Inhibitor Isobutylmethylxanthine Research Articles

Role of cAMP in upregulation of insulin secretion during the adaptation of islets of Langerhans to pregnancy.

Islets undergo a number of upregulatory changes to meet the increased demand for insulin during pregnancy, including an increase in glucose-stimulated insulin secretion with a reduction in the stimulation threshold. Treatment with the lactogenic hormone prolactin (PRL) in vitro has been shown to induce changes in islets similar to those observed during pregnancy. We examined cAMP production in islets treated with PRL to determine if changes in cAMP are involved in the upregulation of insulin secretion. Insulin secretion and cAMP concentrations were measured from islets in response to a suprathreshold (6.8 mmol/l) or high (16.8 mmol/l) glucose concentration in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. Insulin secretion increased by 2.1-, 5.0-, and 5.9-fold at the suprathreshold glucose concentration and by 1.6-, 2.3-, and 2.9-fold at the higher glucose concentration after 1, 3, and 5 days of PRL treatment, respectively. After a similar pattern, cAMP metabolism increased by 1.2-, 1.6-, and 2.1-fold at the suprathreshold glucose concentration and by 1.2-, 1.7-, and 2.2-fold at the high glucose concentration after 1, 3, and 5 days of PRL treatment, respectively. The similar increases in insulin secretion and cAMP concentration suggest that changes in cAMP metabolism are involved in lactogen-induced upregulation of insulin secretion. To gain additional insight into the role of cAMP in the upregulation of islet function after lactogen treatment, we examined the relationship between changes in cAMP concentration and insulin secretion. Under all conditions (differing glucose concentrations and time periods), the increase in insulin release was directly proportional to the increase in cAMP. Thus increased glucose-stimulated insulin secretion from lactogen-treated islets could be accounted for by increased generation of cAMP and did not appear to require any further specific changes in intracellular processes mediated by cAMP. Because the PRL receptor is not directly involved in cAMP metabolism, the lactogen-induced increase in cAMP was most likely due to the increase in glucose metabolism that we have previously demonstrated in PRL-treated islets and in islets during pregnancy.

Aging modulates nitric oxide synthesis and cGMP levels in hippocampus and cerebellum. Effects of amyloid beta peptide.

The biological roles of nitric oxide (NO) and cGMP as inter- and intracellular messengers have been intensively investigated during the last decade. NO and cGMP both mediate physiological effects in the cardiovascular, endocrinological, and immunological systems as well as in central nervous system (CNS). In the CNS, activation of the N-methyl-D-aspartic acid (NMDA) type of glutamatergic receptor induces Ca(2+)-dependent NOS and NO release, which then activates soluble guanylate cyclase for the synthesis of cGMP. Both compounds appear to be important mediators in long-term potentiation and long-term depression, and thus may play important roles in the mechanisms of learning and memory. Aging and the accumulation of amyloid beta (A beta) peptides are important risk factors for the impairment of memory and development of dementia. In these studies, the mechanism of basal- and NMDA receptor-mediated cGMP formation in different parts of adult and aged brains was evaluated. The relative activity of the NO cascade was determined by assay of NOS and guanylate cyclase activities. In addition, the effect of the neurotoxic fragment 25-35 of A beta (A beta) peptide on basal and NMDA receptor-mediated NOS activity was investigated. The studies were carried out using slices of hippocampus, brain cortex, and cerebellum from 3- and 28-mo-old rats. Aging coincided with a decrease in the basal level of cGMP as a consequence of a more active degradation of cGMP by a phosphodiesterase in the aged brain as compared to the adult brain. Moreover, a loss of the NMDA receptor-stimulated enhancement of the cGMP level determined in the presence of cGMP-phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) was observed in hippocampus and cerebellum of aged rats. However, this NMDA receptor response was preserved in aged brain cerebral cortex. A significant enhancement of the basal activity of NOS by about 175 and 160% in hippocampus and cerebellum, respectively, of aged brain may be involved in the alteration of the NMDA receptor response. The neurotoxic fragment of A beta, peptide 25-35, decreased significantly the NMDA receptor-mediated calcium, and calmodulim-dependent NO synthesis that may then be responsible for disturbances of the NO and cGMP signaling pathway. We concluded that cGMP-dependent signal transduction in hippocampus and cerebellum may become insufficient in senescent brain and may have functional consequences in disturbances of learning and memory processes. A beta peptide accumulated during brain aging and in Alzheimer disease may be an important factor in decreasing the NO-dependent signal transduction mediated by NMDA receptors.

