Synaptic membrane proteins as substrates for cyclic AMP-stimulated protein phosphorylation in various regions of rat brain.

Synaptosomal plasma membranes from mammalian brain contain protein kinase activity which phosphorylates endogenous membrane proteins and is stimulated by cyclic AMP. Using polyacrylamide gel electrophoresis it was shown that at least ten proteins in the synaptosomal plasma membrane fraction could be phosphorylated by endogenous cyclic AMP-stimulated protein kinase activity. The number of proteins whose phosphorylation was stimulated by cyclic AMP was strongly influenced by the pH and Mg2+ concentration used in the phosphorylation reaction. A complex pattern of cyclic AMP-stimulated protein phosphorylation was obtained only with synaptosomal plasma membranes and a crude microsomal fraction. Mitochondrial and myelin fractions exhibited no cyclic AMP-stimulated protein kinase activity. Investigation of the distribution of substrates for cyclic AMP-stimulated phosphorylation among various brain regions failed to reveal any regional differences.

Effects of mixed chimeric bone marrow repopulation on platelet storage pool-associated bleeding defects in mouse mutants.

We have previously shown mouse platelet storage pool deficiency (SPD) to be associated with lesions at eight different genetic loci, each of which is sufficient to produce murine SPD. We have also shown that normal bleeding times and normal platelet functions are restored when mice with SPD are transplanted with marrow from normal mice. Conversely, when normal mice are transplanted with mutant marrow, they present symptoms of SPD. In order to determine the amount of normal platelets needed to prevent the prolonged bleeding times associated with SPD, we established stable mixed chimeric mice by transplanting various ratios of normal and mutant marrow into lethally irradiated host animals. The proportion of normal input marrow correlated well with the proportion of normal peripheral red blood cells and platelets determined in chimerae 100 days after transplantation using direct morphology and electrophoretic variants of glucose phosphate isomerase to identify normal and mutant cell populations. The proportions of normal input marrow were also reflected in the proportions of platelets with normal and mutant platelet morphology in the chimerae. This confirms that the platelet abnormality in SPD is intrinsic to the stem cell population from which the platelets are derived. When bleeding times were determined in the mixed chimeric mice, a surprisingly high percentage of normal platelets (greater than 50% and sometimes greater than 75%) were needed to stop bleeding. These results suggest that the mutant platelets in the mixed chimeric mice may interfere with normal platelet aggregation patterns. They also raise some important considerations in devising treatment for SPD. Bleeding episodes in human SPD are normally treated by platelet transfusion. The results suggest that, at least in some cases, transfusions may not be effective. Also, in future gene therapy of this disease, it is like that a functional gene will have to be present in greater than 50% of stem cells for therapy to be effective.

Radiation inactivation analysis of the A1 adenosine receptor of rat brain. Decrease in radiation inactivation size in the presence of guanine nucleotide.

Radiation inactivation analysis of the binding of the A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine to rat brain membranes yielded a radiation inactivation size of 58 kDa. In the presence of GTP gamma S this was reduced to 33 kDa, in good agreement with the size of the ligand-binding subunit detected after photoaffinity labelling. The data indicate that the structural association of A1 adenosine receptors with G-protein components is altered in situ in the presence of guanine nucleotides.

The distribution of adenosine A1 receptors and 5'-nucleotidase in the hippocampal formation of several mammalian species.

The distributions of adenosine A1 receptors, as demonstrated by 3H-cyclohexyladenosine (3H-CHA) binding, and the adenosine-producing enzyme 5'-nucleotidase were examined in the hippocampal formation of the rat, mouse, gerbil, cat, hamster, rabbit, and guinea pig. The enzyme and binding sites were restricted to subregions and often individual layers of this structure. The distribution of 3H-CHA binding was consistent among the species with the strata radiatum and oriens of fields CA1 and CA3 exhibiting the highest levels of binding. A distinct band of 3H-CHA binding was observed in the stratum moleculare of the dentate gyrus; and in most species, this band was restricted to the inner one-third of the stratum moleculare (i.e., proximal to the stratum granulosum). The strata pyramidale, granulosum, and lucidum were in general only weakly positive for 3H-CHA binding. The binding to the stratum lacunosum/moleculare (or the distinct strata lacunosum and moleculare in the rabbit and cat) was moderate. In contrast to the relative consistency of the patterns of 3H-CHA binding in these species, 5'-nucleotidase exhibited wide variations in both the absolute amount of activity and its localization. In all species, the strata granulosum and pyramidale appeared devoid of 5'-nucleotidase activity. The only clear exception to this rule was the CA3 region of the cat where activity was seen between the cell bodies of stratum pyramidale. The strata radiatum and oriens of CA1 were strongly positive in the rat and hamster but only low to moderately stained in the other species examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoradiographic evidence for multiple CNS binding sites for adenosine derivatives.

