Cyclic nucleotide content of ciliary and dorsal root ganglia during embryonic development in the chick

The concentration of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) was measured in developing chick ciliary (CG) and dorsal root ganglia (DRG) as a function of embryonic age from day 8 through day 18 using radioimmunoassays. The concentration of cAMP and cGMP increased in both ganglia from day 8 through day 14. cAMP levels were nearly two-fold higher in DRG than in CG. Normalization of the data for ciliary ganglia to the number of cells per ganglion and calculation of their molar concentrations indicates a 48% increase in cGMP and a 3.2-fold increase in cAMP during the developmental process of natural neuronal cell death.     

Developmental changes of cyclic 3′,5′-guanosine and cyclic 3′,5′-adenosine monophosphates in rat skeletal muscle

Cyclic 3′,5′-guanosine monophosphate (cGMP) and cyclic 3′,5′-adenosine monophosphate (cAMP) concentrations were assayed in the gastrocnemius muscles of young rats. The cGMP concentration was highest in neonatal muscles and gradually declined to the adult level at 28 days after birth. A sharp rise in cAMP concentration was observed between Days 7 and 14 concurrently with the period of differentiation of muscle fibers. A relatively sharp decrease in cAMP concentration occurred between Days 21 and 35, reaching the concentration of adult muscle. The reciprocal changes in cGMP and cAMP concentrations appeared to parallel changes in the differentiation of fiber types.     

Brain natriuretic peptide enhances the endothelium-independent cAMP and vasorelaxant responses of calcitonin gene-related peptide in rat aorta

Calcitonin gene-related peptide (CGRP) causes vasorelaxation in rat aorta involving endothelium/nitric oxide (NO)-dependent elevations of both cAMP and cGMP levels. When endothelium is removed, preincubation with exogenous NO uncovers and potentiates direct (endothelium-independent) cAMP elevations and vasorelaxations caused by CGRP. This enhancing effect of NO potentially involves elevation of cGMP and inhibition of Type III (cGMPinhibitable) phosphodiesterase, causing accumulation of cAMP. However, NO may have other actions. The aim of the present study was to determine if brain natriuretic peptide (BNP), which elevates cGMP levels independent of NO, could enhance cAMP accumulations and vasorelaxations induced by CGRP in rat aortic rings denuded of endothelium. When added separately, neither CGRP (100 nM) nor BNP (10 nM) altered cAMP levels. When added in combination, CGRP (100 nM) and BNP (10 nM) significantly elevated cAMP levels (from control of 0.95 ± 0.08 to 1.53 ± 0.09 pmol/mg protein) at 2 min. BNP (10 nM) elevated cGMP levels 10-fold at 2 min and this response was not altered by co-administration of CGRP (100 nM).Pretreatment with BNP at concentrations as low as 1 nM in endothelium-denuded aortic rings greatly enhanced the direct vasorelaxant effects of CGRP (100 nM) (from control of 0% to 57.6 ± 6.8% relaxation of phenylephrineprecontractions). Our findings indicate that BNP enhances direct (endothelium-independent) cAMP elevations and vasorelaxations caused by CGRP in rat aorta, thus supporting the concept that cGMP inhibits cAMP metabolism and enhances CGRP-induced responses in aortic smooth muscle cells.     

Catecholamine metabolism in rat brain following the intracerebroventricular administration of cyclic nucleotides

Summary The dibutyryl analogues of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were administered into the lateral ventricles and catecholamine metabolites were determined in brain 40 min later.Dibutyryl cAMP elevated the level of homovanillic acid in whole brain and dihydroxyphenyl acetic acid levels in striatum, the dopamine-rich part of the limbic system and hemispheres but neither affected the accumulation of 3-methoxytyramine following inhibition of MAO with pargyline nor dopamine and noradrenaline levels. Normetanephrine accumulating after MAO inhibition was elevated markedly by dibutyryl cAMP. Dibutyryl cGMP was without effect on the catecholamine metabolites investigated.Dibutyryl cAMP appears to stimulate dopamine metabolism within dopaminergic nerve endings but does not stimulate dopamine release. Dibutyryl cAMP-induced activation of noradrenaline metabolism, however, appears to coincide with a stimulation of noradrenaline release.     