Regulatable production of insulin from primary-cultured hepatocytes: insulin production is up-regulated by glucagon and cAMP and down-regulated by insulin

To utilize hepatocytes for insulin-producing surrogate cells, we devised a regulatory secretion system by placing proinsulin DNA under the regulatable promoter for phosphoenolpyruvate carboxykinase (PEPCK). The expression of PEPCK is down-regulated by insulin, and up-regulated by cAMP and glucagon. To express insulin in hepatocytes, we constructed an adenoviral insulin expression system. After infection, the hepatocytes secreted immunoreactive insulin (IRI) at an increasing rate. IRI secretion increased over four-fold upon stimulation with 300 microM cAMP and 500 microM of the cAMP-dependent phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). This increase was also observed with glucagon and IBMX. Production was augmented two-fold by the addition of wortmannin, phosphatidylinositol (PI)-3-kinase inhibitor, suggesting that inhibitory insulin signaling to the PEPCK promoter may be mediated through PI-3-kinase. Addition of exogenous insulin to the culture decreased insulin mRNA expression remarkably on Northern blot. Thus, by using a PEPCK promoter for insulin expression, we were able to up-regulate insulin production from hepatocytes with cAMP and glucagon, and down-regulate with insulin itself.

Topographical relationship between neuronal nitric oxide synthase immunoreactivity and cyclic 3′,5′-guanosine monophosphate accumulation in the brain of the adult Xenopus laevis

Previous immunohistochemical staining procedures of the brain and pituitary in Xenopus laevis, using an antiserum against neuronal nitric oxide (NO) synthase (nNOS) and nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry, have revealed NOS activity in neurons and fibers in a number of brain areas, as well as in fibers in the pituitary. In the present study we have localized the target structures of the NOergic system in the Xenopus brain by visualizing the sites of NO-sensitive cyclic 3′,5′-guanosine monophosphate (cGMP) accumulation, according to a method for cGMP visualization in rat brain slices. Brain slices of unfixed Xenopus are incubated in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine and the NO donor sodium nitroprusside, followed by fixation and cryosectioning. Sections were then processed for immunohistochemistry using rabbit and sheep antisera against cGMP and a sheep antiserum against nNOS. Visualization of single and double labeling of cGMP immunoreactive and/or nNOS immunoreactive structures was performed with combined CY3/fluorescein isothiocyanate fluorescence microscopy. Following this procedure, we provide immunohistochemical evidence for the distribution of cGMP-accumulating neurons in the brain of adult Xenopus. In most brain areas, the distribution of nNOS and cGMP immunoreactive structures (neuron somata and fibers) is distinct and separate, for instance in the dorsal pallium, the lateral thalamic nuclei, the optic tectum, the locus coeruleus and the reticular formation. However, nNOS and cGMP immunoreactive structures are often found in the vicinity of each other, and in the optic tectum even in adjacent neuron fibers and somata. The present observations are in line with the presence of an NO-dependent soluble guanylate cyclase in distinct brain areas of Xenopus laevis, corroborating similar data in the mammalian brain. Further, our observations may add to the understanding of the anatomical connectivity pattern and functional relevance of the NOergic system in the amphibian brain.

Distribution of nitric oxide synthase and nitric oxide-receptive, cyclic GMP-producing structures in the rat brain

