(1/146) Involvement of phosphodiesterase-cGMP-PKG pathway in intracellular Ca2+ oscillations in pituitary GH3 cells.
The present study investigates the potential role of the Ca2+-calmodulin-dependent type I phosphodiesterase (PDE)-cGMP-protein kinase G (PKG) pathway in spontaneous [Ca2+]i oscillations in GH3 cells using fura-2 single cell videoimaging. Vinpocetine (2.5-50 microM), a selective inhibitor of type I PDE, induced a concentration-dependent inhibition of spontaneous [Ca2+]i oscillations in these pituitary cells, and at the same time produced an increase of the intracellular cGMP content. The cell permeable cGMP analog N2,2'-O-dibutyryl-cGMP (dB-cGMP) (1 mM) caused a progressive reduction of the frequency and the amplitude of spontaneous [Ca2+]i oscillations when added to the medium. KT5823 (400 nM), a selective inhibitor of cGMP-dependent protein kinase (PKG), produced an increase of baseline [Ca2+]i and the disappearance of spontaneous [Ca2+]i oscillations. When KT5823 was added before vinpocetine, the PKG inhibitor counteracted the [Ca2+]i lowering effect of the cGMP catabolism inhibitor. Finally, the removal of extracellular Ca2+ or the blockade of L-type voltage-sensitive calcium channels (VSCC) by nimodipine produced a decrease of cytosolic cGMP levels. Collectively, the results of the present study suggest that spontaneous [Ca2+]i oscillations in GH3 cells may be regulated by the activity of type I PDE-cGMP-PKG pathway. (+info)
(2/146) Characterization of the cyclic nucleotide phosphodiesterase subtypes involved in the regulation of the L-type Ca2+ current in rat ventricular myocytes.
The effects of several phosphodiesterase (PDE) inhibitors on the L-type Ca current (I(Ca)) and intracellular cyclic AMP concentration ([cAMP]i) were examined in isolated rat ventricular myocytes. The presence of mRNA transcripts encoding for the different cardiac PDE subtypes was confirmed by RT-PCR. IBMX (100 microM), a broad-spectrum PDE inhibitor, increased basal I(Ca) by 120% and [cAMP]i by 70%, similarly to a saturating concentration of the beta-adrenoceptor agonist isoprenaline (1 microM). However, MIMX (1 microM), a PDE1 inhibitor, EHNA (10 microM), a PDE2 inhibitor, cilostamide (0.1 microM), a PDE3 inhibitor, or Ro20-1724 (0.1 microM), a PDE4 inhibitor, had no effect on basal I(Ca) and little stimulatory effects on [cAMP]i (20-30%). Each selective PDE inhibitor was then tested in the presence of another inhibitor to examine whether a concomitant inhibition of two PDE subtypes had any effect on I(Ca) or [cAMP]i. While all combinations tested significantly increased [cAMP]i (40-50%), only cilostamide (0.1 microM)+ Ro20-1724 (0.1 microM) produced a significant stimulation of I(Ca) (50%). Addition of EHNA (10 microM) to this mix increased I(Ca) to 110% and [cAMP]i to 70% above basal, i.e. to similar levels as obtained with IBMX (100 microM) or isoprenaline (1 microM). When tested on top of a sub-maximal concentration of isoprenaline (1 nM), which increased I(Ca) by (approximately 40% and had negligible effect on [cAMP]i, each selective PDE inhibitor induced a clear stimulation of [cAMP]i and an additional increase in I(Ca). Maximal effects on I(Ca) were approximately 8% for MIMX (3 microM), approximately 20% for EHNA (1-3 microM), approximately 30% for cilostamide (0.3-1 microM) and approximately 50% for Ro20-1724 (0.1 microM). Our results demonstrate that PDE1-4 subtypes regulate I(Ca) in rat ventricular myocytes. While PDE3 and PDE4 are the dominant PDE subtypes involved in the regulation of basal I(Ca), all four PDE subtypes determine the response of I(Ca) to a stimulus activating cyclic AMP production, with the rank order of potency PDE4>PDE3>PDE2>PDE1. (+info)
(3/146) The calcium/calmodulin-dependent phosphodiesterase PDE1C down-regulates glucose-induced insulin secretion.
