Transcriptional and post-transcriptional regulation of FSH receptor in rat granulosa cells by cyclic AMP and activin

The effects of zero magnetic field on human VH-10 fibroblasts and lymphocytes were studied by the method of anomalous viscosity time dependencies (AVTD). A decrease of about 20% in the AVTD peaks was observed within 40 to 80 min of exposure of fibroblasts. This decrease was transient and disappeared 120 min after beginning of exposure. Similar kinetics for the effect of zero field was observed when cells were exposed 20 min and then kept at an ambient field. A 20% decrease of the AVTD peaks (p < 0.005 to 0.05) 40 to 70 min after 20 min exposure to zero field was reproduced in four independent experiments (out of four) with human lymphocytes from the same healthy donor. Contrary to the effects of zero field, irradiation of lymphocytes or fibroblasts with gamma-rays resulted in significant increase of the AVTD peaks immediately after irradiation. We concluded that zero field and gamma-rays caused hypercondensation and decondensation of chromatin, correspondingly. The effect of ethidium bromide served as a positive control and supported this conclusion. The effects of zero field on human lymphocytes were more significant in the beginning of G1-phase than in G0-phase. Thus, human fibroblasts and lymphocytes were shown to respond to zero magnetic field.     

Mediation of cyclic AMP signaling by the first intracellular loop of the gonadotropin-releasing hormone receptor

The gonadotropin-releasing hormone (GnRH) receptor, which is a unique G protein-coupled receptor without a C-terminal cytoplasmic domain, activates both inositol phosphate (InsP) and cAMP signaling responses. The function of the highly basic first intracellular (1i) loop of the GnRH receptor in signal transduction was evaluated by mutating selected residues located in its N and C termini. Replacements of Leu58, Lys59, Gln61, and Lys62 at the N terminus, and Leu73, Ser74, and Leu80 at the C terminus, caused no change in binding affinity. The agonist-induced InsP and cAMP responses of the Q61E and K59Q,K62Q receptors were also unaffected, but the L58A receptor showed a normal InsP response and an 80% decrease in cAMP production. At the C terminus, the InsP response of the L73R receptor was normal, but cAMP production was reduced by 80%. The EC50 for GnRH-induced InsP responses of the S74E and L80A receptors was increased by about one order of magnitude, and the cAMP responses were essentially abolished. These findings indicate that cAMP signaling from the GnRH receptor is dependent on specific residues in the 1i loop that are not essential for activation of the phosphoinositide signaling pathway.     

Transcriptional regulation of aquaporin-2 water channel gene by cAMP

In patients awaiting heart transplantation, end-stage disease of a second organ may occasionally require consideration of simultaneous multiorgan transplantation. Outcome statistics in multiorgan transplant recipients are needed to define optimal utilization of scarce donor resources. Incidence of cardiac allograft rejection, actuarial recipient survival, and cardiac allograft rejection-free survival were evaluated in 82 recipients of 84 simultaneous heart and kidney transplants. Twenty-three of the 82 dual-organ recipients have died with 1, 6, 12, and 24-month actuarial survival rates of 92%, 79%, 76%, and 67%, respectively. The actuarial survival rates in the heart-kidney recipients were similar to those observed in 14,340 isolated heart recipients (United Network for Organ Sharing Scientific Registry) during the same period (92%, 86%, 83%, and 79%, respectively; P=0.20). Clinical data on all episodes of treated rejection in either organ and on immunosuppressive regimens were available on 56 patients; 48% of these patients have had no rejection in either organ, 27% experienced heart rejection alone, 14% experienced kidney rejection alone, and 11% had both heart and kidney allograft rejection. Heart allograft rejection was less common in heart-kidney recipients, as compared with isolated heart transplant recipients; 0, 1, and > or = 2 treated cardiac allograft rejection episodes occurred in 63%, 20%, and 18% of heart-kidney recipients compared with 46%, 27%, and 28% of 911 isolated heart recipients reported by Transplant Cardiologists' Research Database (P=0.02). The rejection-free survival rates at 1, 3, and 6 months were 88%, 74%, and 71% in the double-organ recipients, as compared with 66%, 44%, and 39%, respectively, in the single-organ recipients. Compared with isolated heart transplantation, combined heart-kidney transplantation does not adversely affect intermediate survival and results in a lower incidence of treated cardiac allograft rejection. The findings suggest that combined heart-kidney transplantation may be an acceptable option in a small subset of potential heart transplant recipients with severe renal dysfunction.     