The structures capable of synthesizing cyclic GMP in response to nitric oxide in the rat brain were compared relative to the anatomical localization of neuronal nitric oxide synthase. In order to do this, we used brain slices incubated in vitro, where cyclic GMP-synthesis was stimulated using sodium nitroprusside as a nitric oxide-donor compound, in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. Nitric oxide-stimulated cyclic GMP synthesis was found in cells and fibers, but was especially prominent in varicose fibers throughout the rat brain. Fibers containing the nitric oxide-stimulated cyclic GMP production were present in virtually every area of the rat brain although there were large regional variations in the density of the fiber networks. When compared with the localization of nitric oxide synthase, it was observed that although nitric oxide-responsive and the nitric oxide-producing structures were found in similar locations in general this distribution was complementary. Only occasionally was nitric oxide-mediated cyclic GMP synthesis observed in structures which also contained nitric oxide synthase. We conclude that the nitric oxide-responsive soluble guanylyl cyclase and nitric oxide synthase are usually juxtaposed at very short distances in the rat brain. These findings very strongly support the proposed role of nitric oxide as an endogenous activator of the soluble guanylyl cyclase in the central nervous system and convincingly demonstrate the presence of the nitric oxide–cyclic GMP signal transduction pathway in virtually every area of the rat brain.

Onset and maintenance of metamorphic competence in the marine polychaete Hydroides elegans Haswell in response to three chemical cues

This study documents the order in which larvae of the tube-dwelling polychaete Hydroides elegans Haswell become competent to metamorphose in response to three substances: 30 mM excess K + in seawater, the cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) at 10 −4 M in seawater, and a water-soluble homogenate of adults and their tubes. Four experiments were conducted, three at 25°C and one at 20°C, typically with 10–13 larvae per replicate and five replicates per treatment. Each assay lasted 48 h. Larvae typically became responsive to IBMX at least 24 h before they became responsive to adult homogenate and were always slowest to become responsive to excess K +, suggesting that IBMX acts downstream from the sites at which the other cues operate and that the upstream sites become operational later in development. Competent larvae also responded far more quickly to IBMX — usually within 6–8 h — than to the other cues. Excess K + and cues associated with adult homogenate may take longer (24–48 h) to penetrate to their sites of action. Alternatively, IBMX may stimulate metamorphosis directly, possibly by increasing intracellular concentrations of cAMP, cGMP, or Ca 2+, while the other tested substances may act instead to turn off production of an inhibitory factor, which must then degrade before metamorphosis can occur. In three additional experiments, competent larvae remained responsive to all three inducers during 3–4 days of starvation, indicating that the metamorphic pathway of H. elegans, once developed, is not readily degraded even under severe nutritional stress.

Vasorelaxant effects of SCA40 (a phosphodiesterase III inhibitor) in pulmonary vascular preparations in rats.

1. The novel phosphodiesterase (PDE) inhibitor SCA40 (6-bromo-8(methylamino)imidazo[1,2-a]pyrazine-2-carbonitrile) was examined for its vasorelaxant activity on isolated pulmonary vascular preparations from rats. 2. SCA40 relaxed ring preparations of main and intralobar pulmonary artery precontracted submaximally with either phenylephrine or U46619 (thromboxane-mimetic). Based on negative log EC50 values, SCA40 was six-to 14-fold more potent than the PDE III inhibitor milrinone or the non-selective PDE inhibitor 3-isobutyl-1-methyl xanthine (IBMX). The potency of SCA40 corresponded to its reported potency as a PDE III inhibitor. 3. In isolated perfused lungs, SCA40 reversed the vasoconstriction induced by alveolar hypoxia. It was 49-fold more potent than IBMX. 4. In main pulmonary artery the vasorelaxation induced by SCA40 was not blocked by the large-conductance calcium-activated potassium channel (BKCa) inhibitors iberiotoxin (50 and 100 nmol/L) or charybdotoxin (100 and 300 nmol/L). This was in contrast to data on guinea-pig trachea, where responses to SCA40 were significantly inhibited by charybdotoxin (100 nmol/L). 5. It is concluded that opening of BKCa channels does not contribute to the pulmonary vasorelaxant effects of SCA40 in main pulmonary artery and it is likely that responses reflect the PDE III inhibitory properties of the drug. 6. It is postulated that SCA40 may be useful as a pulmonary vasodilator in disorders such as pulmonary hypertension.

Demonstration of a relaxin receptor and relaxin-stimulated tyrosine phosphorylation in human lower uterine segment fibroblasts.