To understand the role cAMP phosphodiesterases (PDEs) play in the regulation of insulin secretion, we analyzed cyclic nucleotide PDEs of a pancreatic beta-cell line and used family and isozyme-specific PDE inhibitors to identify the PDEs that counteract glucose-stimulated insulin secretion. We demonstrate the presence of soluble PDE1C, PDE4A and 4D, a cGMP-specific PDE, and of particulate PDE3, activities in betaTC3 insulinoma cells. Selective inhibition of PDE1C, but not of PDE4, augmented glucose-stimulated insulin secretion in a dose-dependent fashion thus demonstrating that PDE1C is the major PDE counteracting glucose-dependent insulin secretion from betaTC3 cells. In pancreatic islets, inhibition of both PDE1C and PDE3 augmented glucose-dependent insulin secretion. The PDE1C of betaTC3 cells is a novel isozyme possessing a K(m) of 0.47 microM for cAMP and 0.25 microM for cGMP. The PDE1C isozyme of betaTC3 cells is sensitive to 8-methoxymethyl isobutylmethylxanthine and zaprinast (IC(50) = 7.5 and 4.5 microM, respectively) and resistant to vinpocetine (IC(50) > 100 microM). Increased responsiveness of PDE1C activity to calcium/calmodulin is evident upon exposure of cells to glucose. Enhanced cAMP degradation by PDE1C, due to increases in its responsiveness to calcium/calmodulin and in intracellular calcium, constitutes a glucose-dependent feedback mechanism for the control of insulin secretion. (+info)
(4/146) Effect of type-selective inhibitors on cyclic nucleotide phosphodiesterase activity and insulin secretion in the clonal insulin secreting cell line BRIN-BD11.
1. The cyclic nucleotide phosphodiesterases (PDEs) present in an insulin secreting cell line, BRIN - BD11, were characterized using calcium/calmodulin, IGF-1, isoenzyme-selective PDE inhibitors and RT - PCR. 2. Calmodulin activated cyclic AMP or cyclic GMP PDE activity in pellet and was 3 fold (P=0.002) more potent in activating cyclic nucleotide hydrolysis in pellet compared with supernatant fractions. 3. The PDE1/PDE5 inhibitor zaprinast inhibited both cyclic AMP and cyclic GMP PDE activity in both pellet and supernatant fractions of cell homogenates by a maximum of around 25% (IC(50) 1 - 5 microM), while rolipram (PDE4 selective) inhibited only cyclic AMP hydrolysis. 4. The PDE3-selective inhibitors Org 9935 (0.02 - 10 microM) and siguazodan (0.1 - 10 microM) inhibited cyclic AMP PDE activity in the pellet but not the supernatant fractions of cell homogenates, with a maximum inhibition of about 30%. IGF-1 (2 - 7.5 ng ml(-1)) potently augmented this PDE activity. 5. RT - PCR using specific primers for PDE3B, but not for PDE3A, amplified, from BRIN - BD11 cell total RNA, a 351 base pair product that was >97% homologous with rat adipose tissue PDE3B. 6. IBMX, Org 9935, siguazodan and rolipram (1 - 50 microM), but not zaprinast, each augmented glucose-induced insulin secretion in the presence of 16.7 mM but not 1 mM glucose. 7. These findings, in a clonal insulin secreting cell line, are consistent with an important role for PDE3B in regulating the pool of cyclic AMP relevant to the modulation of glucose-induced insulin secretion. (+info)
(5/146) Differential inhibition of multiple cAMP phosphodiesterase isozymes by isoflavones and tyrphostins.
A series of isoflavone and tyrphostin compounds were found to inhibit the degradation of cAMP by several cyclic nucleotide phosphodiesterase (PDE) isozymes. Specific hydroxyl groups on the isoflavone structure were critical for PDE isozyme-selective inhibition. Replacement of the C-7 hydroxyl group of the isoflavone with a methoxy group raised the IC(50) for PDE1, PDE3, and PDE4. The absence of the C-5 hydroxyl group raised the IC(50) from 5 to >100 microM for PDE4, but actually lowered the IC(50) for PDE3 and PDE1. Replacement of the C-4' hydroxyl group with a methoxy group raised the IC(50) for PDE3 and PDE1, yet only slightly changed the IC(50) for PDE4. Various tyrphostins were also potent inhibitors of PDE1, PDE3, and PDE4. The four-carbon side chained tyrphostins were much less potent; however, a very interesting pattern was observed in which removal of phenolic hydroxyls on the tyrphostin structure increased the potency for PDE1 and PDE3, but not PDE4. These results may help to explain some of the therapeutic and intracellular signaling effects of isoflavones and tyrphostins. Moreover, the isozyme selectivity demonstrated by the isoflavones and tyrphostins can serve as a pharmacophore for the design of specific PDE inhibitors. (+info)
(6/146) Differential changes in the expression of cyclic nucleotide phosphodiesterase isoforms in rat brains by chronic treatment with electroconvulsive shock.
