Antigenic conservation of brain guanylate cyclase during evolution

A comparative study of brain guanylate cyclase from different animal species (including man, bird, fish and amphibian) has been performed using a specific antibody directed against soluble rat brain guanylate cyclase. Analyses were performed on supernatant fractions by the double-immunodiffusion test, by the protein blotting technique after SDS-polyacrylamide gel electrophoresis and by analytical isoelectric focusing on agarose allowing specific immunodetection of isoelectric patterns. Membrane-bound guanylate cyclase from rat brain and soluble guanylate cyclase from several rat tissues cross-reacted with the antibody. All the brain enzymes tested were found to be identical by double-immunodiffusion. The electrophoretic and isoelectrophoretic profiles of the different brain guanylate cyclases were found to exhibit many common features with some differences between mammalian and non-mammalian enzymes. In human brain, guanylate cyclase has been localized in glial and neuronal cells by immunohistochemistry. The results demonstrate that guanylate cyclase has been well conserved during the course of evolution and are consistent with the involvement of guanylate cyclase and cyclic GMP in basic cellular function.     

Guanylate cyclase activity in normal and diseased human muscle.

Guanylate cyclase activity has been studied in muscle of normal subjects and of patients suffering from muscular and neuromuscular diseases. In normal tissue a guanylate cyclase activity was found in both soluble and particulate fractions of homogenate. We found also that the kinetic analysis of the enzyme of soluble differed from that of particulate fraction. A decrease of guanylate cyclase activity in crude homogenate was observed in muscular dystrophies, in neuromuscular atrophies, and in inflammatory forms of muscle disease.     

Calmodulin-dependent adenylate cyclase in rat retina and the response to dopamine

Adenylate cyclase activity from the rat neural retina was highly stimulated with Ca²⁺ and calmodulin. The retinal adenylate cyclase activity was also increased by dopamine, and the activation was not changed with or without Ca²⁺-calmodulin in fractions from the neural retina homogenate after sucrose density gradient centrifugation. The results suggest that the two regulation systems (i.e. dopamine and Ca²⁺-calmodulin) of adenylate cyclase in the rat retina appear to be independent.     

Calmodulin stimulates adenylate cyclase activity and increases dopamine activation in bovine retina

Adenylate cyclase activity in bovine retina is highly responsive to Ca2+ and the endogenous Ca2+-binding protein, calmodulin (CaM). CaM stimulated adenylate cyclase activity in washed particulate fractions of bovine retina by 6.6-fold in a Ca2+-dependent manner. Activation of adenylate cyclase activity by CaM was maximal at 0.12 microM free Ca2+. The apparent Ka for calmodulin stimulation of adenylate cyclase was 67 nM and the apparent Vmax was 116 pmol/min/mg of protein above basal activity. Adenylate cyclase activity in bovine retina was stimulated approximately 50% by guanosine 5'-triphosphate (GTP), but the nonhydrolyzable GTP analogue, guanosine-5'-(beta, gamma-imido)triphosphate (Gpp(NH)p), was able to activate the enzyme nearly 5-fold. CaM and Gpp(NH)p appeared to be partially competitive activators of adenylate cyclase in the retina particulate fraction. Dopamine stimulated adenylate cyclase activity in the presence of GTP with an apparent Ka of 1.0 microM and an apparent Vmax of 66 pmol/min/mg of protein. Ca2+ and CaM increased the apparent Vmax of the dopamine-stimulated adenylate cyclase activity more than 2-fold to a level of 146 pmol/min/mg of protein but did not alter the apparent Ka. This suggests that CaM is an endogenous modulator of dopamine-stimulated adenylate cyclase activity in the retina. CaM-stimulated adenylate cyclase activity may be a common component to retina since we found this activity in retinas from rabbit, rat, and goldfish as well as cow.     

Involvement of adenylate cyclase and tyrosine kinase signaling pathways in response of crayfish stretch receptor neuron and satellite glia cell to photodynamic treatment

