Mechanism of guanosine-induced neuroprotection in rat hippocampal slices submitted to oxygen-glucose deprivation

Guanine derivates have been implicated in many relevant extracellular roles, such as modulation of glutamate transmission, protecting neurons against excitotoxic damage. Guanine derivatives are spontaneously released to the extracellular space from cultured astrocytes during oxygen-glucose deprivation (OGD) and may act as trophic factors, glutamate receptors blockers or glutamate transport modulators, thus promoting neuroprotection. The aim of this study was to evaluate the mechanisms involved in the neuroprotective role of the nucleoside guanosine in rat hippocampal slices submitted to OGD, identifying a putative extracellular binding site and the intracellular signaling pathways related to guanosine-induced neuroprotection. Cell damage to hippocampal slices submitted to 15 min of OGD followed by 2 h of reperfusion was decreased by the addition of guanosine (100 microM) or guanosine-5'-monophosphate (GMP, 100 microM). The neuroprotective effect of guanosine was not altered by the addition of adenosine receptor antagonists, nucleosides transport inhibitor, glutamate receptor antagonists, glutamate transport inhibitors, and a non-selective Na(+) and Ca(2+) channel blocker. However, in a Ca(2+)-free medium (by adding EGTA), guanosine was ineffective. Nifedipine (a Ca(2+) channel blocker) increased the neuroprotective effect of guanosine and 4-aminopyridine, a K(+) channel blocker, reversed the neuroprotective effect of guanosine. Evaluation of the intracellular signaling pathways associated with guanosine-induced neuroprotection showed the involvement of PKA, PKC, MEK and PI-3 K pathways, but not CaMKII. Therefore, this study shows guanosine is acting via K(+) channels activation, depending on extracellular Ca(2+) levels and via modulation of the PKA, PKC, MEK and/or PI-3 K pathways.     

The possible involvement of both calcium and cyclic AMP in the dopaminergic inhibition of prolactin secretion

Dopamine inhibited basal, TRH-, IBMX- and A23187-stimulated prolactin secretion from rat anterior pituitary cells. However, dopamine did not inhibit prolactin secretion stimulated by elevated K+ concentrations. These data are interpreted in terms of dopaminergic inhibition of both cyclic AMP production and Ca2+ influx through agonist-, but not voltage-, dependent Ca2+ channels.     

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.     

K+-Dependent Stimulation of Dopamine Synthesis in Striatal Synaptosomes Is Mediated by Protein Kinase C

Dopamine synthesis rate was measured in striatal synaptosomes. Removal of Na⁺ increased synthesis rate; this was blocked in Ca²⁺-free medium and by addition of the Ca²⁺/calmodulin inhibitor N-6-aminohexyl-5-chloro-1-naphthalenesulfonamide (W7). The increase in dopamine synthesis rate caused by the addition of the phorbol ester 12-O-tetradecanoylphorboI-13-acetate (TPA) was blocked by the protein kinase C inhibitor polymyxin B. K⁺-stimulated synthesis was unchanged in Ca²⁺-free medium or by addition of W7; it was blocked by polymyxin B. The effect of 50 mM K⁺ was additive with that of 8-Br cyclic AMP and of Na⁺ removal; the combined effect of 50 mM K⁺ and TPA was no greater than that of either alone. These results suggest that stimulation of dopamine synthesis in striatal synaptosomes by 50 mM K⁺ is mediated by protein kinase C.     

