Further Characterization of the Long-Term Effect of RU24722 on Tyrosine Hydroxylase in the Rat Locus Coeruleus

Abstract: Recent data have indicated that the long-lasting increase in tyrosine hydroxylase (TH) protein could be differently expressed in the anterior and posterior locus coeruleus (LC) after a single intraperitoneal injection of RU24722, which has been proposed as a potent activator of catecholaminergic systems. In the present study, we have evaluated the dose and time course responses and the effect of a repeated treatment with RU24722 at 3-day intervals on TH protein level in the anterior and posterior rat LC. The results showed that RU24722 induces a long-lasting increase of TH protein level in the anterior and posterior LC that was maximal 3 days following a single injection of 30 mg/kg. The increase in TH protein was maintained at a constant level after repeated administrations of RU24722 at 3-day intervals. Furthermore, we have investigated whether the effect of the drug on TH protein could be modulated via several hormonal systems. The long-term increase of TH steady-state content after RU24722 was still observed 15 days after castration, adrenalectomy, hypophysectomy, and thyroidectomy. The initial steady-state TH protein level was significantly higher in the anterior LC of thyroid- or hypophysectomized and in the posterior LC of hypophysectomized rats. However, this increase was reversed when animals were housed at 28°C.     

Ontogeny of Tyrosine Hydroxylase Concentration in Locus Coeruleus of Newborn Rats: Long-Term Effects of RU24722

The ontogenetic variations of tyrosine hydroxylase (TH) have been studied in locus coeruleus of developing rats. During the first 2 weeks after birth, a large increase in TH content (6.04-23.99 TH units) in the noradrenergic structure was observed, followed by a period of progressive increase of the protein concentration (42 TH units in adult rats). The expression of TH was studied in the same ontogenetic period after treatment by RU24722 (20 mg/kg, i.p.). The long-term increase in TH concentration produced by the drug was found to follow ontogenetic variations. It becomes significant around the middle of the second week after birth and gradually increases until the 24th day of postnatal development, indicating a maturation of the mechanisms involved in the inducing effect.     

Direct Demonstration of the Activation of UDP-N-Acetylgalactosamine: [GM3]N-Acetylgalactosaminyltransferase by Cyclic AMP

Treatment of NG108-15 cells in culture with the opiate peptide [D-Ala2,D-Leu5]enkephalin produces maximal inhibition of cyclic AMP synthesis in less than 15 min. The activity of [GM3]:N-acetylgalactosaminyltransferase is similarly inhibited, but maximal inhibition is not observed for at least 30 min following the addition of [D-Ala2,D-Leu5]enkephalin. Conversely, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine rapidly potentiates the intracellular accumulation of cyclic AMP and, in a more gradual fashion, increases [GM3]:N-acetylgalactosaminyltransferase activity. The reductions in the activity of [GM3]:N-acetylgalactosaminyltransferase that occur following treatment of NG108-15 cells with indomethacin argues for a direct role of cyclic AMP in the observed changed in [GM3]:N-acetylgalactosaminyltransferase activity. By adding low concentrations of cyclic AMP (but not cyclic GMP) to microsomes derived from neonatal rat brain, we were able to demonstrate a dose-dependent phosphorylation of membrane protein and subsequent doubling of [GM3]:N-acetylgalactosaminyltransferase activity.     

Activation of Retinal Tyrosine Hydroxylase In Vitro by Cyclic AMP-Dependent Protein Kinase: Characterization and Comparison to Activation In Vivo by Photic Stimulation