Cyclic nucleotides in platelets of genetically hypertriglyceridemic and hypertensive rats. Thrombin and nitric oxide responses are unrelated to plasma triglyceride levels

Prague hereditary hypertriglyceridemic (HTG) rats constitute a genetic model of hypertension associated with hyperlipidemia and insulin resistance. Various cell alterations, including changes in membrane dynamics, ion transport, and decreased platelet responses to thrombin have been observed in this strain. As hypertriglyceridemia appears to be associated with reduced endothelium-dependent vasodilation and platelet aggregation, we examined whether triglycerides could modulate cell responsiveness through changes in cyclic nucleotides in platelets of HTG rats. From the age of 6 weeks, these hypertensive animals were subjected for 10 weeks to interventions that modified circulating triglycerides levels (2.17+/-0.09 mmol/l), leading to their reduction (gemfibrozil treatment, 0.87+/-0.05 mmol/l) or elevation (high fructose intake, 3.23+/-0.07 mmol/l). Basal cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) contents were 15% and 48% lower in isolated platelets of HTG rats than in those of Lewis controls. cAMP level was further reduced in HTG rats subjected to high fructose intake. Irrespective of their plasma triglyceride levels, the thrombin-induced increase in platelet cGMP levels present in Lewis rats was absent in platelets of HTG rats. In contrast, no strain- or treatment-related differences were observed in the magnitude or kinetics of cGMP response to exogenous nitric oxide (NO). NO-induced cGMP and cAMP changes were associated in an opposite manner with trimethylamino-diphenylhexatriene (TMA-DPH) anisotropy, a biophysical parameter that reflects the microviscosity of the outer part of the cell membrane. Our results indicate that the attenuation of platelet responsiveness to thrombin in HTG rats represents a strain difference that cannot merely be due to a difference in plasma triglyceride levels. Platelet hyporesponsiveness to agonists such as thrombin in HTG rats cannot be explained by a change in levels of inhibitory cyclic nucleotides, since they were actually found to be low and not high.     

Lowered cAMP and cGMP signalling in the brain during levodopa-induced dyskinesias in hemiparkinsonian rats: new aspects in the pathogenetic mechanisms

Dysregulation of dopamine receptors is thought to underlie levodopa-induced dyskinesias in experimental models of Parkinson's disease. It is unknown whether an imbalance of the second messengers, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), is involved in the alterations of levodopa/dopamine signal transduction. We examined cAMP and cGMP signalling in the interconnected cortico-striatal-pallidal loop at the peak of levodopa-induced dyskinesias in rats with 6-hydroxydopamine lesions in the substantia nigra. In addition, we examined the role of phosphodiesterase (PDE) and the rate of cAMP and cGMP degradation on the severity of levodopa-induced dyskinesias in animals pretreated with PDE inhibitor, zaprinast. Unilateral lesion of substantia nigra led to an increase in cAMP but a decrease in cGMP levels in the ipsilateral basal ganglia. After chronic levodopa treatment, cAMP and cGMP were differentially regulated in eukinetic animals: the cAMP level increased in the cortex and striatum but decreased in the globus pallidus of both hemispheres, whereas the cGMP decreased below baseline levels in the contralateral cortico-striatal-pallidal regions. In dyskinetic animals chronic levodopa treatment led to an absolute decrease in cAMP and cGMP levels in cortico-striatal-pallidal regions of both hemispheres. Pretreatment with zaprinast reduced the severity of levodopa-induced dyskinesias, and partly prevented the decrease in cyclic nucleotides compared with pretreatment with saline-levodopa. In conclusion, using a rat model of hemiparkinsonism, we observed a significant reduction in the levels of cyclic nucleotides in both hemispheres at the peak of levodopa-induced dyskinesias. We propose that such a decrease in cyclic nucleotides may partly result from increased catabolism through PDE overactivity.     

CSF cyclic nucleotides and somatostatin in Parkinson's disease

Concentrations of cyclic adenosine 3',5' monophosphate (cAMP) were significantly lower in parkinsonian patients than in controls, but concentrations of guanosine 3',5' monophosphate (cGMP) were not altered. Both cAMP and cGMP levels were lower in patients with more severe symptoms on the left side of the body. Somatostatin-like immunoreactivity (SLI) was similar in parkinsonian patients and controls. Both cAMP and SLI were significantly related to acetylcholinesterase activity.     