Expression of A1 Adenosine Receptors Modulating Dopamine-Dependent Cyclic AMP Accumulation in the Chick Embryo Retina

Dopamine and 2-chloroadenosine independently promoted the accumulation of cyclic AMP in retinas from 16-day-old chick embryos. The two compounds added together either in saturating or subsaturating concentrations were not additive for the accumulation of the cyclic nucleotide in the tissue. This fact was shown to be due to the existence of an adenosine receptor that mediates the inhibition of the dopamine-dependent cyclic AMP accumulation in the retina. Adenosine inhibited, in a dose-dependent fashion, the accumulation of cyclic AMP induced by dopamine in 12-day-old chick embryo retinas, with an IC50 of approximately 1 microM. This effect was not blocked by dipyridamole. N6-(l-Phenylisopropyl)adenosine, (l-PIA) was the most potent adenosine analog tested, showing an IC50 of 0.1 microM which was two orders of magnitude lower than its stereoisomer d-PIA (10 microM). The maximal inhibition of the dopamine-elicited cyclic AMP accumulation by adenosine and related analogs was 70%. The inhibitory effect promoted by adenosine was blocked by 3-isobutyl-1-methylxanthine (IBMX) or by adenosine deaminase. Adenine was not effective; whereas ATP and AMP promoted the inhibition of the dopamine effect only at very high concentrations. Apomorphine was only 30% as effective as dopamine in promoting the cyclic AMP accumulation in retinas from 11- to 12-day-old embryos and 2-chloroadenosine did not interfere with the apomorphine-mediated shift in cyclic AMP levels. In the retinas from 5-day-old posthatched chickens dopamine and apomorphine were equally effective in eliciting the accumulation of cyclic AMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transient Increase of Cyclic AMP Induced by Glutamate in Cultured Neurons from Rat Spinal Cord

Abstract: We demonstrated that glutamate increased the cyclic AMP level in cultured neurons from rat spinal cord. A bath application of glutamate (300 µ M ) elicited a rapid increase of the cyclic AMP concentration reaching a level three times as high as the basal level in ∼3 min, and its content then decreased to the control level in 15 min. The increase was not observed in a Ca²⁺-free medium and was inhibited by an antagonist of NMDA receptors or a voltage-sensitive Ca²⁺ channel blocker. Preincubation with W7 also inhibited the glutamate-evoked cyclic AMP increase. NMDA, aspartate, and high-K⁺ conditions also induced a cyclic AMP increase; however, a decreasing phase did not follow. The decreasing phase was observed when (2 S ,1' S ,2' S )-2-(carboxycyclopropyl)-glycine, a potent agonist for metabotropic glutamate receptors, was combined with NMDA. These results suggest that the cyclic AMP increase is mediated by a Ca²⁺ influx via both NMDA receptors and voltage-sensitive Ca²⁺ channels followed by an activation of the Ca²⁺/calmodulin system, and the decreasing phase observed in the case of glutamate exposure is due to the activation of the metabotropic glutamate receptors.     

K+-Evoked Depolarization Stimulates Cyclic AMP Accumulation in Photoreceptor-Enriched Retinal Cell Cultures: Role of Calcium Influx Through Dihydropyridine-Sensitive Calcium Channels

The effect of membrane depolarization on cyclic AMP synthesis was studied in glia-free, low-density, monolayer cultures of chick retinal photoreceptors and neurons. In photoreceptor-enriched cultures prepared from embryonic day 6 retinas and cultured for 6 days, elevated K+ concentrations increased the intracellular concentration of cyclic AMP and stimulated the conversion of [3H]adenine to [3H]cyclic AMP. The K(+)-evoked increase of cyclic AMP accumulation was blocked by omitting CaCl2 from the incubation medium, indicating a requirement for extracellular Ca2+. Stimulation of cyclic AMP accumulation was also inhibited by nifedipine, methoxyverapamil, Cd2+, Co2+, and Mg2+, and was enhanced by the dihydropyridine Ca2+ channel agonist Bay K 8644. The enhancement of K(+)-evoked cyclic AMP accumulation by Bay K 8644 was antagonized by nifedipine. Thus, Ca2+ influx through dihydropyridine-sensitive channel is required for depolarization-evoked stimulation of cyclic AMP accumulation in photoreceptor-enriched cultures.     

