Distribution and regulation of cyclic nucleotide levels in cerebellum, in vivo.

Abstract— In mouse cerebellum, in vivo. cyclic GMP levels are 7 pmol/mg protein in the vermis and 40% lower in the hemispheres, whereas cyclic AMP levels are 7 9 pmol/mg protein in both regions. In the vermis. most of the cyclic GMP is contained in the molecular layer; cyclic AMP levels are highest in the granular layer. Amphetamine, harmaline. pentylenetetrazol and physical shaking elevate, and diazepam and reserpine depress levels of cyclic GMP in both vermis and hemispheres. Oxotremorine and atropine, respectively, increase and decrease cyclic GMP levels only in vermis. Regardless of the agent used, most of the change (67 89%) in cyclic GMP levels occurs in the molecular layer of the vermis; the remainder occurs in the granular layer. Of the drugs tested, only pentylenetetrazol affects cyclic AMP levels, and this drug increases cyclic AMP levels in both vermis and hemispheres and causes equal elevations in the molecular and granular layers of the vermis. In incubated slices of mouse cerebellum, none of the drugs produces changes in cyclic nucleotide levels which are similar to those in vivo. These data indicate that many drugs and conditions that alter cyclic GMP levels in cerebellum act via a common, but indirect, process. We suggest that cyclic GMP levels in cerebellum are regulated by the activity of both the climbing fiber and mossy fiber cerebellar afferent systems. Increased activity in these afferent pathways causes elevation of cyclic GMP levels in Purkinje cells and perhaps in other cells; decreased activity leads to depressed cyclic GMP levels.     

Morphological and functional studies on the serotoninergic innervation of the inferior olive

The inferior olive of the cat has, with fluorescence histochemistry, been shown to receive a rich serotoninergic innervation. The distribution of this innervation agrees with the topography of spinal afferent termination as well as the olivo-cerebellar climbing fiber projection. This indicates that different olivary compartments are under different serotoninergic influence. The serotoninergic innervation of the dorsal accessory nucleus (DAO) of the inferior olive of the rat has been identified with electron microscopic radioautography after labelling with 3H-serotonin. The serotoninergic varicosities contain microcanaliculi, tubular-vesicular organelles and large granular vesicles. Few of the serotoninergic varicosities engage in typical synaptic junctions. However, non-junctional varicosities often display other ultrastructural indications of polarity and directed transmitted release. Electrophysiological results indicate that the harmaline-induced tremor, as well as the tremor component of the "serotonin-syndrome", depends on the serotoninergic innervation of the inferior olive. Thus, the sensitivity of different olivary compartments to the induction of rhythmic, synchronous activity by harmaline parallels the distribution of serotoninergic innervation. Neurotoxic destruction of the serotoninergic innervation leads to decreased sensitivity to harmaline. Further, the serotonin receptor agonist 5-methoxy-N,N-dimethyltryptamine, as well as monoamine oxidase inhibition + L-tryptophan loading, leads to rhythmic mass climbing fiber activity in the cerebellum and whole body tremor. A neuromodulatory effect of serotonin on the olivary action potential mechanisms is proposed.     

The accumulation of cyclic AMP and cyclic GMP in guinea pig brain slices: Effect of calcium ions,norepinephrine and adenosine