Neuroglial interactions are most profound during development or damage of nerve tissue. We studied the responses of crayfish stretch receptor neurons (SRN) and satellite glial cells to photosensitization with sulfonated aluminum phthalocyanine Photosens. Although Photosens was localized mainly in the glial envelope, neurons were very sensitive to photodynamic treatment. Photosensitization gradually inhibited and then abolished neuron activity. Neuronal and glial nuclei shrank. Some neurons and glial cells lost the integrity of the plasma membrane and died through necrosis after the treatment. The nuclei of other glial cells but not neurons become fragmented, indicating apoptosis. The number of glial nuclei around neuron soma increased, probably indicating proliferation for enhanced neuron protection. Adenylate cyclase (AC) inhibition by MDL-12330A, or tyrosine kinase (TK) inhibition by genistein, shortened neuron lifetime, whereas AC activation by forskolin or protein tyrosine phosphatases (PTP) inhibition by sodium orthovanadate prolonged neuronal activity. Therefore, cAMP and phosphotyrosines produced by AC and TK, respectively, protected SRN against photoinactivation. AC inhibition reduced photodamage of the plasma membrane and subsequent necrosis in neuronal and glial cells. AC activation prevented apoptosis in photosensitized glial cells and stimulated glial proliferation. TK inhibition protected neurons but not glia against photoinduced membrane permeabilization and subsequent necrosis whereas PTP inhibition more strongly protected glial cells. Therefore, both signaling pathways involving cAMP and phosphotyrosines might contribute to the maintenance of neuronal activity and the integrity of the neuronal and glial plasma membranes. Adenylate cyclase but not phosphotyrosine signaling pathways modulated glial apoptosis and proliferation under photooxidative stress.     

[H]5-HT binding sites and 5-HT-sensitive adenylate cyclase in glial cell membrane fraction

Glial cell membrane fractions were prepared using glial cells preparations isolated from horse brain striatum. [³H]5-HT binding was measured by the filtration technique and the adenylate cyclase activity determined by measuring the cAMP production using a radioimmunoassay.Serotonin binds to glial membrane fractions with an affinity corresponding to a dissociation constantK_d = 10 nM. The corresponding site is serotoninergic specific: [³H]5-HT binding is inhibited by 5-HT agonists (5 OH NM-DMT, 5-MeOHT, 5-MeOH-DMT, NN-DMT) or antagonists (cinanserine, cyproheptadine, methysergide, LSD) and not (or poorly) inhibited by non-serotoninergic related drugs. The population of sites binding 5-HT, present in neuronal membrane preparations and determined in parallel assays is distinct from that observed in glial preparations. The glial membrane fractions contains an adenylate cyclase activated by 5-HT with an apparent affinity constant close to 1 μM. It is serotonin-specific and clearly distinct from the DA-stimulated adenylate cyclase present in the same preparation.The sites binding 5-HT and activating the adenylate cyclase with low affinities might be directly related. This system, clearly distinct from the postsynaptosomal serotoninergic receptor, represents presumably a glial serotoninergic receptor; however, it cannot be totally excluded that these sites may refer to presynaptic membranes.     

[3H]5-HT binding sites and 5-HT-sensitive adenylate cyclase in glial cell membrane fraction

Glial cell membrane fractions were prepared using glial cells preparations isolated from horse brain striatum. [³H]5-HT binding was measured by the filtration technique and the adenylate cyclase activity determined by measuring the cAMP production using a radioimmunoassay.Serotonin binds to glial membrane fractions with an affinity corresponding to a dissociation constantK_d = 10 nM. The corresponding site is serotoninergic specific: [³H]5-HT binding is inhibited by 5-HT agonists (5 OH NM-DMT, 5-MeOHT, 5-MeOH-DMT, NN-DMT) or antagonists (cinanserine, cyproheptadine, methysergide, LSD) and not (or poorly) inhibited by non-serotoninergic related drugs. The population of sites binding 5-HT, present in neuronal membrane preparations and determined in parallel assays is distinct from that observed in glial preparations. The glial membrane fractions contains an adenylate cyclase activated by 5-HT with an apparent affinity constant close to 1 μM. It is serotonin-specific and clearly distinct from the DA-stimulated adenylate cyclase present in the same preparation.The sites binding 5-HT and activating the adenylate cyclase with low affinities might be directly related. This system, clearly distinct from the postsynaptosomal serotoninergic receptor, represents presumably a glial serotoninergic receptor; however, it cannot be totally excluded that these sites may refer to presynaptic membranes.     

A functional parameter to study heterogeneity of glial cells in rat brain slices: cyclic guanosine monophosphate production in atrial natriuretic factor (ANF)-responsive cells

Stimulation of guanylate cyclase in vitro by atrial natriuretic factor (ANF) or sodium nitroprusside was studied in rat brain tissue slices biochemically as well as by means of cyclic guanosine monophosphate (cGMP) immunocytochemistry. The ANF-responsive, cGMP-producing cells were studied in the olfactory bulb, the septal area, the hippocampus, the medial amygdala, and the medial preoptic area. These cells, having the ANF-stimulated particulate guanylate cyclase, were characterized as astroglial cells on the basis of their glial fibrillary acidic protein (GFAP) immunostaining, although not all astroglial cells in these areas could be identified as cGMP-immunoreactive cells. Sodium nitroprusside-stimulated soluble guanylate cyclase activity was demonstrated in neuronal cell bodies and varicose fibers and was associated with blood vessel walls. Upon maturation, a significant decrease in cGMP production was found after stimulation by 100 nM ANF-(103-126) in the olfactory bulb, the medial amygdala, and the hippocampus, but not in the septal area; no change was found in these areas in cGMP content after stimulation of cGMP production by 10 microM sodium nitroprusside. Via cGMP immunocytochemistry, no qualitative differences were seen in the ANF-responsive, cGMP-producing cells upon maturation.     