Role of Excitatory Amino Acid Receptors in K+-and Glutamate-Evoked Release of Endogenous Adenosine from Rat Cortical Slices

K+ and glutamate released endogenous adenosine from superfused slices of rat parietal cortex. The absence of Ca2+ markedly diminished K+- but not glutamate-evoked adenosine release. Tetrodotoxin decreased K+- and glutamate-evoked adenosine release by 40 and 20%, respectively, indicating that release was mediated in part by propagated action potentials in the slices. Inhibition of ecto-5'-nucleotidase by alpha,beta-methylene ADP and GMP decreased basal release of adenosine by 40%, indicating that part of the adenosine was derived from the extracellular metabolism of released nucleotide. In contrast, inhibition of ecto-5'-nucleotidase did not affect release evoked by K+ or glutamate, suggesting that adenosine was released as such. Inhibition of glutamate uptake by dihydrokainate potentiated glutamate-evoked release of adenosine. Glutamate-evoked adenosine release was diminished 50 and 55% by the N-methyl-D-aspartate (NMDA) receptor antagonists, DL-2-amino-5-phosphonovaleric acid and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801), respectively. The remaining release in the presence of MK-801 was diminished a further 66% by the non-NMDA receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione, suggesting that both NMDA and non-NMDA receptors were involved in glutamate-evoked adenosine release. Surprisingly, K+-evoked adenosine release was also diminished about 30% by NMDA antagonists, suggesting that K+-evoked adenosine release may be partly mediated indirectly through the release of an excitatory amino acid acting at NMDA receptors.     

Activation of adenosine A1 and A2A receptors modulates dopamine D2 receptor-induced responses in stably transfected human neuroblastoma cells

Adenosine can influence dopaminergic neurotransmission in the basal ganglia via postsynaptic interaction between adenosine A2A and dopamine D2 receptors. We have used a human neuroblastoma cell line (SH-SY5Y) that was found to express constitutively moderate levels of adenosine A1 and A2A receptors (approximately 100 fmol/mg of protein) to investigate the interactions of A2A/D2 receptors, at a cellular level. After transfection with human D2L receptor cDNA, SH-SY5Y cells expressed between 500 and 1,100 fmol of D2 receptors/mg of protein. In membrane preparations, stimulation of adenosine A2A receptors decreased the affinity of dopamine D2 receptors for dopamine. In intact cells, the calcium concentration elevation induced by KCI treatment was moderate, and dopamine had no effect on either resting intracellular free Ca2+ concentration ([Ca2+]i) or KCI-induced responses. In contrast, pretreatment with adenosine deaminase for 2 days dramatically increased the elevation of [Ca2+]i evoked by KCI, which then was totally reversed by dopamine. The effects induced by 48-h adenosine inactivation were mimicked by application of adenosine A1 antagonists and could not be further reversed by acute activation of either A1 or A2A receptors. Acute application of the selective A2 receptor agonist CGS-21680 counteracted the D2 receptor-induced [Ca2+]i responses. The present study shows that SH-SY5Y cells are endowed with functional adenosine A2A and A1 receptors and that A2A receptors exert an antagonistic acute effect on dopamine D2 receptor-mediated functions. In contrast, A1 receptors induce a tonic modulatory role on these dopamine functions.     

Regulation of tyrosine hydroxylase activity and phosphorylation at Ser(19) and Ser(40) via activation of glutamate NMDA receptors in rat striatum