The effect of Ca²⁺ and putative neurotransmitters on formation of cyclic AMP and cyclic GMP has been studied in incubated slices of brain tissue. Cyclic AMP levels in cerebellar slices after about 90 min of incubation ranged from 10 pmol/mg protein in rabbit, to 25 in guinea pig, to 50 in mouse and 200 in rat. Cyclic GMP levels in the same four species showed no correlation with cyclic AMP levels and were, respectively, 1.3, 20, 5 and 30 pmol/mg protein. The absence of calcium during the prolonged incubation of cerebellar slices had little effect on final levels of cyclic AMP, while markedly decreasing final levels of cyclic GMP. Reintroduction of Ca²⁺ resulted in a rapid increase in cerebellar levels of cyclic GMP which was most pronounced for guinea pig where levels increased nearly 7-fold within 5 min. Prolonged incubation of guinea pig cerebral cortical slices in calcium-free medium greatly elevated cyclic AMP levels apparently through enhanced formation of adenosine, while having little effect on final levels of cyclic GMP. Norepinephrine and adenosine elicited accumulations of cyclic AMP and cyclic GMP in both guinea pig cerebral cortical and cerebellar slices. Glutamate, γ-aminobutyrate, glycine, carbachol, and phenylephrine at concentrations of 1 mM or less had little or noe effect on cyclic nucleotide levels in guinea pig cerebellar slices. Prostaglandin E₁ and histamine slightly increased cerebellar levels of cyclic AMP. Isoproterenol increased both cyclic AMP and cyclic GMP. The accumulation of cyclic AMP and cyclic GMP elicited by norepinephrine in cerebellar slices appeared, baed on dose vs. response curves, agonist-antaganonist relationships and calcium dependency, to involve in both cases activation of a similar set of ß-adrenergic receptors. In cerebellar slices accumulations of cyclic AMP and cyclic GMP elicted by norepinephrine and by a depolarizing agent, veratridine, were strongly dependent on the presence of calcium. The stimulatory effects of adenosine on cyclic AMP and cyclic GMP formation were antagonized by theophylline. The lack of correlations between levels of cyclic AMP and cyclic GMP under the various conditions suggested independent activation of cyclic AMP- and cyclic GMP-generating systems in guinea pig cerebellar slices by interactions with Ca²⁺, norephinephrine and adenosine.     

The Nitric Oxide-Cyclic GMP Pathway Plays an Essential Role in Both Promoting Cell Survival of Cerebellar Granule Cells in Culture and Protecting the Cells Against Ethanol Neurotoxicity

Abstract: NMDA has two beneficial effects on primary neuronal cultures of cerebellar granule cells (CGCs) established from 10-day-old rat pups. First, NMDA is neurotrophic and will enhance survival of CGCs in culture in the absence of ethanol. Second, ethanol exposure will induce cell death in CGC cultures, and NMDA can lessen this ethanol-induced cell loss, i.e., NMDA is neuroprotective. Because NMDA can stimulate production of nitric oxide (NO), which can in turn enhance synthesis of cyclic GMP, this study tested the hypothesis that the NO-cyclic GMP pathway is essential for NMDA-mediated neurotrophism and neuroprotection. Inhibiting the synthesis of NO with N ^G-nitro- l -arginine methyl ester eliminated both the NMDA-mediated neurotrophic and neuroprotective effects. Similarly, inhibiting production of cyclic GMP with the agent LY83583 also abolished these effects. The NO generator 2,2'-(hydroxynitrosohydrazono)bisethanamine produced neurotrophic and neuroprotective effects that were similar to those induced by NMDA. Also, 8-bromo-cyclic GMP produced neurotrophic and neuroprotective effects that were quite similar to the effects produced by NMDA. In conclusion, NMDA enhances survival of cerebellar granule cells and protects the cells against ethanol-induced cell death by a mechanism(s) that involves the NO-cyclic GMP pathway.     

N-Methyl-D-Aspartate Receptors and Ethanol: Inhibition of Calcium Flux and Cyclic GMP Production

Measurements of calcium uptake and cyclic GMP production by cerebellar granule cells grown in primary culture demonstrated that ethanol preferentially inhibited N-methyl-D-aspartate (NMDA) receptor-gated cation channel function. Concentrations of ethanol as low as 10 mM inhibited NMDA-stimulated Ca2+ uptake by greater than 30%, and ethanol also inhibited NMDA-stimulated (Ca2+-dependent) cyclic GMP accumulation in a similar, dose-dependent manner. Responses to kainate were significantly less sensitive to ethanol. Studies using various concentrations of NMDA, as well as phencyclidine (PCP) and glycine, suggested that ethanol affected the "coagonist" binding site of the NMDA receptor-channel complex, rather than the PCP recognition site.     