银杏叶提取物抗血小板聚集机制研究

目的 通过体外观察银杏叶提取物(EGB)对血小板磷酸二酯酶(PDE)、核苷酸环化酶[腺苷酸环化酶(AC)、鸟苷酸环化酶(GC)]活性和环核苷酸[环腺苷酸(cAMP)、环鸟苷酸(cGMP)]水平的影响,探讨EGB抗血小板聚集的可能机制.方法 采集2周内未服任何药物的3例健康志愿者肘静脉血(柠檬酸钠抗凝),血小板分离洗涤后分别观察不同浓度EGB对血小板cAMP、cGMP水平及经超声匀浆后分离的PDE、AC、GC活性的影响,并以加同等体积药物溶剂为空白对照.结果 随着EGB浓度的增加,血小板cAMP水平明显增高,PDE3活性明显抑制,变化呈剂量依赖性;大剂量EGB(80 mg/L)对PDE5有轻度抑制作用;不同浓度EGB对cGMP水平和PDE2、AC、GC活性均无明显影响.结论 EGB通过抑制血小板PDE3活性,提升cAMP水平从而起到抗血小板聚集作用.

Abstract：

Objective To investigate the effect of Ginkgo biloba extract (EGB) on changes of activities of platelet phosphodiesterase (PDE) and nucleotide cyclase (adenylate cyclase [AC] and guanylate cyclase [GC]), and levels of cyclic nucleotide (cylic adenosine monophosphate [cAMP] and cyclic guanosine monophosphate [cGMP]), and investigate the mechanism of platelet anti-aggregation with EGB. Methods Blood samples from 3 healthy volunteers, not taken any drugs within 2 weeks of the experiments, were anticoagulated with 3.8% sodium citrate. After isolating and washing, platelet was given various concentrations of EGB, and then applied for measurement of the levels cAMP and cGMP and the activities of PDE, AC and GC isolated from platelet sonic homogenates. Controls were given the same volume of bulk drug solvent. Results EGB dose-dependence was noted: the higher the level of EGB, the higher the cAMP level and the more active the PDE3. EGB at high-dose could slightly inhibit the PDE5 activity. EGB in any dosages could not affect the cGMP level, and activities of PDE2, AC and GC. Conclusion EGB has platelet anti-aggregation effect through inhibiting the PDE3 activity and increasing the cAMP level.     

Production of cAMP by adrenomedullin in human oligodendroglial cell line KG1C: comparison with calcitonin gene-related peptide and amylin.

The actions and the presence of adrenomedullin (AM) were investigated in cultured human oligodendroglial cell line KG1C. AM and AM mRNA were detected in KG1C cells by immunohistochemistry and RT-PCR. mRNAs for calcitonin receptor-like receptor (CRLR) and receptor-activity-modifying proteins (RAMPs) 1, 2 and 3 but not for calcitonin receptors were detected in the cells, while mRNAs for CRLR, calcitonin receptors and all RAMPs were detected in the human cerebellum. Application of AM resulted in time- and concentration-dependent increases in the cAMP level of KG1C cells. Calcitonin gene-related peptide (CGRP) and amylin, peptides structurally related to AM, also increased cAMP. The potencies for the cAMP production of the three peptides were CGRP > or =AM > amylin with EC(50) of 8, 18, 90 nM, respectively. The responses induced by AM were strongly inhibited by the CGRP(1) receptor antagonist human CGRP(8-37), and inhibited also by the AM receptor antagonist human AM(22-52). In contrast, the responses induced by CGRP or amylin were inhibited only by CGRP(8-37) and not by AM(22-52). The responses induced by all three peptides were unaffected by the amylin receptor antagonist human amylin(8-37). The CGRP(2) receptor agonist human [Cys(Acm)(2,7)]CGRP significantly increased the cAMP level but the increase was smaller than that caused by CGRP. This increase in cAMP was unaffected by CGRP(8-37), AM(22-52) or by amylin(8-37). These results suggest that in KG1C cells, AM increases cAMP through AM and CGRP(1) receptors, whereas CGRP does so through CGRP(1) and CGRP(2) receptors, and amylin exerts its effects through CGRP(1) receptors. Collectively, these findings imply that AM released from oligodendroglial cells may play a role in the regulation of oligodendrocytes via autocrine/paracrine through AM receptors and CGRP(1) receptors.     

