effective at inhibiting antigen- or mitogen-driven proliferative responses of human PBMCs. The type IV inhibitor, rolipram, was unique in its ability to inhibit the RW-driven proliferative response and, to a lesser extent, the TT-driven proliferative response. However, rolipram was ineffective at inhibiting the mitogen-driven proliferative response. Only with a combination of type III and type IV inhibitors could the efficacy on the TT response be made to approximate that of the type IV inhibitor alone in the RW-driven system. Although the efficacies of IBMX and rolipram were identical in the RW-driven system, the IC50 value of the latter was 10-fold lower, similar to the difference noted in the TT-driven system between IBMX alone and the combination of rolipram with siguazodan. Dose-response curves generated by using the D- or L-isomers of rolipram were not appreciably different from each other or from the curve generated with racemic rolipram. The addition of supraphysiologic doses of human rIL-2 and rIL-4 was unable to counteract the inhibitory effect of IBMX or rolipram. These data support the hypothesis that modulation of proliferation of PBMCs by selective PDE inhibitors varies in sensitivity with the type of stimulus used, and that the type IV PDE exerts the predominant cAMP-associated regulatory effect on allergen-driven proliferation. Finally, the inhibitory effect induced by rolipram is independent of its stereochemistry and cannot be exclusively attributed to deficits in IL-2 or IL-4.
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To determine whether phosphodiesterase (PDE) is involved in the degradation of cGMP in human erythrocytes, we studied the cell cGMP content in the presence of different PDE inhibitors: zaprinast and dipyridamole, specific inhibitors of cGMP-binding, cGMP-specific PDE (cG-BPDE); vinpocetine, a specific inhibitor of Ca2+, calmodulin-dependent phosphodiesterase (CaM-PDE); an unspecific inhibitor, 3-isobutyl-1-methylxanthine (IBMX). IBMX, zaprinast, and dipyridamole at 30 microM did not affect the intracellular cGMP content. However, vinpocetine at this concentration increased the cGMP content by 102 +/- 14% (p < 0.05). The effect of vinpocetine was dose-dependent, reached the maximal level after 1 min of incubation and flattened at the same level. Ca2+ (10 microM) in the presence of the Ca(2+)-ionophore, A23187 (5 microM), decreased the cGMP content (-23% +/- 4; p < 0.05), which can be explained by the CaM-PDE activation. The Ca(2+)-induced decrease in cGMP was completely inhibited by the CaM antagonist, W-7 (100 microM). These data suggest that erythrocytes contain Ca2+, CaM-PDE.
Zaprinast
IBMX
Vinpocetine
PDE10A
Phosphodiesterase inhibitor
Phosphodiesterase 3
Amrinone
Cyclic nucleotide phosphodiesterase
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Second messenger system
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