Opposite effects of different hexachlorocyclohexane (lindane) isomers on cerebellar cyclic GMP: relation of cyclic GMP accumulation to seizure activity

A number of different depressant and convulsant agents have been shown to alter accumulation of cerebellar cyclic GMP. Since the different hexachlorocyclohexane (HCH) isomers elicit different pharmacological responses in mammals, we examined their effects on the accumulation of cerebellar cyclic GMP. Mice received one of the HCH isomers and were sacrificed for determination of cyclic GMP concentrations one hour later. Gamma-HCH increased cyclic GMP while alpha and delta-HCH decreased it. In addition, alpha and delta-HCH prevented the increase in cyclic GMP due to the gamma isomer. Picrotoxin increased cyclic GMP in a manner similar to that of gamma-HCH while strychnine produced only a small increase. All three HCH isomers inhibited the binding of 3H-TBOB (a ligand for the GABA-A-receptor linked chloride channel) in mouse cerebellum. It is concluded that the different HCH isomers can have different effects on cerebellar cyclic GMP accumulation and that these effects may be mediated through actions at the GABA-A receptor linked chloride channel.     

A probable site of action of diazepam in rat cerebellar GABA system

Diazepam and some neurotropic drugs were examined for their effects on the cyclic guanosine 3′, 5′-monophosphate (cyclic GMP) content in rat cerebellum. Diazepam caused a marked decrease of cerebellar cyclic GMP content in a dose-dependent manner. Elevation of cerebellar gamma-aminobutyric acid (GABA) level by aminooxyacetic acid also caused a decrease in the cyclic GMP content. On the other hand, dl-amphetamine, oxotremorine, picrotoxin, and GABA-reducing agents such as isoniazid or thiosemicarbazide increased the content of this nucleotide. The increase of cyclic GMP content elicited by isoniazid was blocked completely by the premedication of diazepam in doses causing partial reduction of dl-amphetamine or oxotremorine action. Changes of cerebellar GABA level, which were caused by aminooxyacetic acid or thiosemicarbazide, did not influence the effect of diazepam. Moreover, the inhibitory action of diazepam on the picrotoxin-induced increase of cyclic GMP was a competitive type. These results suggest that diazepam facilitates the GABAergic function by acting on a picrotoxin-sensitive site of the GABA receptor complex in vivo.     

Effects of sera types and cell density on the concentration of cyclic nucleotides in normal and simian virus transformed mouse fibroblasts

The effects of serum and cell density on the concentration of cyclic AMP, cyclic GMP in normal mouse fibroblasts cells (3T3 cells) and their Simian Virus 40 transformed derivative (SV3T3 cells) were studied. 3T3 cells grown in 10% foetal bovine serum exhibit density dependent inhibition of growth and associated with this in an increase in the concentration of cyclic AMP, a decrease in the concentration of cyclic GMP and an increase in the ratio (cyclic AMP/cyclic GMP) of the cyclic nucleotides. 3T3 cells grown in 10% newborn calf serum exhibit a higher saturation density and this is associated with a low concentration of cyclic AMP and a high concentration of cyclic GMP. SV3T3 cells grown in either 10% foetal bovine serum or 10% newborn calf serum show high saturation densities and this is associated with a low and decreasing concentration of cyclic AMP and a high concentration of cyclic GMP. When the level of the cyclic AMP in both cell lines was artificially raised by adding dibutyryl cyclic AMP and theophylline to the growth media, the cells grew to low densities.     

Desensitization of cyclic GMP-mediated regulation of fatty acid metabolism in hepatocytes from ethanol-fed rats

The mechanisms by which ethanol causes accumulation of hepatic triacylglycerols are complex. It has been proposed that nitric oxide/cyclic GMP signaling pathway may be involved in regulation of fatty acid metabolism in the liver. Here, we investigated if this mechanism may have a role in adaptation to ethanol consumption. Hepatocytes were isolated from rats fed with an ethanol-containing liquid diet and pair-fed control rats, and incubated with a range of concentrations of 8-bromo-cyclic GMP. In both types of cells, this cyclic GMP analog inhibited in parallel fatty acid synthesis de novo and acetyl-CoA carboxylase activity. Addition of 8-bromo-cyclic GMP also decreased the rate of palmitate esterification to triacylglycerols and phospholipids, whereas palmitate oxidation was increased. However, in all these metabolic effects, hepatocytes from ethanol-fed rats were significantly less sensitive to the addition of 8-bromo-cyclic GMP. In order to know if this may be a more general mechanism of adaptation to ethanol, we also studied the effects on glucose metabolism. Similarly, hepatocytes from ethanol-fed rats showed a decreased sensitivity in the inhibition by 8-bromo-cyclic GMP of glycogen synthesis, fatty acid synthesis and the synthesis of glycerol backbone of hepatic triacylglycerols. These data suggest that ethanol consumption induces a desensitization of the regulatory effects mediated by cyclic GMP in fatty acid metabolism, contributing to triacylglycerol accumulation in the liver.     