Abstract: Tyrosine hydroxylase in rat retina is activated in vivo as a consequence of photic stimulation. Tyrosine hydroxylase in crude extracts of dark-adapted retinas is activated in vitro by incubation under conditions that stimulate protein phosphorylation by cyclic AMP-dependent protein kinase. Comparison of the activations of the enzyme by photic stimulation in vivo and protein phosphorylation in vitro demonstrated several similarities. Both treatments decreased the apparent K _m of the enzyme for the synthetic pterin cofactor 6MPH₄. Both treatments also produced the same change in the relationships of tyrosine hydroxylase activity to assay pH. When retinal extracts containing tyrosine hydroxylase activated either in vivo by photic stimulation or in vitro by protein phosphorylation were incubated at 25°C, the enzyme was inactivated in a time-dependent manner. The inactivation of the enzyme following both activation in vivo and activation in vitro was partially inhibited by sodium pyrophosphate, an inhibitor of phosphoprotein phosphatase. In addition to these similarities, the activation of tyrosine hydroxylase in vivo by photic stimulation was not additive to the activation in vitro by protein phosphorylation. These data indicate that the mechanism for the activation of tyrosine hydroxylase that occurs as a consequence of light-induced increases of neuronal activity is similar to the mechanism for activation of the enzyme in vitro by protein phosphorylation. This observation suggests that the activation of retinal tyrosine hydroxylase in vivo may be mediated by phosphorylation of tyrosine hydroxylase or some effector molecule associated with the enzyme.     

Activation of Tyrosine Hydroxylase in PC12 Cells by the Cyclic GMP and Cyclic AMP Second Messenger Systems

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is subject to regulation by a variety of agents. Previous workers have found that cyclic AMP-dependent protein kinase and calcium-stimulated protein kinases activate tyrosine hydroxylase. We wanted to determine whether cyclic GMP might also be involved in the regulation of tyrosine hydroxylase activity. We found that treatment of rat PC12 cells with sodium nitroprusside (an activator of guanylate cyclase), 8-bromocyclic GMP, forskolin (an activator of adenylate cyclase), and 8-bromocyclic AMP all produced an increase in tyrosine hydroxylase activity measured in vitro or an increased conversion of [14C]tyrosine to labeled catecholamine in situ. Sodium nitroprusside also increased the relative synthesis of cyclic GMP in these cells. In the presence of MgATP, both cyclic GMP and cyclic AMP increased tyrosine hydroxylase activity in PC12 cell extracts. The heat-stable cyclic AMP-dependent protein kinase inhibitor failed to attenuate the activation produced in the presence of cyclic GMP. It eliminated the activation produced in the presence of cyclic AMP. Sodium nitroprusside also increased tyrosine hydroxylase activity in vitro in rat corpus striatal synaptosomes and bovine adrenal chromaffin cells. In all cases, the cyclic AMP-dependent activation of tyrosine hydroxylase was greater than that of the cyclic GMP-dependent second messenger system. These results indicate that both cyclic GMP and cyclic AMP and their cognate protein kinases activate tyrosine hydroxylase activity in PC12 cells.     

Effects of Vasoactive Intestinal Peptide and Other Peptides on Cyclic AMP Accumulation in Intact Pieces and Isolated Horizontal Cells of the Teleost Retina

The effects of vasoactive intestinal peptide (VIP) and several other peptides have been examined on cyclic AMP accumulation in intact pieces and isolated horizontal cells of the teleost (carp) retina. VIP was the most effective peptide examined, inducing a dose-related response, and an approximately fivefold increase in cyclic AMP production when used at a concentration of 10 microM. Porcine histidine isoleucine-containing peptide and secretin, peptides structurally related to VIP, also stimulated cyclic AMP accumulation, but at concentrations of 10 microM induced responses which were only approximately 40% and 10%, respectively, of the response observed with 10 microM VIP. In contrast, several other peptides, including glucagon, neurotensin, somatostatin, luteinizing hormone-releasing hormone, alpha-melanocyte-stimulating hormone, cholecystokinin octapeptide26-33, gastrin-releasing peptide, thyrotropin-releasing hormone, and VIP10-28 were totally inactive. The response to 10 microM VIP was not antagonized by several dopamine antagonists, indicating the presence of a population of specific VIP receptors coupled to adenylate cyclase, distinct from the population of dopamine receptors coupled to adenylate cyclase also known to be present in this tissue. Finally, experiments involving the use of fractions of isolated horizontal cells indicate that these neurons possess a population of VIP receptors coupled to cyclic AMP production which would appear to share a common pool of adenylate cyclase with a population of similarly coupled dopamine receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of the Stress-Induced Synthesis of hsp27 and αB-Crystallin by Cyclic AMP in C6 Rat Glioma Cells