To elucidate the mechanism of relaxin action, we studied the binding characteristics of human relaxin and its effects on intracellular concentrations of cAMP and tyrosine phosphorylation of cellular proteins in a model system of human cervix, human lower uterine segment fibroblasts. Human relaxin labeled with 125I bound specifically to a single class of high-affinity relaxin binding sites, distinct from insulin receptors, with a mean (+/-SEM) dissociation constant (Kd) of 4.36 +/- 1.7 x 10(-9) M and a mean of 3220 +/- 557 binding sites per cell in human lower uterine segment fibroblasts. Relaxin, in quantities that were shown previously to stimulate intracellular levels of cAMP in other cell types, had no effect on intracellular levels of cAMP in human lower uterine segment fibroblasts even in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX). Incubation of the cells with relaxin caused a significant increase in tyrosine phosphorylation of a protein with an apparent Mr of approximately 220 kDa in these cells. In concert with results of recent studies that demonstrated that the Mr of the relaxin receptor is approximately 220 kDa, our data suggest that the phosphorylated protein is likely to be the relaxin receptor.

Serotonin inhibition of steroid-induced meiotic maturation in the teleost Fundulus heteroclitus: role of cyclic AMP and protein kinases.

The transduction of the serotonin (5-HT) signal in Fundulus heteroclitus ovarian follicles leading to the inhibition of oocyte meiosis reinitiation (oocyte maturation) in vitro induced by the naturally occurring maturation-inducing steroid 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17,20 beta P) was investigated. Steroid-induced oocyte maturation was inhibited by 5-HT in a dose-dependent manner; maximum inhibition (90%) was observed with 10(-4) M 5-HT. Groups of follicle-enclosed oocytes were cultured in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and treated with increasing doses of 5-HT. Serotonin was found to slightly increase the levels of follicular 3',5'-cyclic adenosine monophosphate (cAMP) in a dose-dependent manner; 10(-4) M 5-HT induced approximately a 3-fold increase in cAMP with respect to the controls. The changes in cAMP were then evaluated in follicles treated with 17,20 beta P in IBMX-free culture media in the presence or absence of 10(-4) M 5-HT. The exposure of follicles to 17,20 beta P alone produced a small and transient reduction in cAMP (40%) within 1-3 hr of steroid stimulation, and these early changes in cAMP appeared associated with a high incidence of germinal vesicle breakdown (80% GVBD) by 24 hr of incubation. Under these conditions, treatment of follicles with 5-HT also increased significantly the production of cAMP, and when 5-HT was combined with 17,20 beta P, the steroid-mediated reduction in cAMP was prevented and the levels of GVBD inhibited by 95%. Meiosis also was reinitiated with either the protein kinase A (PKA) inhibitor H8 or the protein kinase C (PKC) activator PMA, and the 5-HT inhibitory action on GVBD was found to be 100-fold reduced or completely ineffective, respectively. Preincubation of follicles with the PKC inhibitor GF109203x abolished PMA-induced GVBD in a dose-dependent manner, whereas this inhibitor had no effect on 17,20 beta P-triggered meiotic maturation, indicating that activation of PKC is apparently sufficient but not necessary to reinitiate meiosis. Taken together, these findings suggest that 5-HT may inhibit 17,20 beta P-induced meiotic reinitiation through the activation of a cAMP-PKA transduction pathway and that PKC possibly induces oocyte maturation by a different pathway than the steroid and thus is not affected by 5-HT.

Serotonin inhibition of steroid‐induced meiotic maturation in the teleost Fundulus heteroclitus: Role of cyclic AMP and protein kinases