Cyclic GMP mediates the agonist-stimulated increase in plasma membrane calcium entry in the pancreatic acinar cell

The present studies were performed to determine the role of cyclic GMP in regulating agonist mediated calcium entry in the pancreatic acinar cell. In guinea pig-dispersed pancreatic acini the findings demonstrated that carbachol stimulated a transient 20-40-fold rise in cellular cyclic GMP followed by a sustained 3-4-fold rise in cellular cyclic GMP. The guanylate cyclase inhibitor, 6-anilino-5,8-quinolinedione (LY83583), caused a dose-dependent inhibition of carbachol-stimulated increases in cellular cyclic GMP both during the initial transient large increase in cyclic GMP and the sustained increase in cyclic GMP. LY83583 also inhibited cellular Ca2+ influx during carbachol stimulation and reloading of the agonist-sensitive pool of Ca2+ at the termination of carbachol stimulation with atropine. The effect of the inhibition on reloading of the agonist-sensitive pool was secondary to its effects on the plasma membrane C2+ entry. The addition of dibutyryl cyclic GMP to LY83583-treated acini restored Ca2+ influx across the plasma membrane. Nitroprusside increased both cellular cyclic GMP and the rate of Ca2+ influx. During periods when plasma membrane Ca2+ entry was activated, cellular cyclic GMP levels were increased. These results suggest that agonist-induced increases in cellular cyclic GMP are necessary and sufficient to mediate the effects of the agonist on the plasma membrane Ca2+ entry mechanism.     

Neurochemical Interactions of Competitive N-Methyl-D-Aspartate Antagonists with Dopaminergic Neurotransmission and the Cerebellar Cyclic GMP System: Functional Evidence for a Phasic Glutamatergic Control of the Nigrostriatal Dopaminergic Pathway

Direct intrastriatal injection of N-methyl-D-aspartate (NMDA; 100 micrograms/rat) increased striatal dopamine (DA) release in vivo. However, parenteral administration of (+/-)-3-(2-carboxypiperizin-4-yl)propyl-1-phosphonic acid (CPP) and cis-4-phosphonomethyl-2-piperidine carboxylic acid (CGS-19755) did not alter DA metabolism and release in several brain regions in the rat and mouse. Intracerebroventricular administration of the competitive NMDA antagonists CPP, CGS-19755, 2-amino-5-phosphonopentanoate, and 2-amino-7-phosphonoheptanoate did not alter rat striatal DA metabolism and release but profoundly reduced cerebellar cyclic GMP (cGMP) levels in the same animals. CPP and CGS-19755 decreased basal cerebellar cGMP levels in the mouse with ED50 values of 6 and 1 mg/kg, i.p., respectively. CPP antagonized the harmaline-induced increases in cGMP levels with an ED50 value of 5.0 mg/kg, i.p. CPP (25 mg/kg, i.p.) also decreased basal cGMP levels in mouse cerebellum for up to 3 h, a result suggesting brain bioavailability and a long duration of NMDA receptor antagonism in vivo. These contrasting patterns suggest that NMDA receptors exert a tonic excitatory tone on the guanine nucleotide signal transduction pathway in the cerebellum while exerting a phasic control over nigrostriatal dopaminergic neurotransmission. These results also indicate that competitive NMDA antagonists, unlike phencyclidine receptor agonists, may not mediate biochemical and behavioral effects via dopaminergic mechanisms.     