Adrenomedullin-2/Intermedin Induces cAMP Accumulation in Dissociated Rat Spinal Cord Cells: Evidence for the Existence of a Distinct Class of Sites of Action

Adrenomedullin-2/intermedin is structurally related to the calcitonin family of peptides, which includes calcitonin gene-related peptide (CGRP), adrenomedullin, and amylin. We recently reported that CGRP and adrenomedullin act through distinct receptors to induce cyclic adenosine monophosphate (cAMP) accumulation in dispersed cells from embryonic rat spinal cord. Here, we investigated the apparent affinity and efficacy of adrenomedullin-2/intermedin for these receptors. Adrenomedullin-2/intermedin competed with [(125)I]-CGRP for binding to specific embryonic spinal cord cells with a pIC(50) of 9.73 +/- 0.06. Interestingly, adrenomedullin-2/intermedin competed for specific [(125)I]-adrenomedullin binding in a biphasic manner with pIC(50) of 9.03 +/- 0.22 and 6.45 +/- 0.24, respectively. Cellular levels of cAMP were increased by adrenomedullin-2/intermedin (pEC(50) 7.84 +/- 0.08) when cells were exposed to this peptide for 10 min at 37 degrees C. This effect was partially inhibited by the non-peptide antagonist BIBN4096BS (pA(2) 6.56 +/- 0.12), the adrenomedullin antagonist hAM(22-52) (pA(2) 6.36 +/- 0.30), and the adrenomedullin/CGRP antagonist CGRP(8-37) (pA(2) 7.24 +/- 0.60). More interestingly, a highly significant effect of adrenomedullin-2/intermedin on cAMP accumulation (pEC(50) 7.3 +/- 0.14) was still observed even in the presence of a mixture of saturating concentrations of BIBN4096BS, hAM(22-52), and the amylin antagonist AC187. Taken together, these data provide evidence for the possible existence of a distinct class of receptor sites for adrenomedullin-2/intermedin in embryonic rat spinal cord cells.     

Dysregulation of Cerebellar Adrenomedullin Signaling During Hypertension

Adrenomedullin (AM) is a peptide involved in blood pressure regulation. AM activates three different receptors, the AM type 1 (AM1), type 2 (AM2), and calcitonin gene-related peptide 1 (CGRP1) receptors. AM triggers several signaling pathways such as adenylyl cyclase (AC), guanylyl cyclase (GC), and extracellular signal-regulated kinases (ERK) and modulates reactive oxygen species (ROS) metabolism. Cerebellar AM, AM-binding sites, and its receptor components are altered during hypertension, although it is unknown if these alterations are associated with changes in AM signaling. Thus, we assessed AM signaling pathways in cerebellar vermis of 16-week-old Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). Animals were sacrificed by decapitation, and cerebellar vermis was microdissected under stereomicroscopic control. Tissue was stimulated in vitro with AM. Then the production of cyclic guanosine monophosphate (cGMP), nitric oxide (NO) and cyclic adenosine monophosphate (cAMP) were assessed along with ERK1/2 activation and three antioxidant enzymes’ activity: glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD). Our findings demonstrate that in the cerebellar vermis of normotensive rats, AM increases cGMP, NO, cAMP production, and ERK1/2 phosphorylation, while decreases basal antioxidant enzyme activity. In addition, AM antagonizes angiotensin II (ANG II)-induced increment of antioxidant enzyme activity. Hypertension blunts AM-induced cGMP and NO production and AM-induced decrease of antioxidant enzyme activity. Meanwhile, AM-induced effects on cAMP production, ERK1/2 activation, and AM-ANG II antagonism were not altered in SHR rats. Our results support a dysregulation of several AM signaling pathways during hypertension in cerebellar vermis.     

