Neurotoxicity of cGMP in the vertebrate retina: from the initial research on rd mutant mice to zebrafish genetic approaches.

Zebrafish are an excellent animal model for research on vertebrate development and human diseases. Sophisticated genetic tools including large-scale mutagenesis methodology make zebrafish useful for studying neuronal degenerative diseases. Here, we review zebrafish models of inherited ophthalmic diseases, focusing on cGMP metabolism in photoreceptors. cGMP is the second messenger of phototransduction, and abnormal cGMP levels are associated with photoreceptor death. cGMP concentration represents a balance between cGMP phosphodiesterase 6 (PDE6) and guanylate cyclase (GC) activities in photoreceptors. Various zebrafish cGMP metabolism mutants were used to clarify molecular mechanisms by which dysfunctions in this pathway trigger photoreceptor degeneration. Here, we review the history of research on the retinal degeneration (rd) mutant mouse, which carries a genetic mutation of PDE6b, and we also highlight recent research in photoreceptor degeneration using zebrafish models. Several recent discoveries that provide insight into cGMP toxicity in photoreceptors are discussed.

Chronic hyperalgesic priming in the rat involves a novel interaction between cAMP and PKCepsilon second messenger pathways.

Toward the goal of defining new pharmacological targets for the treatment of chronic pain conditions, in previous studies we established a model, termed 'hyperalgesic priming,' in which an acute inflammatory stimulus causes a long-lasting latent susceptibility to hyperalgesia induced by subsequent exposures to the inflammatory mediator, prostaglandin E2 (PGE2). Those investigations suggested the hypothesis that priming induces a novel linkage between the PGE2-activated second messenger cascade and the epsilon isoform of protein kinase C (PKCepsilon). In the present study, comparison of dose-response relations for hyperalgesia produced by PGE2, forskolin, 8-Br-cAMP, or the protein kinase A (PKA) catalytic subunit, in primed versus normal animals, demonstrated that priming-induced enhancement of the PGE2-activated second messenger cascade occurs downstream to adenylate cyclase and upstream to PKA. Therefore, PGE2-induced hyperalgesia in the primed animal is enhanced by the recruitment of a novel cAMP/PKCepsilon signaling pathway in addition to the usual cAMP/PKA pathway. These observations suggest that pharmacological disruption of the novel interaction between cAMP and PKCepsilon might provide a route toward the development of highly specific methods to reverse cellular processes that underlie chronic pain states.

Intracellular cyclic nucleotide analogues inhibit in vitro mitogenesis and activation of fibroblasts derived from obstructed rat kidneys.

As several studies indirectly suggest that inhibiting the intracellular breakdown of cyclic nucleotides may inhibit fibrogenesis, this study used membrane permeable cyclic nucleotide analogues to examine the role of cAMP and cGMP signaling pathways in the regulation of renal fibroblast function. Fibroblasts were isolated by explant outgrowth culture of rat kidneys post unilateral ureteric obstruction. Subcultured cells were exposed to 10- 1,000 microM of the cyclic nucleotide analogues 8-bromo-cAMP (8br-cAMP) and 8-bromo-cGMP (8br-cGMP). Functional parameters examined included mitogenesis (thymidine incorporation), collagen synthesis (proline incorporation), myofibroblast differentiation (Western blotting for alpha-smooth muscle actin; alpha-SMA) and expression of CTGF (Northern blotting), a TGF-beta(1)-driven immediate early response gene. Serum-stimulated mitogenesis was decreased 27 +/- 4% by 100 microM 8br-cAMP (p < 0.01), 49 +/- 6% by 1,000 microM 8br-cAMP (p < 0.001) and 43 +/- 7% by 1,000 microM 8br-cGMP (p < 0.01). 1,000 microM 8br-cAMP and 8br-cGMP reduced basal collagen synthesis by 80 +/- 5 and 60 +/- 21% respectively (both p < 0.05). Maximum dose of 8br-cAMP but not 8br-cGMP inhibited basal expression of the differentiation marker alpha-SMA by 43 +/- 33 (p < 0.05), resulted in a more rounded cell morphology and reduced expression of CTGF by 39 +/- 24% (p < 0.05). Measurement of mitochondrial activity confirmed that effects were independent of cell toxicity. In conclusion, cyclic nucleotides inhibit fibrogenesis in vitro. Strategies which elevate intracellular cyclic nucleotide concentrations may therefore be therapeutically valuable in preventing the proliferation and activation of fibroblasts in progressive renal disease.