The binding of the adenosine analogues, 5'-N-ethylcarboxamido[3H]adenosine and N6-cyclohexyl[3H]adenosine, to the rat brain was examined utilizing light-microscopic autoradiographic techniques. While associated with many of the same structures, binding sites for these compounds showed distinct differences in both their patterns of distribution and their capacities to be inhibited by the adenosine analogue, R-phenylisopropyladenosine. Previous studies have shown that, at nanomolar concentrations, cyclohexyladenosine and R-phenylisopropyladenosine bind rather exclusively to the A1 type of adenosine receptors. In contrast, 5'-N-ethylcarboxamidoadenosine has almost equal affinity for A1 and A2 sites. Taking advantage of these characteristics, non-A1 binding sites were resolved by examining 5'-N-ethylcarboxamidoadenosine binding in the presence of micromolar concentrations of unlabeled R-phenylisopropyladenosine. The autoradiographically demonstrated distribution of R-phenylisopropyladenosine-insensitive 5'-N-ethylcarboxamidoadenosine binding sites differed significantly from that of the cyclohexyladenosine binding sites. Such non-A1 binding sites were concentrated in the striatum, nucleus accumbens, medial geniculate, olfactory tubercle, amygdala and certain thalamic nuclei. In contrast to the distribution of A1 adenosine receptor sites, R-phenylisopropyladenosine-insensitive 5'-N-ethylcarboxamidoadenosine binding was only low to moderate in the hippocampus, cerebellum and superior colliculus, regions which are strongly positive for cyclohexyladenosine binding. The present study provides the first autoradiographic evidence for multiple adenosine binding sites in the brain by demonstrating that the adenosine analogue 5'-N-ethylcarboxamidoadenosine can bind to a site or sites distinct from the A1 adenosine receptor site. The 5'-N-ethylcarboxamidoadenosine binding site which is not displaced by low concentrations of R-phenylisopropyladenosine may correspond to an A2 adenosine receptor site and/or an as yet uncharacterized type of adenosine receptor.

The cellular localization of adenosine receptors in rat neostriatum.

Using quantitative autoradiography of ligand binding sites combined with lesions of specific neuronal pathways, the cellular locations of A1 and A2 adenosine receptors, as well as a third binding site for the adenosine receptor ligand, [3H]N-ethylcarboxamidoadenosine, and a nucleoside transporter were investigated in rat neostriatum. Intrastriatal kainic acid administration resulted in the loss of 50% of A1 adenosine receptors and virtually abolished ligand binding to A2 receptors. A small reduction in [3H]cyclohexyladenosine binding to striatal A1 receptors was found after lesioning the corticostriatal input. A2 receptor sites were unaffected by this treatment. Destruction of dopaminergic neurons using 6-hydroxydopamine or the raphestriatal serotoninergic input using 5,7-dihydroxytryptamine affected neither A1 nor A2 binding sites. These results indicate the localization of both A1 and A2 adenosine receptors on neurons intrinsic to the neostriatum and probably postsynaptic to the dopaminergic input. In addition, a binding site for [3H]N-ethylcarboxamidoadenosine which is not affected by the adenosine receptor agonist, R-phenylisopropyladenosine, was also partly abolished after kainic acid injection. In contrast, no significant change in the binding of the nucleoside transporter ligand, [3H]nitrobenzylthioinosine, was observed after any lesions, indicating the widespread association of this site with various cell types.

Stimulation of P2Y-purinoceptors on astrocytes results in immediate early gene expression and potentiation of neuropeptide action.

The action of adenosine-5'-O-(2-thiodiphosphate), a non-hydrolysable purine analogue and potent P2Y1-purinoceptor agonist, was studied on immediate early gene expression in rat astrocyte cultures. A rapid and transient increase in c-fos, junB, c-jun and Tis11 messenger RNA was observed in cultured astrocytes after treatment with adenosine-5'-O-(2-thiodiphosphate). Maximal induction of immediate early gene expression was obtained within 30 min of stimulation and c-fos was the most sensitive indicator of P2Y-purinoceptor activation. Calcitonin gene-related peptide has also been shown to be a potent inducer of c-fos messenger RNA in cultured astroglial cells. The combined stimulation of astrocytes with calcitonin gene-related peptide and adenosine-5'-O-(2-thiodiphosphate) resulted in the potentiated expression of c-fos messenger RNA. The superinduction of immediate early gene expression by calcitonin gene-related peptide and extracellular ATP in cultured astrocytes might result from intracellular signal transduction cross-talk, since adenosine-5'-O-(2-thiodiphosphate) was found to increase calcitonin gene-related peptide-induced cyclic AMP accumulation by 35%. Phorbol 12-myristate 13-acetate also increased calcitonin gene-related peptide-evoked cyclic AMP accumulation and led to the induction of immediate early gene expression, suggesting that protein kinase C might be at least in part involved in purinergic cross-talk. Our results demonstrate synergistic roles for extracellular ATP and calcitonin gene-related peptide in the transcriptional activation of astroglial cells.