Cyclic nucleotide phosphodiesterase isozymes in neuroblastoma cells

Adenosine 3',5'-cyclic monophosphate (cAMP) content of neurons is determined not only by the rate of synthesis but also by the rate of hydrolysis by cyclic nucleotide phosphodiesterases. Multiple forms of cyclic nucleotide phosphodiesterase exist in brain and other tissues, and these may be regulated by various hormones and neuromodulators. The present study examines this regulation in a cloned line of neuroblastoma cells (N18TG2). A biphasic Lineweaver-Burk plot of cAMP hydrolysis revealed two Kms approximating 5 and 25 microM. Lineweaver-Burk plots of cGMP hydrolysis were linear over a range of 1 microM to 1 mM and exhibited a Km of 37 microM. Neither cAMP nor cGMP competed for hydrolysis of the alternative cyclic nucleotide. No evidence for an allosteric activation of cAMP phosphodiesterase by cGMP was found. Calcium regulation of phosphodiesterase was not found in spite of preparation of the cell extract with several protease inhibitors, and addition of exogenous calmodulin. No effect of calmodulin antagonists (calmidazolium, W7, or trifluoperazine) was observed in vitro or in situ. Growth of the cells in the presence of 200 nM 3,5,3'-triiodothyronine (T3) resulted in an increased hydrolysis of cAMP but of cGMP. This increase was attributed to an increase in Vmax with no change in either high or low Km. This response was blocked by cycloheximide, suggesting that the thyroid hormone effect requires protein synthesis. The thyroid hormone response in neuroblastoma cells is compared with the results of other studies of thyroid hormone effects on phosphodiesterase in other tissues in vivo.     

Change in Intracellular Cyclic Nucleotide Content Accompanies Relaxation of the Isolated Canine Internal Anal Sphincter

Changes in cyclic nucleotide content arc associated with relaxation of lower esophageal sphincter (LES) smooth muscle. Although agents that increase cyclic AMP (cAMP) or cyclic CMP (cGMP) can relax the LES, relaxation produced by activation of enteric neurons is associated with, an increase in intracellular cGMP. To evaluate these changes in another sphincteric area of the gut, ice determined the effects of electrical field stimulation (EFS) (65 V, I ms) and of several relaxant agents that alter cyclic nucleotide content in isolated strips of canine internal anal sphincter. Each strip was contracted with norepinephrine (3 μM). During maximum contraction, tissues were relaxed by EFS or by addition of relaxant agents. EFS produced, a frequency-related relaxation, accompanied by a significant, increase, in cGMP; however, cAMP increased only slightly at the maximum frequency tested (8 Hz). Both EFS-induced relaxation and increased cGMP content were blocked 1 μM tetrodotoxin. Cumulative addition of sodium nitroprusside, forskolin, 8-bromoguanosine 3',5'-cylic monophosphate, or 8-bromoadenosine 3',5'-cyclic monophosphate relaxed the internal anal sphincter in a concentration-dependent manner. Sodium nitroprusside-induced relaxations were accompanied by concentration-dependent increases in cGMP content, whereas forskolin-induced relaxations were accompanied by concentration-dependent increases in cAMP content. In conclusion, both cAMP and cCMP appear to be intracellular mediators of relaxation in the canine internal anal sphincter. In addition, relaxation produced by activation of intrinsic inhibitory neurons is associated with an elevation of intracellular cGMP. Together with our previous findings in the LES, these results suggest that cCMP may be the intracellular mediator of neuronally induced, relaxation of gut sphincteric regions.     

Effect of intracellular injection of cyclic adenosine monophosphate on the calcium current in identified snail neurons

The effect of intracellular cyclic adenosine monophosphate (cAMP) injection and extracellular theophylline application on calcium current were investigated in Helix RPa3 and LPa3 neurons. It was found that iontophoretic cAMP injection (current 10-35 nA, duration about 1 min) caused a decrease in the amplitude of calcium current which restored to the initial level following the cessation of injection. Its current-voltage characteristic was not displaced along the potential axis in the presence of cAMP injection indicating that the calcium current decrease was due to the fall of maximum calcium conductance. Extracellular theophylline application in concentration of 1 mM/1 caused a decrease in the calcium current amplitude by 50-75% from the initial level. The hypothesis is discussed about the participation of cytoplasmic factors in regulation of calcium current in mollusc neurons.     