The transduction of the serotonin (5-HT) signal in Fundulus heteroclitusovarian follicles leading to the inhibition of oocyte meiosis reinitiation (oocyte maturation) in vitro induced by the naturally occurring maturation-inducing steroid 17α,20β-dihydroxy-4-pregnen-3-one (17,20βP) was investigated. Steroid-induced oocyte maturation was inhibited by 5-HT in a dose-dependent manner; maximum inhibition (90%) was observed with 10−4 M 5-HT. Groups of follicle-enclosed oocytes were cultured in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and treated with increasing doses of 5-HT. Serotonin was found to slightly increase the levels of follicular 3′,5′-cyclic adenosine monophosphate (cAMP) in a dose-dependent manner; 10−4 M 5-HT induced approximately a 3-fold increase in cAMP with respect to the controls. The changes in cAMP were then evaluated in follicles treated with 17,20βP in IBMX-free culture media in the presence or absence of 10−4 M 5-HT. The exposure of follicles to 17,20βP alone produced a small and transient reduction in cAMP (40%) within 1–3 hr of steroid stimulation, and these early changes in cAMP appeared associated with a high incidence of germinal vesicle breakdown (80% GVBD) by 24 hr of incubation. Under these conditions, treatment of follicles with 5-HT also increased significantly the production of cAMP, and when 5-HT was combined with 17,20βP, the steroid-mediated reduction in cAMP was prevented and the levels of GVBD inhibited by 95%. Meiosis also was reinitiated with either the protein kinase A (PKA) inhibitor H8 or the protein kinase C (PKC) activator PMA, and the 5-HT inhibitory action on GVBD was found to be 100-fold reduced or completely ineffective, respectively. Preincubation of follicles with the PKC inhibitor GF109203x abolished PMA-induced GVBD in a dose-dependent manner, whereas this inhibitor had no effect on 17,20βP-triggered meiotic maturation, indicating that activation of PKC is apparently sufficient but not necessary to reinitiate meiosis. Taken together, these findings suggest that 5-HT may inhibit 17,20βP-induced meiotic reinitiation through the activation of a cAMP-PKA transduction pathway and that PKC possibly induces oocyte maturation by a different pathway than the steroid and thus is not affected by 5-HT. Mol. Reprod. Dev. 49:333–341, 1998. © 1998 Wiley-Liss, Inc.

Potent Inhibition of the Proinflammatory Responses of Human Polymorphonuclear Cells by Prostaglandin E 1 but not by PGI 2 or Carbacyclin

The effects of prostaglandin E 1 (PGE 1) on adhesion of activated human polymorphonuclear cells (HPMNs) to human umbilical vein endothelial cells (HUVECs) and on chemotaxis of HPMNs were compared with those of prostaglandin I 2 (PGI 2) or its chemically stable analog carbacyclin. Although HUVECs treated with PGI 2 inhibited only fMLP-activated HPMN adhesion, both HUVECs and HPMNs treated with PGE 1 equally inhibited HPMN adhesion, thus indicating the appropriate reactions to endothelium-leukocyte interaction by PGE 1. In the presence of a nonspecific phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), PGE 1 inhibited HPMN chemotaxis induced by either fMLP or LTB 4, whereas both PGI 2 and carbacyclin had no effect. To examine whether the response of PMNs to PGs is species-specific, intracellular cAMP accumulation in response to PGs was determined in human, guinea-pig, and rat isolated PMNs. Although PGE 1 increased cAMP levels in all PMNs tested, both PGI 2 and carbacyclin increased cAMP only in rat PMNs. These results demonstrate that HPMN cAMP formation can be affected by PGE 1 but not by PGI 2. The findings help our understanding of the clinical use of PGs in patients with peripheral arterial occlusive diseases.

Human erythrocytes contain Ca2+, calmodulin-dependent cyclic nucleotide phosphodiesterase which is involved in the hydrolysis of cGMP.

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.

Inhibition of recombinant human cardiac L-type Ca 2+ channel α1C subunits by 3-isobutyl-1-methylxanthine

Inhibition of ion channels by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and related compounds has been demonstrated in various cell types, including the neuromuscular junction, GH 3 cells and vascular smooth muscle cells. These effects may be unrelated to the actions of these compounds on cellular metabolism, intracellular Ca 2+ stores and phosphodiesterase inhibition. In this study, the inhibition of recombinant human cardiac L-type Ca 2+ channel α 1C subunits by IBMX was examined using the whole-cell configuration of the patch clamp technique. Inhibition was repeatable, voltage-independent and associated with increased apparent channel inactivation. The actions of IBMX were unaffected in the presence of inhibitors of protein kinases A and G. The non-xanthine phosphodiesterase inhibitor rolipram had a small inhibitory effect on currents, but this was also unaffected by a protein kinase A inhibitor. These effects of IBMX could not be attributed to release of Ca 2+ from intracellular stores. Our findings indicate that methylxanthines can inhibit the cardiac L-type Ca 2+ channel α 1C subunit in the absence of auxiliary subunits by an undetermined, possibly direct mechanism.