Endothelin-1 as a protective factor against beta-adrenergic agonist-induced apoptosis in cardiac myocytes.

OBJECTIVES: The purpose of this study was to investigate the regulation of beta-adrenergic agonist-induced apoptosis by endothelin-1 (ET-1) in cardiac myocytes. BACKGROUND: Numerous hormonal factors including norepinephrine and ET-1 are activated in patients with heart failure. These factors may be involved in the positive and negative regulation of myocardial cell apoptosis observed in failing hearts. Recently, it has been shown that norepinephrine can induce myocardial cell apoptosis via a beta-adrenergic receptor-dependent pathway. METHODS: Primary cardiac myocytes were prepared from neonatal rats. These cells were stimulated with the beta-adrenergic agonist isoproterenol (ISO) in the presence or absence of ET-1. RESULTS: The administration of 10(-7) mol/liter of ET-1 completely blocked Iso-induced apoptosis. An endothelin type A receptor antagonist, FR139317, negated the inhibitory effect of ET-1 on apoptosis, while the endothelin type B receptor antagonist BQ788 did not show such a negation. Endothelin-1 also inhibited apoptosis induced by a membrane-permeable cAMP analogue (8-Br-cAMP), which bypassed Gi. The effect of ET-1 was neutralized by an MEK-1-specific inhibitor (PD098059), a phosphatidylinositol 3'-kinase inhibitor (wortmannin) and its downstream pp70 S6-kinase inhibitor, rapamycin. CONCLUSIONS: These findings suggest that ET-1 represents a protective factor against myocardial cell apoptosis in heart failure and that this effect is mediated mainly through endothelin type A receptor-dependent pathways involving multiple downstream signalings in cardiac myocytes.

Intrathecal injection of cell-permeable analogs of cyclic 3',5'-guanosine monophosphate produces hyperalgesia in mice.

Several recent studies suggest that spinal cord levels of cyclic 3',5'-guanosine monophosphate (cGMP) may participate in the development of hyperalgesia. The purpose of this study was to directly evaluate whether cell permeable analogues of cGMP evoke a thermal hyperalgesia (using a hot-plate assay) when administered intrathecally in mice. Our results indicate that two cell permeable forms of cGMP evoke a dose dependent hyperalgesia when administered intrathecally in mice. Additionally, this hyperalgesia was selective since neither non-cell permeant cGMP nor guanosine had any effect on the latency of paw withdrawal when compared to the vehicle injected controls. These data indicate that cGMP is involved in the facilitation of thermal hyperalgesia at the level of the spinal cord.

Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells.

Endothelium-derived relaxing factor has been recently identified as nitric oxide. The purpose of this study was to determine if vasodilator drugs that generate nitric oxide inhibit vascular smooth muscle mitogenesis and proliferation in culture. Three chemically dissimilar vasodilators, sodium nitroprusside, S-nitroso-N-acetylpenicillamine and isosorbide dinitrate, dose-dependently inhibited serum-induced thymidine incorporation by rat aortic smooth muscle cells. Moreover, 8-bromo-cGMP mimicked the antimitogenic effect of the nitric oxide-generating drugs. The antimitogenic effect of S-nitroso-N-acetylpenicillamine was inhibited by hemoglobin and potentiated by superoxide dismutase, supporting the view that nitric oxide was the ultimate effector. Sodium nitroprusside and S-nitroso-N-acetylpenicillamine significantly decreased the proliferation of vascular smooth muscle cells. Moreover, the inhibition of mitogenesis and proliferation was shown to be independent of cell damage, as documented by several criteria of cell viability. These results suggest that endogenous nitric oxide may function as a modulator of vascular smooth muscle cell mitogenesis and proliferation, by a cGMP-mediated mechanism.