Nitric Oxide Induces Morphological Changes in Cultured Neurohypophysial Astrocytes

Cultured pituicytes, derived from the neurohypophysis of adult rats, have previously been reported to change from a non-stellate form to a stellate form when incubated in medium containing a β -adrenoreceptor agonist. This study was designed to determine whether the same morphological change could be induced by direct activation of adenylate cyclase or of soluble guanylate cyclase. The fraction of stellate cells was normally low (<0.25) when the pituicytes were incubated (90  min) in a HEPES buffered salt solution (HBSS); most pituicytes had an amorphous protoplasmic appearance. The fraction of stellate cells was significantly increased when pituicytes were incubated in HBSS supplemented with isoproterenol (10  μM) or forskolin (5  μM) or with either of the nitric oxide donors nitroprusside (10–25  μM) and 3-morpholinosydnonimine (SIN-1; 10  μM). The effect of forskolin was mimicked by 8-bromo cyclic AMP, a membrane permeable analog of cyclic AMP, but not by the inactive forskolin analog 1, 9 dideoxyforskolin. The effect of nitroprusside was blocked by methylene blue, an inhibitor of soluble guanylate cyclase, and was mimicked by 8-bromo cyclic GMP, a membrane permeable analog of cyclic GMP. These results demonstrate that activation of adenylate cyclase and also of soluble guanylate cyclase can induce pituicytes to undergo morphological changes in vitro . The data suggest that the activity of both enzymes may be important in control of the plastic relationship that exists between neuronal and glial elements in the neurohypophysis in vivo .     

Developmental changes in carbonic anhydrase and adenylate cyclase in quaking mice

The present investigation focuses on the developmental changes in the activity and levels of carbonic anhydrase, adenylate cyclase and 3′,5′-cyclic adenosine monophosphate (cAMP) in the Quaking mouse mutant in different regions of the brain.Carbonic anhydrase activity was found to be lower than normal in the forebrain of the C57/B Quaking mouse. The deficit in forebrain carbonic anhydrase was restricted to subcortical structures and was reflected equally in the membrane and soluble fractions, indicating that neither pool was affected selectively. However, no difference in carbonic anhydrase activity was observed in purified myelin from Quaking and control mice. Investigation of the changes in carbonic anhydrase activity as a function of age showed a cessation in enzyme accumulation in Quaking mice at around 20 days postnatally, suggesting an abnormality in cellular development. A tritiated acetazolamide binding assay was used to quantitate the amount of enzyme present. The amount of carbonic anhydrase parallelled enzyme activity, suggesting that the defect in the Quaking animals was at the level of the control of enzyme synthesis.Similar studies on cyclic AMP metabolism showed a higher than normal adenylate cyclase activity in the upper brain stem region of Quaking mice of ages between 19 and 40 days. Adenylate cyclase activity was stimulated by norepinephrine in both control and Quaking animals. The increased adenylate cyclase activity in the Quaking mice was in contrast to a lower cyclic AMP level and could not be accounted for by an alteration in phosphodiesterase activity.     

Protective action by methylprednisolone, allopurinol and indomethacin against stroke-induced damage to adenylate cyclase in gerbil cerebral cortex

Adenylate cyclase activity was investigated in either homogenate or particulate fractions from the frontal cerebral cortex of the gerbil following five experimental conditions of bilateral ischemia. After periods of 15 min ischemia, 15 min ischemia plus 15 min of recirculation or 60 min ischemia the enzyme generally displayed enhanced responses to GTP, norepinephrine (NE), dopamine (DA), NE + GTP and DA + GTP. Pretreatment of the gerbils with methylprednisolone, allopurinol or indomethacin did not significantly influence the outcome of these findings. When the animals were subjected to 60 min ischemia plus 15 min of reflow, enzyme responses to the stimulatory agents including forskolin and NaF were all reduced. Pretreatment with methylprednisolone, allopurinol or indomethacin prevented the damage to adenylate cyclase in the 60 min ischemia plus 15 min reflow animals. When animals were made ischemic for 15 min followed by one week of recovery, enzyme sensitivity to GTP, calmodulin-Ca++, NE, combinations thereof and forskolin were reduced in only the particulate fractions. Enzyme damage was reversed following methylprednisolone. Enzyme damage may result from generation of free radicals during reflow and drugs that either inhibit synthesis pathways generating free radicals, stabilize cell membranes or act as free radical scavengers may be therapeutically beneficial under specific conditions of stroke.     