The activity of tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of dopamine, is stimulated by phosphorylation. In this study, we examined the effects of activation of NMDA receptors on the state of phosphorylation and activity of tyrosine hydroxylase in rat striatal slices. NMDA produced a time-and concentration-dependent increase in the levels of phospho-Ser(19)-tyrosine hydroxylase in nigrostriatal nerve terminals. This increase was not associated with any changes in the basal activity of tyrosine hydroxylase, measured as DOPA accumulation. Forskolin, an activator of adenylyl cyclase, stimulated tyrosine hydroxylase phosphorylation at Ser(40) and caused a significant increase in DOPA accumulation. NMDA reduced forskolin-mediated increases in both Ser(40) phosphorylation and DOPA accumulation. In addition, NMDA reduced the increase in phospho-Ser(40)-tyrosine hydroxylase produced by okadaic acid, an inhibitor of protein phosphatase 1 and 2A, but not by a cyclic AMP analogue, 8-bromo-cyclic AMP. These results indicate that, in the striatum, glutamate decreases tyrosine hydroxylase phosphorylation at Ser(40) via activation of NMDA receptors by reducing cyclic AMP production. They also provide a mechanism for the demonstrated ability of NMDA to decrease tyrosine hydroxylase activity and dopamine synthesis.     

Cyclic AMP-mediated regulation of striatal glutamate release: interactions of presynaptic ligand- and voltage-gated ion channels and G-protein-coupled receptors

The presynaptic regulation of striatal glutamate transmission was investigated using D-[3H]aspartate and mouse striatal slices. Functional changes in voltage-dependent and glutamate receptor-gated ion channels were elicited by pharmacologically modifying intracellular cyclic AMP formation via G-protein-coupled receptor stimulation. The kainate (KA)-evoked release was potentiated by the stimulatory G-protein (G(s))-coupled beta-adrenoceptor agonist isoproterenol (ISO) in a concentration-dependent manner. This effect was mimicked by the specific calmodulin (CaM) antagonists trifluoperazine and calmidazolium. Tetrodotoxin (TTX), a blocker of Na(+) channels, did not affect the basal release but inhibited to the same degree the releases evoked by kainate alone and by kainate and isoproterenol together. Vinpocetine, a blocker of voltage-dependent Na(+) channels, did not alter the basal or the evoked release. The Na(+) channel activator veratridine enhanced the basal release in a concentration-dependent manner and isoproterenol attenuated this effect. The opposite effects of isoproterenol on the kainate- and veratridine-evoked releases may reflect prevention of the cyclic AMP-protein kinase A (PKA) phosphorylation cascade in striatal glutamatergic signal transduction. In addition, the calmidazolium-induced potentiation of kainate-evoked release was thwarted by LY354740 and L-2-amino-4-phosphonobutanoate, agonists of the inhibitory G-protein (G(i))-coupled metabotropic group II and III glutamate receptors (mGluRs). Vinpocetine, which inhibits the CaM-dependent phosphodiesterase (PDE1), was likewise inhibitory. In turn, selective agonists and antagonists of the G(q)-protein-coupled group I mGluRs and (S)-3,5-dihydroxyphenylglycine (3,5-DHPG) and (RS)-1-aminoindan-1,5-dicarboxylate (AIDA), which modulate the intracellular Ca(2+) levels, did not alter the kainate-evoked release.The beta-adrenoceptor-mediated cyclic AMP accumulation seems to downregulate Na(+) channels but to enhance glutamate release by means of upregulation of kainate receptors. This regulation of presynaptic ligand- and voltage-gated ion channels is affected by the cAMP-protein kinase A-dependent phosphorylation cascade and controlled by G(i)-protein-coupled mGluRs.     

Rat fat-cells have three types of adenosine receptors (Ra, Ri and P). Differential effects of pertussis toxin.