Electroconvulsive shock (ECS) has been suggested to affect cAMP signaling pathways to exert therapeutic effects. ECS was recently reported to increase the expression of PDE4 isoforms in rat brain, however, these studies were limited to PDE4 family in the cerebral cortex and hippocampus. Thus, for comprehensive understanding of how ECS regulates PDE activity, the present study was performed to determine whether chronic ECS treatment induces differential changes in the expression of all the PDE isoforms in rat brains. We analyzed the mRNA expression of PDE isoforms in the rat hippocampus and striatum using reverse transcription polymerase chain reaction. We found chronic ECS treatment induced differential changes in the expression of PDE isoform 1, 2, 3, 4, 5 and 7 at the rat hippocampus and striatum. In the hippocampus, the expression of PDE1A/B (694%), PDE4A (158%), PDE4B (323 %), and PDE4D (181%) isoforms was increased from the controls, but the expression of PDE2 (62.8%) and PDE7 (37.8%) decreased by chronic ECS treatment. In the striatum, the expression of PDE1A/B (179%), PDE4A (223%), PDE4B (171%), and PDE4D (327%) was increased by chronic ECS treatment with the concomitant decrease in the expression of PDE2 (78.4%) and PDE3A (67.1%). In conclusion, chronic ECS treatment induces differential changes in the expression of most PDE isoforms including PDE1, PDE2, PDE3, PDE4, PDE5, and PDE7 in the rat hippocampus and striatum in an isoform- and brain region-specific manner. Such differential change is suggested to play an important role in regulation of the activity of PDE and cAMP system by ECS. (+info)
(7/146) "cAMP-specific" phosphodiesterase contributes to cGMP degradation in cerebellar cells exposed to nitric oxide.
Nitric oxide (NO) functions as a diffusible messenger in the central nervous system and elsewhere, exerting many of it physiological effects by activating soluble guanylyl cyclase, so increasing cellular cGMP levels. Hydrolysis of cyclic nucleotides is achieved by phosphodiesterases (PDEs) but the enzyme isoforms responsible for degrading cGMP in most cells have not been identified. We have devised a method for quantitatively monitoring the rate of breakdown of cGMP within intact cells and have applied it to rat cerebellar cell suspensions previously stimulated with NO. In contrast to previous findings in cultured cerebellar cells, there was no evidence from the use of selective inhibitors that PDE 1 participated importantly in cGMP hydrolysis. Moreover, procedures expected to increase PDE 1 activity by raising cytosolic Ca2+ concentrations (neurotransmitter agonists, Ca2+ ionophore) failed to influence cGMP breakdown. Instead, through the use of inhibitors selective for different PDE families, two isoforms were implicated: a "cGMP-specific" PDE (PDE 5), inhibited by sildenafil and zaprinast, and a "cAMP-specific" PDE (PDE 4), inhibited by low concentrations of rolipram and Ro-20-1724 and by milrinone. An explanation is offered for a participation of PDE 4 based on the high estimated intracellular cGMP concentration (approximately 800 microM) and the low affinity of the enzyme for cGMP. In accordance with predictions, recombinant PDE 4 was shown to hydrolyze high cGMP concentrations in a rolipram-sensitive manner. The widespread use of rolipram to test for a specific involvement of cAMP in cellular phenomena must therefore be questioned. (+info)
(8/146) Anti-inflammatory and immunomodulatory potential of the novel PDE4 inhibitor roflumilast in vitro.
From a series of benzamide derivatives, roflumilast (3-cyclo-propylmethoxy-4-difluoromethoxy-N-[3,5-di-chloropyrid-4-yl]-benzamide) was identified as a potent and selective PDE4 inhibitor. It inhibits PDE4 activity from human neutrophils with an IC(50) of 0.8 nM without affecting PDE1 (bovine brain), PDE2 (rat heart), and PDE3 and PDE5 (human platelets) even at 10,000-fold higher concentrations. Roflumilast is almost equipotent to its major metabolite formed in vivo (roflumilast N-oxide) and piclamilast (RP 73401), however, more than 100-fold more potent than rolipram and Ariflo (cilomilast; SB 207499). The anti-inflammatory and immunomodulatory potential of roflumilast and the reference compounds was investigated in various human leukocytes using cell-specific responses: neutrophils [N-formyl-methyl-leucyl-phenylalanine (fMLP)-induced formation of LTB(4) and reactive oxygen species (ROS)], eosinophils (fMLP- and C5a-induced ROS formation), monocytes, monocyte-derived macrophages, and dendritic cells (lipopolysaccharide-induced tumor necrosis factor-alpha synthesis), and CD4+ T cells (anti-CD3/anti-CD28 monoclonal antibody-stimulated proliferation, IL-2, IL-4, IL-5, and interferon-gamma release). Independent of the cell type and the response investigated, the corresponding IC values (for half-maximum inhibition) of roflumilast were within a narrow range (2-21 nM), very similar to roflumilast N-oxide (3-40 nM) and piclamilast (2-13 nM). In contrast, cilomilast (40-3000 nM) and rolipram (10-600 nM) showed greater differences with the highest potency for neutrophils. Compared with neutrophils and eosinophils, representing the terminal inflammatory effector cells, the relative potency of roflumilast and its N-oxide for monocytes, CD4+ T cells, and dendritic cells is substantially higher compared with cilomilast and rolipram, probably reflecting an improved immunomodulatory potential. The efficacy of roflumilast in vitro and in vivo (see accompanying article in this issue) suggests that roflumilast will be useful in the treatment of chronic inflammatory disorders such as asthma and chronic obstructive pulmonary disease. (+info)