Absolute requirement of cyclic nucleotide in the activation of the G141Q mutant cAMP receptor protein from Escherichia coli

cAMP receptor protein (CRP), when interacting with cAMP, controls the expression of a network of catabolite-sensitive genes in Escherichia coli. To understand the molecular events that lead to the activation of CRP, a combined approach of site-directed mutagenesis and thermodynamic analysis was employed to study a member of a specific class of CRP mutant, CRP, which activates the in vivo expression of CRP-dependent operons in cya- strains in the absence of exogenous cAMP. Results from in vitro studies show that the CRP mutant G141Q absolutely requires cAMP for interacting with specific DNA. A quantitative comparison of the thermodynamic parameters governing ligand binding and DNA-protein complex formation in the presence of different cyclic nucleotides leads to the conclusion that this CRP mutant is activated only in the presence of cyclic nucleotides. The specificity toward cyclic nucleotides exhibited by wild-type CRP is lost in this mutant. Furthermore, the binding affinity of the ligand for the first binding site of the mutant is essentially the same as that of wild-type CRP regardless of the identity of the cyclic nucleotide. Hence, the observed in vivo activation of CRP-dependent operons by G141Q in the absence of exogenous cAMP is most likely the consequence of the replacement of cAMP by other cyclic nucleotides to activate the mutant. It is also possible that trace levels of cAMP present in the cya- strain could account for the in vivo activation of the mutant. Furthermore, these results indicate that this CRP mutant does not assume the activated conformation in the absence of cyclic nucleotides, in contrast to the current model derived from results of in vivo studies.     

Differential regulation of phospholipase A2 (PLA2)-dependent Ca2+ signaling in smooth muscle by cAMP- and cGMP-dependent protein kinases. Inhibitory phosphorylation of PLA2 by cyclic nucleotide-dependent protein kinases

Both cAMP- and cGMP-dependent protein kinases inhibit agonist-stimulated phospholipase C-beta (PLC-beta) activity and inositol 1,4,5-trisphosphate-dependent Ca2+ release in vascular and visceral smooth muscle. In smooth muscle of the intestinal longitudinal layer, however, the initial steps in Ca2+ mobilization involve activation of cytosolic PLA2 (cPLA2) and arachidonic acid (AA)-dependent stimulation of Ca2+ influx. The present study examined whether cAMP- and cGMP-dependent protein kinases are capable of regulating these processes also. Agents that activated cAMP-dependent protein kinase (5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole 3',5'-cyclic monophosphothioate (Sp-isomer) and isoproterenol), cGMP-dependent protein kinase (8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate and Na nitroprusside), or both kinases (vasoactive intestinal peptide and isoproterenol >1 microM) induced phosphorylation of cPLA2 and inhibition of agonist-stimulated cPLA2 activity. Phosphorylation and inhibition of cPLA2 activity by cAMP- and cGMP-dependent protein kinases were blocked by the corresponding selective inhibitors (cAMP-dependent protein kinase, N-[2(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide hydrochloride (H-89) and myristoylated protein kinase inhibitor () amide; cGMP-dependent protein kinase, (8R,9S, 11S)-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8, 11-epoxy-1H,8H,11H,-2,7b,11a-trizadizobenzo(a,g)cycloocta(c, d, e)-trinden-1-one (KT-5823)). In contrast, AA-stimulated Ca2+ influx was inhibited by agents that activated cGMP-dependent protein kinase only; the inhibition was selectively blocked by KT-5823. The study provides the first evidence of inhibitory phosphorylation of cPLA2 in vivo by cAMP- and cGMP-dependent protein kinases. Inhibition of cPLA2 activity and AA-induced Ca2+ influx partly account for the ability of cAMP-dependent protein kinase and/or cGMP-dependent protein kinase to cause relaxation. Their importance resides in their location at the inception of the Ca2+ signaling cascade.     

Synergistic activation of mitogen-activated protein kinase by cyclic AMP and myeloid growth factors opposes cyclic AMP's growth-inhibitory effects