Heparin suppresses cyclosporine-induced endothelin-1 synthesis in rat endothelial cells.

Cyclosporine stimulates vasoconstrictor endothelin-1 (ET-1) synthesis. This study examined the effect of heparin on cyclosporine-induced ET-1 synthesis in Wistar rat aortic endothelial cells in culture. Cyclosporine (0.01-5 mumol/L) stimulated ET-1 mRNA expression in a dose-dependent manner. A nitric oxide synthesis inhibitor, NG-monomethyl-L-arginine (L-NMMA) (10(-5) mol/L), did not affect cyclosporine-induced ET-1 mRNA expression. Heparin (1-20 U/ml) suppressed cyclosporine-induced ET-1 mRNA expression in a dose-dependent manner. The inhibitory effect of heparin was blunted in the presence of either L-NMMA (10(-5) mol/L) or calmodulin inhibitors such as N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) (5 x 10(-5) mol/L) or calmidazolium (5 x 10(-5) mol/L) in the presence of the phosphodiesterase inhibitor 3-isobutyl 1-methylxanthine (0.1 mmol/L). These results suggest that heparin suppresses cyclosporine-induced ET-1 mRNA expression via both NO- and calmodulin-dependent pathways.

Expression of CD54 (intercellular adhesion molecule-1) and the beta 1 integrin CD29 is modulated by a cyclic AMP dependent pathway in activated primary rat microglial cell cultures.

Microglial cell activation plays a central role in acute and chronic inflammatory processes associated with neurodegeneration. As macrophage activation is generally associated with the up-regulation of specific surface antigens, the expression of CD54, and CD29 were evaluated on CD11b positive neonatal rat microglial cell cultures by flow cytometry. These cells when exposed to lipopolysaccharide, LPS, and gamma interferon, IFN gamma, exhibited a 2-3 fold increase in CD54 expression, an increase in CD29 and no change in CD11b. Maximal increases in CD54 and CD29 staining on CD11b positive microglial cells were apparent 20-24 h after LPS and IFN gamma while nitrite production reflecting inducible nitric oxide synthase activity, continued to increase. The increases in CD29 and CD54 staining were inhibited in a dose dependent manner by agents which increased intracellular cAMP levels including 100 microM 8-bromoadenosine 3':5'-cyclic monophosphate but not 8-bromoadenosine monophosphate, the phosphodiesterase inhibitor isobutyl methylxanthine and by direct activation of adenylate cyclase with forskolin. Concomitant with the dose dependent decreases in CD29 and CD54 staining were increases in intracellular cAMP and reduced TNF secretion. These results suggest that regulation of CD29 and CD54 expression on activated microglial cells involves a cAMP dependent pathway.

Rolipram, a selective phosphodiesterase type-IV inhibitor, prevents induction of heat shock protein HSP-70 and hsp-70 mRNA in rat retrosplenial cortex by the NMDA receptor antagonist dizocilpine.

The non-competitive NMDA receptor antagonists, such as (+)-MK-801 (dizocilpine), cause the expression of heat shock protein HSP-70 and pathomorphological damage in the retrosplenial cortex of the rat brain. However, the precise mechanism(s) underlying the neurotoxicity of NMDA receptor antagonists is unknown. The present study was undertaken to examine the role of phosphodiesterase type IV in the expression of heat shock genes induced by dizocilpine. Heat shock protein HSP-70, which is known as a sensitive marker of neuron injury, was induced in the retrosplenial cortex of the rat brain 24 h after a single administration of dizocilpine (1 mg/kg). Pretreatment with the specific phosphodiesterase type IV inhibitor rolipram (2.5, 5 or 10 mg/kg, 15 min before dizocilpine) attenuated the expression of HSP-70 and hsp-70 mRNA induced by dizocilpine (1 mg/kg) in a dose-dependent manner. Furthermore, another phosphodiesterase type IV inhibitor, Ro 20-1724 (5 or 10 mg/kg, 15 min before dizocilpine), and a non-selective phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) (5 or 10 mg/kg, 15 min before dizocilpine), significantly attenuated the expression of HSP-70 protein and hsp-70 mRNA induced in the retrosplenial cortex by dizocilpine. However, the induction of the immediate early gene c-fos and microglial activation in the retrosplenial cortex after administration of dizocilpine was not attenuated by pretreatment with rolipram (5 or 10 mg/kg, 15 min before dizocilpine). Moreover, histopathological study indicated that pretreatment with rolipram (5 or 10 mg/kg, 15 min before dizocilpine) did not prevent the formation of vacuoles caused by treatment with dizocilpine. The present findings suggest that phosphodiesterase type IV may play a significant role in the expression of HSP-70 protein and hsp-70 mRNA in the rat retrosplenial cortex after administration of dizocilpine, and that phosphodiesterase type IV may not play a role in the neurotoxicity of NMDA receptor antagonists such as dizocilpine.