Control of opiate receptor-adenylate cyclase interactions by calcium ions and guanosine-5′-triphosphate

Adenylate cyclase of homogenates of NG108-15 neuroblastoma x glioma hybrid cells is activated by low concentrations of Ca2+ ions and is inhibited by higher (greater than 0.1 mM) concentrations of Ca2+ ions. Activation of either opiate receptors by 10 microM morphine or alpha-adrenergic receptors by 10 microM norepinephrine inhibits adenylate cyclase by 55% in the absence of Ca2+ ions, and inhibits the Ca2+-dependent activation of adenylate cyclase by more than 90%. Concentrations of Ca2+ ions greater than 0.1 mM inhibit adenylate cyclase and also reduce the extent inhibition of adenylate cyclase by morphine but not by norepinephrine. Guanosine-5'-triphosphate (0.1-1 microM) is required for inhibition of adenylate cyclase by morphine. The results show that morphine inhibits adenylate cyclase by a guanosine-5-triphosphate-dependent process and that the extent of inhibition of adenylate cyclase by morphine or norepinephrine is a function of the Ca2+ ion concentration and the proportion of adenylate cyclase molecules that are activated or inhibited by Ca2+ ions.     

Axonal transport of adenylate cyclase activity in normal and axotomized frog sciatic nerve

Adenylate cyclase activity accumulated proximal to a constriction placed around the frog sciatic nerve. The rate of accumulation was linear between 8 and 24 h following placement of the constriction; accumulation rate declined substantially after 24 h. Accumulation of activity distal to the constriction in normal nerve was not significantly different from control for the first 72 h, but increased at 5 days. These data are interpreted as indicating that adenylate cyclase is transported from the cell body to the nerve terminals in normal frog nerve, but not in the reverse direction. Following axon transection, anterograde transport of adenylate cyclase activity declined, but a transient retrograde transport of adenylate cyclase activity appeared. In addition, adenylate cyclase activity accumulated in the proximal transected nerve stump during the period when Schwann cell proliferation and the initiation of nerve regeneration both appear. The pattern of response of adenylate cyclase activity to nerve injury suggests that the adenylate cyclase: cAMP system could play some role in peripheral nerve regeneration.     

Neuronal localization of dopamine-sensitive adenylate cyclase within the rat olfactory tubercle

We have used selective chemical lesions to localize dopamine-sensitive adenylate cyclase within the rat olfactory tubercle. Stereotaxic injections were made directly into the tubercle with kainic acid, or with 6-OH dopamine. Animals were sacrificed 3 days later by decapitation and homogenates of the olfactory tubercle were assayed for dopamine-sensitive adenylate cyclase. The kainic acid lesion reduced the dopamine-sensitive adenylate cyclase activity by 80% as compared to values from sham lesioned or unlesioned controls. Homogenates of 6-OH dopamine-treated tubercles were slightly greater in dopamine-sensitive adenylate cyclase activity than untreated controls. Sections from kainic acid-treated tubercles showed extensive neuronal losses with increased numbers of glial cells. Examination of 6-OH dopamine-treated tissue by glyoxylic acid-induced histofluorescence established the loss of dopaminergic terminals. The marked decrease of dopamine-sensitive adenylate cyclase accompanying the selective loss of neurons (kainic acid treatment) but not accompanying the loss of dopaminergic terminals (6-OH dopamine treatment) suggests that this enzyme occurs in the neurons and not in the glia or in the dopaminergic terminals of this region.     

Endogenous activation of guanylate cyclase in synaptosomal soluble fraction from rat brain

Guanylate cyclase in crude mitochrondrial (P2) soluble fraction prepared from rat brain, obtained by a hypo-osmotic treatment of P2, showed extremely higher activity than that in the same fraction from other organs. In addition the soluble fraction obtained from synaptosomes (P2 - B) contained the highest enzyme activity among other subfractions of the cerebral P2 examined. Guanylate cyclase activity in the synaptosomal soluble fraction was, however, markedly suppressed by various compounds reacting with free radicals. These results suggest that guanylate cyclase in the synaptosomal soluble fraction may be activated endogenously be a free radical and involved in the regulatory mechanisms for cyclic guanosine monophosphate (cyclic GMP) at presynaptic terminals.     