Colony-stimulating factors (CSFs) promote the proliferation, differentiation, commitment, and survival of myeloid progenitors, whereas cyclic AMP (cAMP)-mediated signals frequently induce their growth arrest and apoptosis. The ERK/mitogen-activated protein kinase (MAPK) pathway is a target for both CSFs and cAMP. We investigated how costimulation by cAMP and colony-stimulating factor-1 (CSF-1) or interleukin-3 (IL-3) modulates MAPK in the myeloid progenitor cell line, 32D. cAMP dramatically increased ERK activity in the presence of CSF-1 or IL-3. IL-3 also synergized with cAMP to activate ERK in another myeloid cell line, FDC-P1. The increase in ERK activity was transmitted to a downstream target, p90(rsk). cAMP treatment of 32D cells transfected with oncogenic Ras was found to recapitulate the superactivation of ERK seen with cAMP and CSF-1 or IL-3. ERK activation in the presence of cAMP did not appear to involve any of the Raf isoforms and was blocked by expression of dominant-negative MEK1 or treatment with a MEK inhibitor, PD98059. Although cAMP had an overall inhibitory effect on CSF-1-mediated proliferation and survival, the inhibition was markedly increased if ERK activation was blocked by PD98059. These findings suggest that upregulation of the ERK pathway is one mechanism induced by CSF-1 and IL-3 to protect myeloid progenitors from the growth-suppressive and apoptosis-inducing effects of cAMP elevations.     

Involvement of cyclic AMP generation in the inhibition of respiratory burst by 2-phenyl-4-quinolone (YT-1) in rat neutrophils

The inhibitory effect of 2-phenyl-4-quinolone (YT-1) on respiratory burst in rat neutrophils was investigated, and the underlying mechanism of action was assessed. YT-1 caused a concentration-dependent inhibition of the rate of O2.- release from rat neutrophils in response to formylmethionyl-leucyl-phenylalanine (fMLP), but not to phorbol 12-myristate 13-acetate (PMA), with an IC50 value of 60.7+/-8.2 microM. A comparable effect was also demonstrated in the inhibition of O2 consumption. Unlike superoxide dismutase, YT-1 had no effect on O2.- generation in the xanthine-xanthine oxidase system and during dihydroxyfumaric acid autoxidation. The fMLP-induced inositol trisphosphate (IP3) formation was unaffected by YT-1. In addition, YT-1 did not affect the initial spike of [Ca2+]i, but it accelerated the rate of [Ca2+]i decline in cells in response to fMLP. YT-1 was found to have little effect on the activity of neutrophil cytosolic protein kinase C (PKC). YT-1 increased the cellular cyclic AMP level, while having no effect on the cyclic GMP level. In addition, YT-1 increased neutrophil cytosolic protein kinase A (PKA) activity, but had no direct effect on the enzyme activity of pure porcine heart PKA. When neutrophils were treated with (8R,9S,11S)-(-)-9-hydroxy-9-hexoxycarbonyl-8-methyl-2,3,9,10-tetra hydro-8,11-epoxy- 1H,8H,11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde]trinde n-1-one, (KT 5720), a PKA inhibitor, the inhibition of O2.- generation by YT-1, as well as by prostaglandin E1 (PGE1) and dibutyryl cyclic AMP, was attenuated effectively. YT-1 did not activate the adenylate cyclase associated with neutrophil particulate fraction but inhibited the cytosolic phosphodiesterase (PDE) activity in a concentration-dependent manner. Neutrophils treated with YT-1 had a more pronounced increase in cellular cyclic AMP level by PGE1. Moreover, the ability of PGE1 to inhibit the respiratory burst in neutrophils was greatly enhanced by YT-1. These results suggest that the increase in cellular cyclic AMP levels by YT-1 through the inhibition of PDE (probably PDE4 isoenzyme) activity is involved in its inhibition of fMLP-induced respiratory burst in rat neutrophils.     

Synthesis and action of the cyclic AMP antagonist, prostaglandylinositol cyclic phosphate (cyclic PIP), in Dictyostelium discoideum

PURPOSE: To examine the incidence of and risk factors for retinal detachment during a 10-year follow-up on intracapsular cataract extraction (ICCE). METHODS: Retrospective analysis of medical records of 1041 eyes operated on with ICCE in the years 1984-86. A complete follow-up was achieved, and actuarial methods were used in the risk assessment. The risk time averaged 82 months. RESULTS: Twenty-two (2.1%) eyes developed retinal detachment; half of the cases occurred during the first postoperative year, but new cases emerged throughout the period. The cumulative incidence of RD amounted to 2.8%; 95% confidence interval: 1.5-4.2%. Multivariate Cox regression analysis identified younger age at surgery (relative risk for each 10-year increase in age=0.6; 95% confidence interval: 0.39-0.95) and male gender (relative risk=2.5; 95% confidence interval 1.04-6.04) as significant risk factors for RD. Eleven eyes, 50% of eyes with RD and 1% of the total number of eyes, lost useful vision in spite of retinal surgery. CONCLUSION: Even though the risk for both RD and an unfavourable result following retinal surgery may be smaller using contemporary surgical techniques, the present study indicates a substantial morbidity following cataract surgery, when a sufficiently long observation period is considered.