Abstract: The possible participation of cyclic AMP in the stress-induced synthesis of two small stress proteins, hsp27 and αB-crystallin, in C6 rat glioma cells was examined by specific immunoassays, western blot analysis, and northern blot analysis. When C6 cells were exposed to arsenite (50–100 µM for 1 h) or heat (42°C for 30 min), expression of hsp27 and αB-crystallin was stimulated, with levels of the two proteins reaching a maximum after 10–16 h of culture. Induction of hsp27 was markedly enhanced when cells were exposed to arsenite in the presence of isoproterenol (20 µM) or epinephrine (20 µM) but not in the presence of phenylephrine. The stimulatory effects of isoproterenol and epinephrine were blocked completely by propranolol, an antagonist of β-adrenergic receptors. Cholera toxin (2 µg/ml), forskolin (20 µM), and dibutyryl cyclic AMP (2.5 mM), all of which are known to increase intracellular levels of cyclic AMP, also stimulated the arsenite- or heat-induced accumulation of hsp27. Treatment of cells with each of these modulators alone did not result in the induction of hsp27. The level of hsp70 in C6 cells, as estimated by western blot analysis, was also enhanced by arsenite or heat stress. However, induction of hsp70 by stress was barely stimulated by isoproterenol. By contrast, induction of αB-crystallin by heat or arsenite stress was suppressed when isoproterenol, cholera toxin, forskolin, or dibutyryl cyclic AMP was present during the stress period. Northern blot analysis of the expression of mRNAs for hsp70, hsp27, and αB-crystallin showed that the modulation of the stress-induced accumulation of the three hsps by the various agents was regulated at the level of the corresponding mRNA. These results indicate that stress responses of hsp70, hsp27, and αB-crystallin in C6 rat glioma cells are regulated differently and, moreover, that when the level of cyclic AMP increases in cells, the response to stress of hsp27 is stimulated but that of αB-crystallin is suppressed.     

Chronic Antidepressant Administration Alters the Subcellular Distribution of Cyclic AMP-Dependent Protein Kinase in Rat Frontal Cortex

The influence of chronic administration of antidepressants on cyclic AMP-dependent protein kinase activity was examined in rat frontal cortex. Chronic administration of imipramine, tranylcypromine, or electroconvulsive seizures decreased cyclic AMP-dependent protein kinase activity in soluble fractions by approximately 25%, whereas enzyme activity was increased in the particulate fractions by approximately 20%. In contrast, enzyme activity in crude homogenates was not altered. This effect appears to be specific to antidepressant drugs, because representatives of several other classes of psychotropic drugs-namely, haloperidol, morphine, and diazepam--failed to alter either soluble or particulate levels of cyclic AMP-dependent protein kinase activity in this brain region following chronic administration. When the total particulate fraction was subfractionated, it was found that chronic imipramine treatment significantly increased the activity of cyclic AMP-dependent protein kinase in crude nuclear fractions but not in crude synaptosomal or microsomal fractions. Taken together, the data raise the possibility that chronic antidepressant treatments may stimulate the translocation of cyclic AMP-dependent protein kinase from the cytosol to the nucleus. This effect would represent a novel action of antidepressants that could contribute to the long-term adaptive changes in brain thought to be essential for the clinical actions of these treatments.     