Activation of rat adipocyte R1 adenosine receptors by phenylisopropyladenosine (PIA) decreased cyclic AMP and lipolysis; this effect was blocked in cells from pertussis-toxin-treated rats. In contrast, the ability of 2',5'-dideoxyadenosine to decrease cyclic AMP was not affected by pertussis-toxin treatment. Addition of adenosine deaminase to the medium in which adipocytes from control animals were incubated resulted in activation of lipolysis. Interestingly, adipocytes from toxin-treated rats (which had an already increased basal lipolysis) responded in an opposite fashion to the addition of adenosine deaminase, i.e. the enzyme decreased lipolysis, which suggested that adenosine might be increasing lipolysis in these cells. Studies with the selective agonists N-ethylcarboxamidoadenosine (NECA) and PIA indicated that adenosine increases lipolysis and cyclic AMP accumulation in these cells and that these actions are mediated through Ra adenosine receptors. Adenosine-mediated accumulation of cyclic AMP was also observed in cells preincubated with pertussis toxin (2 micrograms/ml) for 3 h. In these studies NECA was also more effective than PIA. Our results indicate that there are three types of adenosine receptors in fat-cells, whose actions are affected differently by pertussis toxin, i.e. Ri-mediated actions are abolished, Ra-mediated actions are revealed and P-mediated actions are not affected.     

Regulation of GH3-cell function via adenosine A1 receptors. Inhibition of prolactin release, cyclic AMP production and inositol phosphate generation.

We examined the mechanism by which adenosine inhibits prolactin secretion from GH3 cells, a rat pituitary tumour line. Prolactin release is enhanced by vasoactive intestinal peptide (VIP), which increases cyclic AMP, and by thyrotropin-releasing hormone (TRH), which increases inositol phosphates (IPx). Analogues of adenosine decreased prolactin release, VIP-stimulated cyclic AMP accumulation and TRH-stimulated inositol phospholipid hydrolysis and IPx generation. Inhibition of InsP3 production by R-N6-phenylisopropyladenosine (R-PIA) was rapid (15 s) and was not affected by the addition of forskolin or the removal of external Ca2+. Addition of adenosine deaminase or the potent adenosine-receptor antagonist, BW-A1433U, enhanced the accumulation of cyclic AMP by VIP, indicating that endogenously produced adenosine tonically inhibits adenylate cyclase. The potency order of adenosine analogues for inhibition of cyclic AMP and IPx responses (measured in the presence of adenosine deaminase) was N6-cyclopentyladenosine greater than R-PIA greater than 5'-N-ethylcarboxamidoadenosine. This rank order indicates that inhibitions of both cyclic AMP and InsP3 production are mediated by adenosine A1 receptors. Responses to R-PIA were blocked by BW-A1433U (1 microM) or by pretreatment of cells with pertussis toxin. A greater amount of toxin was required to eliminate the effect of R-PIA on inositol phosphate than on cyclic AMP accumulation. These data indicate that adenosine, in addition to inhibiting cyclic AMP accumulation, decreases IPx production in GH3 cells, possibly by directly inhibiting phosphoinositide hydrolysis.     

Involvement of glutamate receptors of the NMDA type in the modulation of acetylcholine and glutamate overflow from the guinea pig ileum during in vitro hypoxia and hypoglycaemia

The involvement of NMDA glutamate receptors in the effects of glucose/oxygen deprivation (in vitro ischaemia) on spontaneous endogenous acetylcholine and glutamate overflow from the guinea pig ileum was studied. Neurotransmitter overflow was measured by HPLC. Deprivation of glucose in the medium slightly reduced acetylcholine overflow, and did not significantly influence glutamate overflow. During oxygen deprivation and glucose/oxygen deprivation, acetylcholine overflow augmented with a biphasic modality: an early peak was followed by a long lasting increase, whereas glutamate overflow increased with a rapid and sustained modality. The effects of glucose/oxygen deprivation on both acetylcholine and glutamate overflow were abolished after reperfusion with normal oxygenated medium. Acetylcholine and glutamate overflow induced by glucose/oxygen deprivation were significantly reduced in the absence of external Ca(2+) as well as by the addition of the mitochondrial Na(+)-Ca(2+) exchanger blocker, CGP 37157, and of the endoplasmic reticulum Ca(2+)/ATPase blocker, thapsigargin. +/-AP5, an NMDA receptor antagonist, and 5,7-diCl-kynurenic acid, an antagonist of the glycine site associated to NMDA receptor, markedly depressed glucose/oxygen deprivation-induced acetylcholine and glutamate overflow as well. Our results suggest that in vitro simulated ischaemia evokes acetylcholine and glutamate overflow from the guinea pig ileum, which is partly linked to an increase in intracellular Ca(2+) concentration dependent on both Ca(2+) influx from the extracellular space and Ca(2+) mobilization from the endoplasmic reticulum and mitochondrial stores. During glucose/oxygen deprivation, ionotropic glutamate receptors of the NMDA type exert both a positive feedback modulation of glutamate output and contribute to increased acetylcholine overflow.     