Whole brain spheroid cultures as a model to study the development of nitric oxide synthase-guanylate cyclase signal transduction

Whole brain spheroids provide a suitable model to study neurodevelopment. In the literature a role for the nitric oxide (NO)-cyclic guanosine 3',5'-monophosphate (cGMP) signalling pathway during development has frequently been suggested. In this study we investigated whether functional cGMP pathways were present in differentiated spheroids. In 3-week-old spheroids soluble guanylate cyclase was stimulated with N-methyl D-aspartic acid or sodium nitroprusside (NO donor). The results showed that the NO synthase-cGMP pathway is present in the culture system. Soluble guanylate cyclase-dependent cGMP formation was found in NO synthase containing neurons, in neurons of the GABAergic, glutamatergic and cholinergic system, and in astroglia and oligodendroglia. Activation of particulate guanylate cyclase by atrial natriuretic peptide also triggered an increase in cGMP production. Particulate guanylate cyclase was found in astroglia and in microglia as well as in glutamic acid decarboxylase and calbindin containing structures and neuronal NO synthase containing neurons. Chronic inhibition of NO synthase during culture development had no effect on soluble or particulate guanylate cyclase functioning. Similarly, inhibition of soluble guanylate cyclase during culture development did not have any effect on NO synthase and particulate guanylate cyclase functioning. It is concluded that NO synthase and both soluble and particulate guanylate cyclase are present in whole brain spheroid cultures and that their activity can be influenced by several stimuli. The spheroid culture system constitutes a suitable model to study the NO-cGMP pathway during brain development in mammals.     

Platelet adenylate cyclase and phospholipase C activity in posttraumatic stress disorder

Adenylate cyclase and phospholipase C activity were examined in platelet membranes obtained from 19 male subjects with combat-related posttraumatic stress disorder (PTSD) and 35 age- and gender-matched healthy controls. Basal and forskolin-stimulated adenylate cyclase activity were significantly lower in the PTSD group whereas aluminum chloride plus sodium fluoride (AlCl3/NaF)- and prostaglandin E1 (PGE1)-stimulated responses were normal. There was no difference in phospholipase C activity between the two groups. The lower basal and forskolin-stimulated adenylate cyclase responses replicate a previous report and suggest that PTSD may be associated with an abnormality of the catalytic subunit of the receptor-adenylate cyclase complex.     

Presence of soluble and particulate guanylyl cyclase in the same hippocampal astrocytes

The localisation of particulate and soluble guanylyl cyclase was studied in hippocampal astrocytes. Counting the colocalisation of cGMP immunoreactivity with the astrocytic marker glial fibrillary acidic protein after stimulation of brain slices with sodium nitroprusside (0.1 mM) or atrial natriuretic peptide (100 nM), we were able to show that at least 67% of the hippocampal astrocytes contained both guanylyl cyclase isoforms. In addition, it was shown that a large number of atrial natriuretic peptide, brain-derived natriuretic peptide or sodium nitroprusside responsive cells contain the beta1-subunit of the soluble guanylyl cyclase. The results show that, in at least a subset of hippocampal astrocytes, soluble and particulate guanylyl cyclases are simultaneously present in the same cells.     

Effect of β2-glycoprotein I on the activity of adenylate cyclase in platelet membranes

Washed human platelets were preincubated with 0–300 μg/ml human β₂-glycoprotein I and the effect of this on the adenylate cyclase activity (EC.4.6. 1. 1.) was studied. Adenylate cyclase activity could be increased 2–3 fold. The same degree of activation was seen when low concentrations of prostaglandin E₁ (1 μM) had been present concommitant with β₂-glycoprotein I during preincubation. The dose-response curves of the adenylate cyclase activity measured as a function of the β₂-glycoprotein I concentration were S-shaped in the absence of prostaglandin E₁ and hyperbolic in its presence. The results suggest a biological function of β₂-glycoprotein I as a compound conserving and activating the membrane-bound adenylate cyclase.     

Adenylate cyclase and guanylate cyclase of normal and denervated skeletal muscle.

Cyclic nucleotides mediate the intracellular effects of various extracellular influences. To explore the possibility that nerve-muscle influences are mediated by cyclic nucleotides, we studied the effect of denervation on the cyclase enzymes of rabbit gastrocnemius using the contralateral, unoperated limb as the control. Adenylate cyclase activity decreased dramatically after denervation, while guanylate cyclase activity increased several times in all tissue fractions studied. Neither enzyme demonstrated cholinergic responsiveness. The dramatic changes in cyclase activities following denervation could result from a role of these enzymes in the mediation of nerve-muscle influences.