The Differential Response of Protein Kinase A to Cyclic AMP in Discrete Brain Areas Correlates with the Abundance of Regulatory Subunit II

Abstract: We analyzed the expression and relative distribution of mRNA for the regulatory subunits (RIα, RIIα, and RIIβ) and of 150-kDa RIIβ-anchor proteins for cyclic AMP (cAMP)-dependent protein kinase (PKA) into discrete brain regions. The subcellular distribution of both holoenzyme and free catalytic subunit was evaluated in the same CNS areas. In the neocortex and corpus striatum high levels of RIIβ paralleled the presence of specific RII-anchoring proteins, high levels of membrane-bound PKA holoenzyme, and low levels of cytosolic free catalytic activity (C-PKA). Conversely, in brain areas showing low RIIβ levels (cerebellum, hypothalamus, and brainstem) we found an absence of RII-anchoring proteins, low levels of membrane-bound holoenzyme PKA, and high levels of cytosolic dissociated C-PKA. Response to cAMP stimuli was specifically evaluated in the neocortex and cerebellum, prototypic areas of the two different patterns of PKA distribution. We found that cerebellar holoenzyme PKA was highly sensitive to cAMP-induced dissociation, without, however, a consistent translocation of C-PKA into the nucleus. In contrast, in the neocortex holoenzyme PKA was mainly in the undissociated state and poorly sensitive to cAMP. In nuclei of cortical cells cAMP stimulated the import of C-PKA and phosphorylation of cAMP-responsive element binding protein. Taken together, these data suggest that RIIβ (whose distribution is graded throughout the CNS, reaching maximal expression in the neocortex) may represent the molecular cue of the differential nuclear response to cAMP in different brain areas, by controlling cAMP-induced holoenzyme PKA dissociation and nuclear accumulation of catalytic subunits.     

Direct Effects of Chronic Ethanol Exposure on β-Adrenergic and Adenosine-Sensitive Adenylate Cyclase Activities and Cyclic AMP Content in Primary Cerebellar Cultures

The direct effects of chronic ethanol exposure on adenylate cyclase activity and cyclic AMP content were investigated in primary cerebellar cultures. By morphological criteria these cultures mainly contain granule cells with some astrocytes, and each cell type appears to contain both beta-adrenergic and adenosine-sensitive adenylate cyclase systems. Chronic treatment of the primary cerebellar cultures with 120 mM ethanol for 6 days caused a reduction in the stimulation of cyclic AMP content by isoproterenol and by the adenosine analogue 2-chloroadenosine. Kinetic analysis indicated that the chronic ethanol treatment decreased maximal activation of adenylate cyclase, as well as increased the EC50 values for norepinephrine and 2-chloroadenosine. Activation of norepinephrine-stimulated adenylate cyclase activity by in vitro ethanol was significantly enhanced after the chronic ethanol exposure. However, the chronic treatment did not alter activation of the 2-chloroadenosine-stimulated enzyme by in vitro ethanol. A similar difference in the response to in vitro ethanol after the chronic treatment was observed when cyclic AMP content of the intact cells was measured. The present data indicate that chronic ethanol exposure causes a selective increase in the sensitivity of adenylate cyclase to ethanol in some brain cells and a more generalized desensitization of receptor-stimulated cyclic AMP production.     

Accumulations of Cyclic AMP in Adenine-Labeled Cell-free Preparations from Guinea Pig Cerebral Cortex: Role of α-Adrenergic and H1-Histaminergic Receptors

Norepinephrine, histamine, adenosine, glutamate, and depolarizing agents elicit accumulations of radioactive cyclic AMP from adenine-labeled nucleotides in particulate fractions from Krebs-Ringer homogenates of guinea pig cerebral cortex. The particulate fractions contain sac-like entities, which apparently are associated with a significant portion of the membranal adenylate cyclase. Particulate fractions from sucrose homogenates are a less effective source of such responsive entities. Activation of the adenine-labeled cyclic AMP-generating systems by norepinephrine is by means of alpha-adrenergic receptors, while activation by histamine is through H1- and H2-histaminergic receptors. Adenosine responses are potentiated by the amines and are antagonized by alkylxanthines. Glutamate and depolarizing agents appear to elicit accumulations of cyclic AMP via "release" of endogenous adenosine. It is proposed, based on the virtual absence of an alpha-adrenergic or H1-histaminergic response in the presence of a combination of potent adenosine and H2-histaminergic antagonists, that alpha-adrenergic and H1-histaminergic receptor mechanisms do not activate adenylate cyclase directly in brain slices or Krebs-Ringer particulate fractions, but merely facilitate activation by beta-adrenergic, H2-histaminergic, or adenosine receptors.     