Change of Cyclic AMP Level in Synaptosomes from Cerebral Cortex; Increase by Adenosine Derivatives

The endogenous level of cyclic AMP in incubated synaptosomes from cerebral cortex of guinea pigs was investigated after the addition of various agents to the incubation medium. It appeared that the synaptosomal suspension already contained exogenous adenosine. Preincubation with theophylline or with adenosine deaminase (ADase) decreased both the exogenous level of adenosine and the intrasynaptosomal level of cyclic AMP. The level of cyclic AMP was reincreased by the addition of adenosine agonists, especially 2-chloroadenosine. This increase was antagonized by deoxyadenosine and was not inhibited by dipyridamole. These results suggest that the adenosine derivatives in the synaptic cleft regulate the level of cyclic AMP in nerve terminals through adenosine receptor on the presynaptic membrane. ADP, ATP, dopamine, and histamine also stimulate the formation of cyclic AMP in the ADase-treated synaptosomes.     

Glutamate agonists and [3H]GABA release from rat hippocampal slices: Involvement of metabotropic glutamate receptors in the quisqualate-evoked release

The effects of glutamate agonists and their selective antagonists on the Ca²⁺-dependent and independent releases of [³H]GABA from rat coronal hippocampal slices were studied in a superfusion system. The Ca²⁺-dependent release evoked by glutamate, kainate and N-methyl-D-aspartate (NMDA) gradually declined with time despite the continuous presence of the agonists. Quisqualate (QA) caused a sustained release which exhibited no tendency to decline within the 20-min period of stimulation. This release was enhanced in Ca²⁺-free medium. The release evoked by QA in Ca²⁺-containing medium was significantly inhibited by (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohept-5,10-imine hydrogen maleate (MK-801) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), showing that QA activates NMDA receptors directly or indirectly through (RS)--amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. The inhibition of MK-801 was slightly diminished and that of CNQX totally abolished in Ca²⁺-free medium. Verapamil inhibited the QA-activated release in both Ca²⁺-containing and Ca²⁺-free media. The effect of QA but not that of AMPA was blocked in Ca²⁺-free medium by L(+)-2-amino-3-phosphonopropionate (L-AP3), a selective antagonist of the metabotropic glutamate receptor. It is suggested that the sustained release of GABA is also mediated partly by activation of metabotropic receptors and mobilization of Ca²⁺ from intracellular stores.     

In Vivo Evidence that Nonneuronal β-Adrenoceptors as Well as Dopamine Receptors Contribute to Cyclic AMP Efflux in Rat Striatum

Abstract: We applied in vivo microdialysis to assess the effects of dopaminergic and β-adrenergic receptor stimulation on cyclic AMP efflux in rat striatum under chloral hydrate anesthesia. Dopamine (up to 1 mM) infused for 20 min through the probe did not increase cyclic AMP, whereas both the selective dopamine D₁ agonist SKF 38393 and D₂ antagonist sulpiride produced modest increases. It is interesting that the β-adrenoceptor agonist isoproterenol produced a marked increase (204.7% of basal level at 1 mM) which was antagonized by the β-adreno-ceptor antagonist propranolol. Pretreatment with a glial selective metabolic inhibitor, fluorocitrate (1 mM), by a 5-h infusion through the probe attenuated basal cyclic AMP efflux by 30.3% and significantly blocked the response to isoproterenol. By contrast, striatal injection of a neuro-toxin, kainic acid (2.5 μg), 2 days before the dialysis experiment did not affect basal cyclic AMP or the response to isoproterenol, but blocked the response to SKF 38393. These data demonstrate that β-adrenoceptors as well as dopamine receptors contribute to cyclic AMP efflux in rat striatum in vivo. They also suggest that basal and β-adre-noceptor-stimulated cyclic AMP efflux are substantially dependent on intact glial cells.     