Adenosine increases human platelet levels of cGMP through nitric oxide: possible role in its antiaggregating effect

Adenosine is an endogenous antiaggregating substance that influences the platelet responses through specific A-type receptors that activate adenylate cyclase increasing the levels of 3',5'-cyclic adenosine monophosphate (cAMP). In this study, we investigated whether adenosine can also influence the levels of 3',5'-cyclic guanosine monophosphate (cGMP) and decrease the aggregating response of human platelets to adenosine-5-diphosphate (ADP) through this nucleotide. In platelet samples from healthy volunteers, we evaluated the effect of adenosine on ADP-induced aggregation and cyclic nucleotide synthesis. Some experiments were repeated in the presence of dipyridamole (inhibitor of adenosine uptake and phosphodiesterase activity), N(G)-monomethyl-L-arginine (L-NMMA, nitric synthase inhibitor), ionomycin (calcium ionophore), and ambroxol (2-amino-3,5-dibromo-N-[trans-4-hydroxycyclohexyl]benzylamine, inhibitor of nitric oxide (NO)-dependent activation of guanylate cyclase). Adenosine decreased the response to ADP in a concentration-dependent way (analysis of variance, ANOVA: P<.0001): cAMP levels increased from 30.0 +/- 2.0 (control) to 46.0 +/- 3.0 pmol/10(9) platelets (in the presence of 15 mumol/l adenosine) and cGMP levels increased from 5.6 +/- 1.0 (control) to 10.9 +/- 2.0 pmol/10(9) platelets (in the presence of 15 mumol/l adenosine). Also, nucleotide levels measured at the end of aggregation were higher in platelet samples exposed to adenosine than in controls. Dipyridamole at 40 mumol/l slightly increased adenosine's effects on both nucleotides. L-NMMA blunted the effect of adenosine on cGMP both in unstimulated samples and in aggregated platelets without any effect on cAMP synthesis. Platelet exposure to L-NMMA and ambroxol partially prevented adenosine's effect on ADP-induced aggregation. In conclusion, adenosine, which enhances intraplatelet cAMP levels, was determined to also cause an increase in cGMP concentrations through a mechanism that involves NO synthesis. This effect plays a direct role in the adenosine-induced antiaggregation.     

Calcitonin Gene-related Peptide Stimulates the Induction of c-fos Gene Expression in Rat Astrocyte Cultures

The action of calcitonin gene-related pepide (CGRP) was studied on c-fos gene expression in rat astrocyte cultures. A strong and transient increase in c-fos mRNA was observed in cultured astrocytes after treatment with CGRP. Quantitative Northern blot analysis revealed an increase of c-fos mRNA within 15 min, a peak after 30 min with a 10 - 15 fold increase over unstimulated cells and a subsequent decline. Induction of the c-fos gene by CGRP was concentration-dependent, half maximal stimulation of c-fos mRNA being obtained with 100 nM CGRP. The CGRP effect appeared to be mediated by a CGRP receptor and calcitonin was found to mimic only weakly the action of CGRP on cultured astrocytes. Calcitonin transiently induced c-fos gene expression with a similar time course to CGRP, but its effect was much less pronounced. Agents affecting the intracellular cyclic AMP level, forskolin and Ro 20-1724, stimulated c-fos mRNA in a strong and transient fashion with a temporal sequence similar to the response to CGRP. Further, the phosphodiesterase inhibitor Ro 20-1724 potentiated the action of CGRP on c-fos mRNA induction, suggesting a role for cyclic AMP in the action of CGRP. The present results indicate that CGRP may play a physiological role as a regulator of astrocyte gene expression.     

Restoration of potential-dependent anti-brain antibody-inhibited calcium current by cyclic adenosine monophosphate

A two-microelectrode voltage clamp method was used for measuring the voltage-dependent calcium current (ICa) in isolated nonidentified snail neurons. Intracellular injection of cyclic adenosine monophosphate (cAMP, 10nA, 5 min) and extracellular application of dibutyryl-cAMP (dcAMP, I mmol/l, 10-20 min) did not change the normal ICa or reversibly decreased the ICa amplitude by 10-20%. Extracellular application of antibodies against S-100 proteins resulted in a Ca2+-dependent inactivation of the ICa: the ICa amplitude dropped to 15 +/- 12% of its initial level; cAMP and dcAMP restored an amplitude of the inhibited ICa up to 50 +/- 11%. It is shown that the effect of cAMP on ICa of intact neurons depends on the cytoplasmic Ca2+-level.     