The effect of various drug pretreatments on the convulsions and cerebellar cyclic nucleotide changes induced by the convulsant 4-isopropyl-2,6,7-trioxa-1-phosphatbicyclo(2,2,2)octane-1-oxide(IPTBO)

The effects of pretreatment of mice with some drugs which modify GABAergic neurotransmission on the convulsions and cerebellar cyclic nucelotide level changes induced by the subsequent intracerebroventricular administration of 4-isopropyl-2,6,7-trioxa-1-phosphabicyclo(2,2,2)octane-1-oxide (IPTBO) have been studied.Muscimol, pentobarbitone and diazepam reduced the number of animals convulsing and prolonged the time to onset of convulsions in those animals which did, whereas baclofen and γ-acetylenic GABA had no effect on the number of animals convulsing but did prolong seizure latency. All the drugs except baclofen prevented the increase in cyclic GMP concentrations following IPTBO treatment. Pentobarbitone, baclofen and γ-acetylenic GABA caused significant increases in the concentrations of cyclic AMP, whereas there was no marked effect of treatment with IPTBO alone on the levels of this nucleotide: however, IPTBO reduced the extent of the increases produced by the drugs.The results are discussed in the context of the mechanism of action of IPTBO and the involvement of GABAergic neurotransmission in convulsant and anticonvulsant activities.     

Ethanol inhibition of antigen-induced histamine release from guinea pig lung: correlation with intracellular levels of cyclic nucleotides.

The effect of ethanol on histamine release from lungs of sensitized guinea pigs was studied in conjunction with measurements of tissue concentrations of cyclic AMP and cyclic GMP. Addition of antigen $\text{in vitro}$ elicited a rapid increase in cyclic AMP and cyclic GMP and stimulated release of histamine. Ethanol (2%) inhibited antigen-induced release of histamine over 95% and completely inhibited the increase in both cyclic nucleotides. The activity of cyclic AMP-dependent protein kinase was only slightly affected by ethanol.Metiamide blocked the ovalbumin stimulated increase in cyclic AMP but not cyclic GMP. Pyrilamine did not prevent the rise in either cyclic nucleotide. This suggests that the antigen-induced rise in cyclic AMP is an indirect result of histamine released from the tissue. The inability of H₁ and H₂ receptor antagonists to affect antigen-induced elevation of cyclic GMP in sensitized lung fragments suggests that an elevation in cyclic GMP might be either a primary event in the mediator release sequence or secondary to the release of a mediator other than histamine. The ability of ethanol to inhibit mediator release might be due to its capacity to attenuate the antigen-induced elevation of cyclic GMP in sensitized lung.     

Immunohistochemical localization of cyclic GMP in rat cerebellum.

The technique of cyclic nucleotide fluorescence immunohistochemistry has been applied for the specific localization of cyclic GMP in rat cerebellum. We report immunofluorescence associated with fibres and membranes, contrasting with previously reported cytoplasmic localization of cyclic AMP in different cell populations, using a similar technique. We have been unable to detect changes in cyclic GMP staining in response to post-mortem changes, harmaline and pentobarbitone administration. A role of cyclic GMP is suggested in membrane ion transport.     

Effect of ethanol upon isoproterentol stimulation of growth and secretion in the mouse parotid gland

Isoproterenol (0.3 mmole/kg body wt.), when injected into the mouse intraperitoneally, increases the weight by 35% and stimulates DNA synthesis 30-fold in the parotid gland. The induction of both hypertrophy and hyperplasia is completely inhibited by ethanol at a dose of 200 mmole/kg body wt. but is almost unaffected by 60 mmole/kg. The full inhibiton of both growth parameters is observed when ethanol is administered up to 5 hr after isoproterenol. Partial inhibition is observed when ethanol is given as long as 15 hr after isoproterenol. It contrast ethanol did not alter the secretion of α-amylase in response to isoproterenol. Ethanol had no effect upon the rise in cyclic GMP level caused by isoproterenol but augmented the rise in cyclic GMP In agreement with these $\text{in}$$\text{vivo}$ observations, low concentrations of ethanol activated adenylate cyclase $\text{in}$$\text{vitro}$, however guanylate cyclase activity was quite strongly inhibited. Although high levels of ethanol (300 mmole/kg) inhibited the induction of both ornithine decarboxylase and S-adenosylmethionine decarboxylase little inhibition was seen at 200 mmole/kg suggesting that the interference with polyamine metabolism is not the mechanism of the ethanol effect upon isoproterenol-induced parotid growth.     