P.6.075 Risperidone in the treatment of neurological disorders

The effects of the microtubule stabilizing agent, deuterium oxide, on in vitro rat antral gastrin release were examined under basal conditions and during stimulation with isobutyl methylxanthine and bombesin plus isobutyl methylxanthine. Basal gastrin release from antral mucosal fragments was unaffected by increasing media concentration of deuterium oxide (12.5 to 75% v/v) during 1 h incubations. Gastrin release stimulated by isobutyl methylxanthine (0.1 mM), a potent inhibitor of phosphodiesterase activity, was inhibited completely by 12.5% deuterium oxide. Bombesin (1 × 10−8 M) in the presence of IBMX (0.1 mM) stimulated gastrin release (29.7 ± 1.9% of total gastrin). This was significantly greater than gastrin released under control conditions and with IBMX alone: 12.0 ± 1.1 (P < 0.001) and 20.2 ± 2.6% of total gastrin (P < 0.02), respectively. Partial inhibition of bombesin-IBMX stimulated gastrin release was achieved with 12.5% and 25% deuterium oxide and stimulation of gastrin release was inhibited completely by 50% deuterium oxide. In contrast to these results, gastrin release stimulated by the calcium ionophore A23187 was not inhibited by 50% deuterium oxide. Additional studies were performed to assess reversibility of the effects of deuterium oxide on stimulated gastrin release. Antral tissue exposed to initial culture medium containing deuterium oxide (50%) and bombesin-IBMX for 60 min was exchanged for medium without deuterium oxide. Restimulation of antral tissue during the second hour of culture resulted in gastrin release that was comparable to that observed in cultures not exposed to deuterium oxide during the first hour of culture. Reversibility of the effects of deuterium oxide suggest that a functional alteration in microtubular function is restored by removal of heavy water from the culture medium. Results of these experiments indicate that deuterium oxide is capable of inhibiting gastrin release stimulated by the peptide hormone bombesin and by the phosphodiesterase inhibitor isobutyl methylxanthine. Furthermore, these results suggest that increased levels of intracellular calcium achieved by the action of ionophore A23187 prevent microtubular stabilization by deuterium oxide.

Role for anions in pulmonary endothelial permeability.

beta-Adrenergic stimulation reduces albumin permeation across pulmonary artery endothelial monolayers and induces changes in cell morphology that are mediated by Cl- flux. We tested the hypothesis that anion-mediated changes in endothelial cells result in changes in endothelial permeability. We measured permeation of radiolabeled albumin across bovine pulmonary arterial endothelial monolayers when the extracellular anion was Cl-, Br-, I-, F-, acetate (Ac-), gluconate (G-), and propionate (Pr-). Permeability to albumin (Palbumin) was calculated before and after addition of 0.2 mM of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), which reduces permeability. In Cl-, the Palbumin was 3.05 +/- 0.86 x 10(-6) cm/s and fell by 70% with the addition of IBMX. The initial Palbumin was lowest for Pr- and Ac-. Initial Palbumin was higher in Br-, I-, G-, and F- than in Cl-. A permeability ratio was calculated to examine the IBMX effect. The greatest IBMX effect was seen when Cl- was the extracellular anion, and the order among halide anions was Cl- > Br- > I- > F-. Although the level of extracellular Ca2+ concentration ([Ca2+]o) varied over a wide range in the anion solutions, [Ca2+]o did not systematically affect endothelial permeability in this system. When Cl- was the extracellular anion, varying [Ca2+]o from 0.2 to 2.8 mM caused a change in initial Palbumin but no change in the IBMX effect. The anion channel blockers 4-acetamido-4'-isothiocyanotostilbene-2, 2'-disulfonic acid (0.25 mM) and anthracene-9-carboxylic acid (0.5 mM) significantly altered initial Palbumin and the IBMX effect. The anion transport blockers bumetanide (0.2 mM) and furosemide (1 mM) had no such effects. We conclude that extracellular anions influence bovine pulmonary arterial endothelial permeability and that the pharmacological profile fits better with the activity of anion channels than with other anion transport processes.