Expression of olfactory-type cyclic nucleotide-gated channels in rat cortical astrocytes

Cyclic nucleotide-gated (CNG) channels are nonselective cation channels activated by cyclic AMP (cAMP) or cyclic GMP (cGMP). They were originally identified in retinal and olfactory receptors, but evidence has also emerged for their expression in several mammalian brain areas. Because cGMP and cAMP control important aspects of glial cell physiology, we wondered whether CNG channels are expressed in astrocytes, the most functionally relevant glial cells in the CNS. Immunoblot and immunofluorescence experiments demonstrated expression of the CNG channel olfactory-type A subunit, CNGA2, in cultured rat cortical astrocytes. In patch-clamp experiments, currents elicited in these cells by voltage ramps from -100 to +100 mV in the presence of the cGMP analogue, dB-cGMP, were significantly reduced by the CNG channel blockers, L-cis-diltiazem (LCD) and Cd(2+) . The reversal potentials of the LCD- and Cd(2+) -sensitive currents were more positive than that of K(+) , as expected for a mixed cation current. Noninactivating, voltage-independent currents were also elicited by extracellular application of the membrane permeant cGMP analogue, 8-Br-cGMP. These effects were blocked by LCD and were mimicked by natriuretic peptide receptor activation and inhibition of phosphodiesterase activity. Voltage-independent, LCD-sensitive currents were also elicited by 8-Br-cGMP in astrocytes of hippocampal and neocortical brain slices. Immunohistochemistry confirmed a broad distribution of CNG channels in astrocytes of the rat forebrain, midbrain, and hindbrain. These findings suggest that CNG channels are downstream targets of cyclic nucleotides in astrocytes, and they may be involved in the glial-mediated regulation of CNS functions under physiological and pathological conditions.     

急性脑梗死患者血小板磷酸二酯酶活性变化及共影响因素研究

目的 观察急性脑梗死患者血小板磷酸二酯酶(PDE)亚型活性变化,探讨影响其因素.方法 对30例急性脑梗死患者分别于发病第1、4、8、15天检测血小板PDE活性、环核苷酸含量、[Ca2+]I水平,并以10例年龄相当的健康体检者为正常对照.结果 与对照组比较,急性脑梗死患者发病第1、4、8天时血小板PDE2、PDE3亚型活性降低,环腺苷酸(cAMP)含量降低,胞浆[Ca2+]I水平升高,差异均有统计学意义(P<0.05);PDE5亚型活性及环鸟苷酸(cGMP)含量则无明显变化.相关分析显示,PDE2活性、PDE3活性与cAMP含量、[Ca2+]I水平均无相关关系,cAMP含量与[Ca2+]I水平间呈负相关关系(R2=0.921,P<0.05).结论 脑梗死急性期血小板胞浆cAMP含量降低,[Ca2+]I水平升高,血小板处于活化状态;血小板PDE2、PDE3通过降低活性、减轻cAMP降低程度、抑制血小板活化而在脑梗死急性期发挥保护作用.     

Inactivation of platelet PDE2 by an affinity label: 8-[(4-bromo-2, 3-dioxobutyl)thio]cAMP

Cyclic GMP-stimulated cyclic nucleotide phosphodiesterase (PDE2) is the second most abundant of this class of enzymes in platelets. PDE2 probably plays an important role in the regulation of elevated intracellular concentrations of cAMP and cGMP in platelets inhibited by prostacyclin and/or nitric oxide. The cAMP and cGMP PDEs have catalytic domains with 28-40% identity, but vary in their substrate specificity and affinity. As a first step toward the goal of identifying important amino acids in the substrate binding site pocket, we have employed the affinity analog 8-[(4-bromo-2, 3-dioxobutyl)thio]adenosine-3'5' cyclic monophosphate (8-BDB-TcAMP) to inactivate PDE2 and observe the pattern of protection by substrates and their products. Incubation of purified platelet PDE2 with 8-BDB-TcAMP (2-10 mM) resulted in a time-dependent, irreversible inactivation of the enzyme with a second-order rate constant of 0.013 min(-1) mM(-1). Both substrates, cAMP and cGMP, as well as the products of hydrolysis by PDE2, AMP and GMP, exhibited concentration-dependent protection against inhibition by 8-BDB-TcAMP, but no protection was noted with ADP or ATP, which are not hydrolyzed by the enzyme. This compound, 8-BDB-TcAMP, and similar affinity reagents should prove useful in delineating amino acids in the active site of PDE2.