Regulation of 3', 5'-cyclic guanosine monophosphate content in deep cerebellar nuclei.

The 3',5'-cyclic guanosine monophosphate (cGMP) content of deep cerebellar nuclei is decreased following activation of γ-aminobutyrate (GABA) receptors by muscimol and diazepam and is increased following a reduction of GABA content caused by isoniazid. Since previous work has shown that, in deep cerebellar nuclei, GABA receptor stimulation decreases the firing rate of neurones (Ito, Yoshida, Obata, Kawai &;Udo, 1970), it appears that the cGMP content of deep cerebellar nuclei may be an index of the activity of these neurones. Neither the destruction nor the specific activation of climbing fibers changes the cGMP content of deep cerebellar nuclei. Morphine and haloperidol decrease the cGMP content of deep cerebellar nuclei without changing the activity of enzymes that metabolize cGMP in cell free preparations or by activating GABA receptors or changing GABA content. Thus, it is tempting to conclude that a decrease in mossy fiber activity could mediate the decrease in the cGMP content of deep cerebellar nuclei induced by morphine and haloperidol injected parenterally.     

The Effect of Renal Transplantation on Plasma Atrial Natriuretic Peptide

The changes in plasma atrial natriuretic peptide (ANP) werestudied in four adult patients after cadaveric renal transplantation.In three patients who achieved good renal function, the correctionof volume overload, as reflected by reduction in weight andright atrial pressure, was associated with a steady fall inplasma ANP and a parallel decrease in both fractional excretionof sodium and plasma cyclic guanosine monophosphate. The fourthpatient. with severe acute rejection, developed severe peripheraloedema, and fractional sodium excretion remained low despitehigh values of ANP.     

Cyclic AMP and growth regulation in rat glioma cells in tissue culture

Peripheral neuropathy accompanied by decreased axonal conduction velocity is a common complication of diabetes. Skeletal muscle weakness and wasting also occur in the severe, uncontrolled form of the disease. For these reasons, it has been hypothesized that diabetes may cause denervation-like changes in skeletal muscle. In the present study, sarcolemmal membranes were characterized in control, early, and late stages of streptozotocin diabetes. After 3 weeks, axonal conduction velocities decreased 25% and muscle weight decreased 50% in diabetic rats. No significant changes were observed in Na⁺,K⁺ (Mg²⁺)-ATPase or adenylate cyclase activities during diabetes. The number of β-adrenergic receptor sites, however, increased from 0.39 pmol/mg in control sarcolemma to 0.46 and 0.58 pmol/mg after 6 and 18 days of diabetes, respectively, with no change in the affinity constant. Membrane phospholipid and polypeptide compositions were unaltered during diabetes, whereas the cholesterol content increased 23% in 18-day diabetic sarcolemma. There was also a 2.5-fold increase in glycosylation of a 72,000-molecular weight glycopeptide after 18 days of diabetes. Previous studies showed that denervation results in a decrease in sodium fluoride- and catecholamine-stimulated adenylate cyclase activities, an increase in Na⁺,K⁺ (Mg²⁺)-ATPase activity, no change in the number or affinity of β-adrenergic receptor sites, and no change in membrane glycosylation (1977. Exp. Neurol.56: 102–114). These findings demonstrate that diabetic and denervated skeletal muscle sarcolemma are distinctly different with respect to a number of their biochemical properties.     

Cholinergic agonists and dibutyryl cyclic guanosine monophosphate inhibit the norepinephrine-induced accumulation of cyclic adenosine monophosphate in the rat cerebral cortex

Addition of cholinergic agonists, namely carbamylcholine (carbachol), acetylcholine, eserine, eserine plus acetylcholine and eserine plus choline chloride, and dibutyryl cyclic guanosine moue-phosphate inhibited the norepinephrine-induced accumulation of cyclic adenosine monophosphate in incubated slices of rat cerebral cortex. Methacholine was ineffective and atropine did not overcome the action of carbachol on the stimulation of cyclic adenosine monophosphate synthesis by norepinephrine. Carbachol stimulated the production of cyclic guanosine monophosphate in the tissue slices white norepinephrine did not influence cyclic guanosine monophosphate levels to a significant degree.The data are in keeping with the ‘Yin-Yang’ hypothesis in which under particular situations the intracellular levels of cyclic guanosine monophosphate may modulate cyclic adenosine monophosphate concentrations.     