Protein Kinase C Activation Enhances K+-Stimulated Endogenous Dopamine Release from Rat Striatal Synaptosomes in the Absence of an Increase in Cytosolic Ca2+

The possibility that protein kinase C modulates neurotransmitter release in brain was investigated by examining the effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on Ca2+ transport and endogenous dopamine release from rat striatal synaptosomes. TPA (0.16 and 1.6 microM) significantly increased dopamine release by 24 and 33%, respectively, after a 20-min preincubation with TPA followed by 60 s of depolarization with 30 mM KCl. Depolarization-induced 45Ca2+ uptake, measured simultaneously with dopamine release, was not significantly increased by TPA. Neither 45Ca2+ uptake nor dopamine release was altered under resting conditions. When the time course of K+-stimulated 45Ca2+ uptake and dopamine release was examined, TPA (1.6 microM) enhanced dopamine release after 15, 30, and 60 s, but not 1, 3, or 5 s, of depolarization. A slight increase in 45Ca2+ uptake after 60 s of depolarization was also seen. The addition of 30 mM KCl to synaptosomes which had been preloaded with the Ca2+-sensitive fluorophore fura-2 increased the cytosolic free Ca2+ concentration ([Ca2+]i) from 445 nM to 506 nM after 10 s of depolarization and remained elevated after 60 s. TPA had no effect on [Ca2+]i under depolarizing or resting conditions. Replacing extracellular Ca2+ with 100 microM EGTA reduced K+-stimulated (60 s) endogenous dopamine release by 53% and decreased [Ca2+]i to 120 nM. In Ca2+-free medium, 30 mM KCl did not produce an increase in the [Ca2+]i. TPA (1.6 microM) did not alter the [Ca2+]i under resting or depolarizing conditions, but did increase K+-stimulated dopamine release in Ca2+-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine Receptor Activation and the Regulation of Tyrosine Hydroxylase Activity in PC 12 and PC 18 Cells

We compared the response of rat PC12 cells and a derivative PC18 cell line to the effects of adenosine receptor agonists, antagonists, and adenine nucleotide metabolizing enzymes. We found that theophylline (an adenosine receptor antagonist), adenosine deaminase, and AMP deaminase all decreased basal cyclic AMP content and tyrosine hydroxylase activity in the PC12 cells, but not in PC18 cells. Both cell lines responded to the addition of 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine, adenosine receptor agonists, by exhibiting an increase in tyrosine hydroxylase activity and cyclic AMP content. The latter finding indicates that both cell lines contained an adenosine receptor linked to adenylate cyclase. We found that the addition of dipyridamole, an inhibitor of adenosine uptake, produced an elevation of cyclic AMP and tyrosine hydroxylase activity in both cell lines. Deoxycoformycin, an inhibitor of adenosine deaminase, failed to alter the levels of cyclic AMP or tyrosine hydroxylase activity. This suggests that uptake was the primary inactivating mechanism of adenosine action in these cells. We conclude that both cell types generated adenine nucleotides which activate the adenosine receptor in an autocrine or paracrine fashion. We found that PC12 cells released ATP in a calcium-dependent process in response to activation of the nicotinic receptor. We also measured the rates of degradation of exogenous ATP, ADP, and AMP by PC12 cells. We found that the rates of metabolism of the former two were at least an order of magnitude greater than that of AMP. Any released ATP would be rapidly metabolized to AMP and then more slowly degraded to adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)