Inhibition of cerebellar nitric oxide synthase and cyclic GMP production by melatonin via complex formation with calmodulin

Constitutive rat cerebellar nitric oxide synthase (NOS) activity is shown to be inhibited by physiological concentrations of the pineal hormone melatonin. The inhibition was dose-dependent and was coupled to an inhibition of the cyclic GMP production activated by L-arginine. Results also show that calmodulin appears to be involved in this process because its presence in the incubation medium was able to prevent the effect of melatonin on both NOS activity and cyclic GMP production. Moreover, polyacrylamide gel electrophoresis studies suggest that melatonin can interact with calmodulin modifying the binding of the peptide to the synthetic NOS peptide encompassing the calmodulin-binding domain of constitutive NOS from rat cerebellum, the natural mechanism by which calmodulin activates cerebellar NOS. J. Cell. Biochem. 65:430–442. © 1997 Wiley-Liss, Inc.     

Evaluation of the pentose phosphate pathway from 14CO2 data. Fallibility of a classic equation when applied to non-homogeneous tissues.

Two cyclic nucleotide phosphodiesterase (PDE) activities were identified in pig aortic endothelial cells, a cyclic GMP-stimulated PDE and a cyclic AMP PDE. Cyclic GMP-stimulated PDE had Km values of 367 microM for cyclic AMP and 24 microM for cyclic GMP, and low concentrations (1 microM) of cyclic GMP increased the affinity of the enzyme for cyclic AMP (Km = 13 microM) without changing the Vmax. This isoenzyme was inhibited by trequinsin [IC50 (concn. giving 50% inhibition of substrate hydrolysis) = 0.6 microM for cyclic AMP hydrolysis in the presence of cyclic GMP; IC50 = 0.6 microM for cyclic GMP hydrolysis] and dipyridamole (IC50 = 5 microM for cyclic AMP hydrolysis in the presence of cyclic GMP; IC50 = 3 microM for cyclic GMP hydrolysis). Cyclic AMP PDE exhibited a Km of 2 microM for cyclic AMP and did not hydrolyse cyclic GMP. This activity was inhibited by trequinsin (IC50 = 0.2 microM), dipyridamole (IC50 = 6 microM) and, selectively, by rolipram (IC50 = 3 microM). Inhibitors of cyclic GMP PDE (M&B 22948) and of low Km (Type III) cyclic AMP PDE (SK&F 94120) only weakly inhibited the two endothelial PDEs. Incubation of intact cells with trequinsin and dipyridamole induced large increases in cyclic GMP, which were completely blocked by LY-83583. Rolipram, SK&F 94120 and M&B 22948 did not significantly influence cyclic GMP accumulation. Dipyridamole enhanced the increase in cyclic GMP induced by sodium nitroprusside. Cyclic AMP accumulation was stimulated by dipyridamole and trequinsin with and without forskolin. Rolipram, although without effect alone, increased cyclic AMP in the presence of forskolin, whereas M&B 22948 and SK&F 94120 had no effects on resting or forskolin-stimulated levels. These results suggest that cyclic GMP-stimulated PDE regulates cyclic GMP levels and that both endothelial PDE isoenzymes contribute to the control of cyclic AMP.     

A low molecular weight factor from sea urchin eggs elevates sperm cyclic nucleotide concentrations and respiration rates.

A factor associated with sea urchin eggs that increases sperm cyclic nucleotide concentrations and respiration rates was identified as having a low molecular weight. The factor was more potent at elevating cyclic GMP concentrations than cyclic AMP concentrations, and represents the first demonstration of a factor associated with eggs that is capable of causing elevations of sperm cyclic GMP. Concentration-response curves of the crude mixture of egg factors to increase sperm cyclic AMP and cyclic GMP concentrations and respiratory rates were very similar, and comparable losses of these three activities were observed after extensive dialysis and heat treatment of the crude egg factors. The factor was partly purified by ethanol precipitation of a large molecular weight egg jelly component, and by charcoal adsorption and LH-20 chromatography of the resultant ethanol-soluble material. The factor was not extracted into a variety of organic solvents and had an apparent molecular weight of between 1000 and 2000, as estimated by gel filtration.     