Adenylate cyclase responses in rat brain after unilateral postnatal X-irradiation

When unilateral x-irradiation was directed to the area of the posterior cerebral cortex overlying the hippocampus of the neonatal rat, histologic damage was observed in the hippocampus (previous observations), pia-arachnoid (cellular proliferation), and retina (two layers of bipolar cells and damage to visual receptor area and diminished number of ganglion cells). Light microscopy did not reveal any remarkable cellular alterations in parenchymal or capillary tissue of the posterior cerebrum. The most prominent deficits in norepinephrine sensitivity of adenylate cyclase occurred in the pia-arachnoid > hippocampus > posterior cerebrum. The associated capillaries were unchanged and in the retina the enzyme displayed an enhanced sensitivity to dopamine.     

亚硒酸钠对HL—60细胞生长,分化和环核苷酸水平的影响

研究亚硒酸钠对人急性白血病细胞（ＨＬ－６０细胞，ＡＭＬ－Ｍ２型白血病细胞）生长、分化及胞内环核苷酸水平的影响。方法ＨＬ－６０细胞用ＲＰＭＩ１６４０培养液培养，对比研究加与不加亚硒酸钠的差别，环磷酸腺苷（ｃＡＭＰ）、环磷酸鸟苷（ｃＧＭＰ）水平的测定用放射免疫分析法。结果１．５×１０－５ｍｏｌ／Ｌ或３．０×１０－５ｍｏｌ／Ｌ的亚硒酸钠能显著抑制ＨＬ－６０细胞的生长，且呈剂量依赖性，１．５×１０－５ｍｏｌ／Ｌ的亚硒酸钠能促进ＨＬ－６０细胞的分化，对静息的胞内ｃＧＭＰ水平有明显的降低作用，对照组（０．０７±０．０２９）ｐｍｏｌ／１０６ｃｅｌｓ，３０ｍｉｎ，亚硒酸钠组（０．０６±０．０１７）ｐｍｏｌ／１０６ｃｅｌｓ，３０ｍｉｎ（Ｐ＜０．０５）。对肾上腺素刺激的胞内ｃＧＭＰ水平也有明显的降低作用，对照组（０．０７±０．０１８）ｐｍｏｌ／１０６ｃｅｌｓ，３０ｍｉｎ，亚硒酸钠组（０．０５±０．０１４）ｐｍｏｌ／１０６ｃｅｌｓ，３０ｍｉｎ（Ｐ＜０．０５）。结论亚硒酸钠的抑瘤作用可能与降低细胞内ｃＧＭＰ水平有关。     

Cellular pharmacokinetics and pharmacodynamics of dipyridamole in vascular smooth muscle cells

Hemodialysis arteriovenous grafts are often plagued by stenosis at the vein-graft anastomosis, which is due to the proliferation of venous smooth muscle cells (SMCs). Perivascular delivery of dipyridamole, a potent antiproliferative agent, has been proposed for the prevention of graft stenosis. In order to develop an optimal delivery system for dipyridamole, we examined its pharmacokinetics and pharmacodynamics in human and porcine venous and arterial SMCs in vitro. SMCs were incubated with dipyridamole for various durations, and visualized for the uptake and release by fluorescence microscopy, which were further quantified by fluorospectrometry. The antiproliferative effect of dipyridamole was examined by cell counting or the methylthiazoletetrazolium (MTT) dye-reduction assay. Cytotoxicity was examined by the lactate dehydrogenase (LDH)-release assay. The kinetics of dipyridamole transport through the cell membrane was compatible with a passive diffusion mechanism. Dipyridamole inhibited SMC proliferation in a dose-dependent manner and was more effective in venous than arterial cells in both species. The inhibition was completely reversible at 15microg/ml upon drug removal from the medium. At 25microg/ml, however, the effect was partially irreversible, which might be attributed to the cytotoxicity of dipyridamole. These data support the need for sustained delivery of dipyridamole to achieve the long-term inhibition of SMC proliferation in the prevention of stenosis since SMCs are continuously stimulated at the anastomosis of hemodialysis arteriovenous grafts.     