Cyclic AMP Inhibits Activation of Mitogen-Activated Protein Kinase and Cell Proliferation in Response to Growth Factors in Cultured Rat Cortical Astrocytes

Abstract: The cyclic AMP (cAMP)-induced inhibitory effect on cell proliferation was examined through inhibition of mitogen-activated protein kinase (MAP kinase) activation in cultured rat cortical astrocytes. Basic fibroblast growth factor (bFGF) at 10 ng/ml maximally stimulated MAP kinase activity, which peaks during 10 min and prolonged for 24 h. Likewise, DNA synthesis was maximally potentiated with 10 ng/ml bFGF and correlated with MAP kinase activity in a dose-dependent manner. Dibutyryl cAMP (dbcAMP) at 1 m M and isoproterenol at 10 µ M inhibited MAP kinase activation and DNA synthesis potentiation with bFGF and platelet-derived growth factor to the control level in cultured astrocytes and C6 glioma cells. The stimulation with bFGF caused a prominent translocation of MAP kinase from the cytosol to the nucleus after 1 h in astrocytes. Treatment of the cells with dbcAMP and isoproterenol completely prevented the translocation of MAP kinase. In experiments with ³²P-labeled cultured astrocytes, phosphorylation of Raf-1 was apparently stimulated with bFGF. Treatment with dbcAMP or isoproterenol had a greatly inhibitory effect on the stimulation of Raf-1 phosphorylation with bFGF. Consistent with the effect on Raf-1 phosphorylation, dbcAMP and isoproterenol completely prevented bFGF-induced phosphorylation of MAP kinase kinases, target proteins of Raf-1. Our observations suggest that cAMP-induced suppression of cell growth in astrocytes is due to the inhibitory effect on activation of MAP kinase and its translocation to the nucleus and that the site of the cAMP action is located at Raf-1 or the upstream site of Raf-1.     

Effects of Different Types of Stimulation on Cyclic AMP Content in the Rabbit Carotid Body: Functional Significance

Cyclic AMP levels in rabbit carotid bodies incubated under control conditions, 100% O2- or 95% O2/5% CO2- equilibrated medium, are close to 1 pmol/mg wet tissue (range 0.4-2.43 pmol/mg). Isobutylmethylxanthine (0.5 mM) increases cyclic AMP levels by a factor of 14 and 8 in HEPES- and CO2/CH3O(-)-buffered medium, respectively. Forskolin (0.5-10 microM) applied during 30 min increases cyclic AMP levels in a dose-dependent manner. Incubation of carotid bodies at low O2 tensions resulted in an elevation of cyclic AMP levels both in the absence and in the presence of isobutymethylxanthine. In the latter conditions cyclic AMP increase was maximum at an O2 tension of 46 mm Hg and tended to decrease at extremely low PO2. In isobutylmethylxanthine-containing Ca2(+)-free medium, cyclic AMP increased linearly with decreasing PO2 from 66 to 13 mm Hg; the absolute cyclic AMP levels attained in Ca2(+)-free medium were smaller than those observed in Ca2(+)-containing medium at any PO2. The differences between Ca2(+)-free and Ca2(+)-containing media appear to be due to the action of released neurotransmitters in the latter conditions, because dopamine and norepinephrine, which are known to be released by hypoxia in a Ca2(+)-dependent manner, increase cyclic AMP in the carotid body. Low pH/high PCO2 and high [K+]e increase cyclic AMP levels only in Ca2(+)-containing medium. Forskolin potentiates the release of catecholamines induced by low PO2. These results suggest that cyclic AMP plays an important role in the modulation of the chemoreception process.     