Effects of adenosine 3' : 5'-monophosphate and guanosine 3' : 5'-monophosphate on calcium uptake and phosphorylation in membrane fractions of vascular smooth muscle.

The effects of adenosine 3' : 5'-monophosphate (cyclic AMP), guanosine 3' : 5'-monophosphate (cyclic GMP) and exogenous protein kinase on Ca uptake and membrane phosphorylation were studied in subcellular fractions of vascular smooth muscle from rabbit aorta. Two functionally distinct fractions were separated on a continuous sucrose gradient: a light fraction enriched in endoplasmic reticulum (fraction E) and a heavier fraction containing mainly plasma membranes (fraction P). While cyclic AMP and cyclic GMP had no effect on Ca uptake in the absence of oxalate, both cyclic nucleotides inhibited the rate of oxalate-activated Ca uptake when used at concentrations higher than 10(-5) M. The addition of bovine heart protein kinase to either fraction produced an increase in the rate of oxalate-activated Ca uptake which was further augmented by cyclic AMP. Cyclic GMP caused smaller stimulations of protein kinase-catalyzed Ca uptake than cyclic AMP. Mg-dependent phosphorylation, attributable to endogenous protein kinase(s), was inhibited in fraction E by low concentrations (10(-8) M) of both cyclic AMP and cyclic GMP. In fraction P, an inhibition by cyclic AMP occurred also at a concentration of 10(-8) M, while with cyclic AMP a concentration of 10(-5) M was required for a similar inhibition. Bovine heart protein kinase stimulated the phosphorylation of the membrane fractions much more than Ca uptake. In fraction E, in the presence of bovine protein kinase, both cyclic AMP and cyclic GMP stimulated phosphorylation up to 200%. Under these conditions, no stimulation was observed in fraction P. These results are compatible with the hypothesis that in vascular smooth muscle soluble rather than particulate protein kinases are involved in the regulation of intracellular Ca concentration.     

Effects of isoproterenol,theophylline and carbachol on cyclic nucleotide levels and relaxation of bovine tracheal smooth muscle

The effect of theophylline and isoproterenol on bovine tracheal smooth muscle tension and cyclic AMP levels was investigated. Concentrations of isoproterenol (4 × 10^?6 M) and theophylline (10 mM) that relaxed carbachol-contracted tracheal muscle by 85–95% did not significantly elevate control levels of cyclic AMP. In the absence of carbachol, several-fold increases in cyclic AMP were caused by isoproterenol although no elevations by theophylline were measurable. However, when isoproterenol and theophylline were administered together, theophylline potentiated the rise in cyclic AMP caused by isoproterenol. Phosphodiesterase studies in tracheal muscle showed the presence of a high and a low K_m enzyme which were inhibited by theophylline. Cyclic GMP levels were elevated in muscles contracted by carbachol as well as in carbachol-contracted muscles that had been relaxed by theophylline. In non-tension studies, in which the tracheal muscle was not under isometric tension, carbachol or theophylline alone increased cyclic GMP and together they synergistically elevated cyclic GMP. Atropine blocked the elevation caused by carbachol but not that caused by theophylline. In contrast to theophylline, isoproterenol did not elevate cyclic GMP, and in carbachol-contracted muscles that had been relaxed by isoproterenol, cyclic GMP levels were no different from control. Also, in non-tension studies, isoproterenol decreased basal cyclic GMP and antagonized the increase in cyclic GMP due to carbachol.The results indicate that whole-tissue levels of cyclic AMP and cyclic GMP do not correlate with the state of tracheal smooth muscle tension. Cyclic GMP levels do not clearly correlate with either contraction or relaxation. The inhibition by carbachol of increases in cyclic AMP due to isoproterenol and the inhibition by isoproterenol of increases in cyclic GMP due to carbachol provide evidence for a reciprocal cholinergic-adrenergic antagonism of cyclic AMP and cyclic GMP levels. The antagonism did not appear to be due to either cyclic nucleotide affecting the elevation of the other since the levels of both cyclic nucleotides were depressed.