Cyclic nucleotide metabolism in mouse brain during seizures induced by bicuculline or dibutyryl cyclic guanosine monophosphate

In adult male C57 black mice, intracortical injection of bicuculline (10 μg) elevated cGMP concentrations and produced contralateral clonic seizures. After intraperitoneal injection, bicuculline (10 mg/kg) elevated cGMP concentrations at the earliest time at which paroxysmal EEG activity could be detected. Changes in cAMP concentration followed those events. Further elevations of cGMP concentrations (and now of cAMP as well) were observed during clonic and tonic seizures. Intracortical injections of dibutyryl cGMP produced behavioral and EEG seizures. These data suggest that cGMP plays an important role in bicuculline seizures.     

Up-regulation of opiate receptors by opiate antagonists in neuroblastoma-glioma cell culture: The possibility of interaction with guanosine triphosphate-binding proteins

Neuroblastoma-glioma NG108-15 cells that were cultured for 48 h with the opiate antagonist, naloxone, respond to the guanosine 5′-triphosphate (GTP) analogue guanosine 5′-[β,γ-imido]-triphosphate (GMP-PNP) in the binding assay as the control, non-treated, cells. This was observed when the guanyl nucleotide was tested in the presence or absence of sodium chloride and also after subcellular fractionation of the membranes on a sucrose gradient which separated between two receptor-containing fractions. The findings suggest that the increase in δ type enkephalin receptors in naloxone-treated NG108-15 cells does not reflect an alteration in the interaction between the receptor and the adenylate cyclase-GTP-binding protein system.     

Activation of PDE2 and PDE5 by specific GAF ligands: delayed activation of PDE5

BACKGROUND AND PURPOSE

By controlling intracellular cyclic nucleotide levels, phosphodiesterases (PDE) serve important functions within various signalling pathways. The PDE2 and PDE5 families are allosterically activated by their substrate cGMP via regulatory so-called GAF domains. Here, we set out to identify synthetic ligands for the GAF domains of PDE2 and PDE5.

EXPERIMENTAL APPROACH

Using fluorophore-tagged, isolated GAF domains of PDE2 and PDE5, promising cGMP analogues were selected. Subsequently, the effects of these analogues on the enzymatic activity of PDE2 and PDE5 were analysed.

KEY RESULTS

The PDE2 ligands identified, 5,6-DM-cBIMP and 5,6-DCl-cBIMP, caused pronounced, up to 40-fold increases of the cAMP- and cGMP-hydrolysing activities of PDE2. The ligand for the GAF domains of PDE5, 8-Br-cGMP, elicited a 20-fold GAF-dependent activation and moreover revealed a time-dependent increase in PDE5 activity that occurred independently of a GAF ligand. Although GAF-dependent PDE5 activation was fast at high ligand concentrations, it was slow at physiologically relevant cGMP concentrations; PDE5 reached its final catalytic rates at 1 µM cGMP after approximately 10 min.

CONCLUSIONS AND IMPLICATIONS

We conclude that the delayed activation of PDE5 is required to shape biphasic, spike-like cGMP signals. Phosphorylation of PDE5 further enhances activity and conserves PDE5 activation, thereby enabling PDE5 to act as a molecular memory balancing cGMP responses to nitric oxide or natriuretic peptide signals.