Suppression of drug-induced epileptiform discharges by cyclic AMP in rat hippocampus.

The effect of cyclic adenosine 3',5'-monophosphate (cAMP) on epileptiform activity in rat hippocampal slices was investigated. Bath-applied cAMP reversibly decreased the frequency of extracellularly recorded discharges in the CA3 subfield induced by bethanechol- or theophylline-containing solutions. Because cAMP was presumed to be relatively membrane impermeant, we developed and tested the hypothesis that this cAMP-mediated effect occurred extracellularly through the catabolic conversion of cAMP to 5'-AMP and, in turn, to adenosine, a known inhibitory neuromodulator. Three predictions derived from this catabolic hypothesis were tested. First, blockers of the enzymes involved were predicted to antagonize this effect of cAMP. In contrast, the coapplication of a cAMP-phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), or a 5'-nucleotidase inhibitor, adenosine 5'-[alpha, beta-methylene] diphosphate (AMP-CP), enhanced the cAMP-induced suppressive effect. Second, the nonhydrolyzable cAMP analogs, dibutyryl- and 8-bromo-cAMP, were predicted to be ineffective. Low concentrations (5-40 microM) of these two derivatives, however, also suppressed bethanechol-induced discharges, while, at a higher concentration (100 microM), both analogs increased discharge frequencies. Third, enzymatic catabolism of adenosine was predicted to antagonize cAMP's effect, but coapplying adenosine deaminase (10 U/mL) did not diminish this action. Because these data did not support the catabolic hypothesis, other, as yet undefined, mechanisms must be responsible for the discharge-suppressant effect of cAMP.

Effect of Recombinant Interleukin-6 and Thrombopoietin on Isolated Guinea Pig Bone Marrow Megakaryocyte Protein Phosphorylation and Proplatelet Formation

ABSTRACT: Guinea pig bone marrow megakaryocytes were isolated and cultured on collagen gels to promote proplatelet formation. In control cultures 15.6% of the cells formed proplatelets. Both IL6 and TPO stimulated dose dependent increases in the percent of proplatelet forming cells up to 26.7% at 100 ng/ml IL6 and 26.8% at 100 ng/ml TPO. IL1 and IL3 had no effect on proplatelet formation. IL3 in combination with IL6 and TPO blocked the increase in proplatelet formation observed with IL6 or TPO alone. IL3 was also found to stimulate thymidine incorporation in megakaryocytes. The role of phosphorylation in proplatelet formation was studied using certain inhibitors. The tyrosine kinase inhibitor genestien had no effect on proplatelet formation at concentrations up to 100 μg/ml. The phosphatase inhibitors calyculin A and okadaic acid both inhibited proplatelet formation. The phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) was a potent stimulator of proplatelet formation. Studies on protein phosphorylation revealed that IL6, but not TPO, stimulated phosphorylation of JAK1, JAK2 and MAP kinase. TPO did stimulate tyrosine phosphorylation of Tyk-2. Although IBMX stimulated proplatelet formation, it inhibited phosphorylation of JAKs and MAP kinase. Adhesion of megakaryocytes to collagen gel also inhibited phosphorylation of JAK1 and JAK2, while MAP kinase phosphorylation was unaffected. These data show that IL6 and TPO stimulate megakaryocyte proplatelet formation. In addition, although these cytokines increase phosphorylation of signal transduction proteins in the JAK/STAT pathway, it appears that a different signal transduction pathway regulated by a combination of phosphatase activity and cAMP levels, leads to proplatelet formation.