Lysophosphatidic Acid-Induced Neurite Retraction in PC12 Cells: Neurite-Protective Effects of Cyclic AMP Signaling

Abstract: Effects of the cyclic AMP second messenger system were studied on the retraction of neurites elicited by the phospholipid mediator lysophosphatidic acid (LPA) in PC12 cells. LPA stimulation inhibited adenylyl cyclase, indicating that the LPA receptor couples to the heterotrimeric G_i proteins. However, pertussis toxin or expression of dominant negative Ras did not prevent neurite retraction. In contrast, cholera toxin, forskolin, and application of dibutyryl-cyclic AMP prevented neurite retraction. The neurite-protective effect of forskolin was blocked by Rp -adenosine 3',5'-phosphorothioate. Forskolin and dibutyryl-cyclic AMP both failed to protect neurites in A126-1B2 and 123.7 cells, which lack cyclic AMP-activated protein kinase. Data indicate that elevation of cyclic AMP levels triggers a cyclic AMP-activated protein kinase-dependent mechanism that opposes the functioning of the morphoregulatory signaling activated by LPA. ADP-ribosylation of Rho by the Clostridium botulinum C-3 toxin in 123.7 cells caused neuronal differentiation, indicated by neurite extension, and blocked LPA-induced neurite retraction. LPA activates G_q- and G_i-linked signaling in parallel; therefore, a morphoregulatory signaling network hypothesis is proposed versus the simplistic approach of a signaling pathway. The signaling network integrates the receptor-activated individual, sequential, and parallel signaling events into an interactive network whose individual components may fulfill required and permissive functions encoding the cellular response.     

Pattern of crypt cell proliferation in the pre- and post-closure ileum of the neonatal rat: Effects of sympathectomy

Summary The ultrastructural localization of adenylate cyclase was accomplished cytochemically in the neurointermediate lobe of Sprague-Dawley rats. The main concentration of the reaction product was found on the plasmalemma of neurosecretory nerve fibers, their terminals and plasma membranes of pituicytes. Positive reaction for adenylate cyclase was found less regularly in endothelial cells, pericapillary spaces and processes of the basal lamina. The septum between the pars nervosa and the pars intermedia showed heavy deposits of the reaction product, especially around the neurosecretory nerve fibers but also around other types of nerve fibers. Reaction for adenylate cyclase was not seen in the cells of the pars intermedia. When the substrate (ATP) was omitted, no reaction product was found. These findings support the suggestion of an involvement of cyclic AMP in the release mechanism of neurohypophysial hormones from the neurosecretory nerve terminals, and possibly also their transfer into blood vessels and perivascular channels.Supported by M.R.C. (Canada)Carreer Investigator of the Medical Research Council of Canada     

Adenosine and Cyclic AMP in Rat Cerebral Cortical Slices: Effects of Adenosine Uptake Inhibitors and Adenosine Deaminase Inhibitors

The endogenous levels of adenosine functionally linked to cyclic AMP systems in rat cerebral cortical slices are regulated by both adenosine deaminase and adenosine uptake systems. 2'-Deoxycoformycin (2'-DCF), an adenosine deaminase inhibitor, slightly increased basal, adenosine, and norepinephrine-elicited accumulations of cyclic AMP, whereas dipyridamole, an uptake inhibitor, had an even greater effect on cyclic AMP accumulations under the same conditions. Combinations of 2'-DCF and dipyridamole elicited a greater effect than either compound alone. Neither 2'-DCF nor dipyridamole significantly augmented accumulations of cyclic AP elicited by a depolarizing agent, veratridine, suggesting that the adenosine "released" during neuronal depolarization of brain slices is not as subject to inactivation by uptake or deamination as endogenous adenosine in control brain slices. The accumulation of cyclic AMP elicited by a combination of norepinephrine and veratridine was greater than additive. The response to a pure beta-adrenergic agonist, isoproterenol, was not potentiated by 2'-DCF, dipyridamole, or veratridine, consonant with minimal interaction of endogenous adenosine with beta-adrenergic systems.