1,3-Dipropyl-8-cyclopentylxanthine (DPCPX) inhibition of [3H]N-ethylcarboxamidoadenosine (NECA) binding allows the visualization of putative non-A1 adenosine receptors.

The binding of the adenosine receptor agonists, [3H]N-ethylcarboxamidoadenosine (NECA) and [3H]cyclohexyladenosine (CHA) to membrane preparations and to cryostat sections of the rat brain was examined. The xanthine derivative, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) was ca. 500-fold more effective at A1 than at A2 sites. [3H]CHA binding to A1 adenosine receptors was virtually eliminated by the inclusion of DPCPX (50 nM), while [3H]NECA binding was only partially inhibited. The pattern of DPCPX-insensitive [3H]NECA binding sites was strikingly different from that of A1 receptors and is believed to represent an association with A2 type adenosine receptors and perhaps another or several, previously undescribed non-A1 sites.

Heterogeneity of binding sites for N-ethylcarboxamido[3H]adenosine in rat brain: effects of N-ethylmaleimide.

Binding sites for N-ethylcarboxamido[3H]adenosine (NECA) in rat brain membranes and cryostat sections were examined in relation to their sensitivities to displacement by unlabeled NECA and R(-)-phenylisopropyladenosine (R-PIA). In membrane fractions from cerebral cortex, cerebellum, hippocampus and striatum, nanomolar concentrations of these adenosine receptor agonists displaced [3H]NECA such that R-PIA was more effective than NECA, consistent with the presence of an A1-adenosine receptor. At concentrations of displacing agent higher than 1 microM, R-PIA was unable to displace [3H]NECA further in cerebral cortex, cerebellum and hippocampus. In striatum, a second R-PIA-sensitive component of [3H]NECA binding was evident which was more sensitive to NECA than to R-PIA, i.e. it showed the characteristics of an A2-adenosine receptor. In striatum, however, R-PIA was also unable to displace [3H]NECA binding completely. Similar results were obtained in quantitative autoradiographic studies. Preincubation of cryostat sections with N-ethylmaleimide (NEM) abolished both the A1- and R-PIA-insensitive binding components such that both NECA and R-PIA were able to displace [3H]NECA binding completely. The remaining sites showed IC50 values of 0.13 and 3.68 microM for NECA and R-PIA, respectively. These A2-like [3H]NECA binding sites had a highly specific distribution in the brain, being concentrated in the striatum, nucleus accumbens and olfactory tubercle. The results indicate the presence in brain tissue of at least 3 classes of [3H]NECA binding sites, an A1-site, an A2-site and a third, unidentified R-PIA-insensitive site.

Regulation of the strength of adenosine modulation in the hippocampus by a differential distribution of the density of A1 receptors.

Correlative studies examining: (1) the binding of [3H]cyclohexyladenosine to A1 adenosine receptor sites, and (2) the depressive action of adenosine on evoked activity, were performed in the hippocampal formation. A greater number of A1 sites was observed in the dorsal versus ventral aspect of the hippocampus, and a larger depression of evoked potentials by adenosine was obtained in the dorsal aspect. These observations suggest that a differential density of A1 receptors might control the magnitude of modulatory action by adenosine on hippocampal circuitry.

Autoradiographic localization of adenosine A1 receptors in brainstem of fetal sheep.

Quantitative autoradiographic techniques were used to localize adenosine A1 receptors at the light microscopic level with the antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine [( 3H]DPCPX) in the brainstem of fetal sheep. Since adenosine has been proposed as a neuromodulator, which effects the depression of fetal breathing movements during hypoxia, attention was directed to respiratory neuronal areas. The highest density of A1 receptors in respiratory related groups was found in an area of the rostral ventrolateral medulla, which is ventral to the facial nucleus, caudal to the superior olive and lateral to the rostral inferior olive. Intermediate densities were seen in the medial and lateral parabrachial nuclei. Adenosine A1 receptor density was low in the areas of the nucleus of the solitary tract and the nucleus ambiguous. These data suggest that moderate hypoxia in the fetus may depress respiration by withdrawing a tonic stimulus at the level of the ventral lateral medullary chemoreceptors.

Properties of binding sites for [3H]cyclohexyladenosine in the hippocampus and other regions of rat brain: a quantitative autoradiographic study.

The properties of binding sites for the adenosine receptor ligand, [3H]cyclohexyladenosine ([3H]CHA), were investigated in rat brain using quantitative autoradiography. Scatchard analysis of the binding data showed that there were no significant differences between Kd values for [3H]CHA in any of the regions investigated. The highest concentrations of [3H]CHA binding sites were found in the cerebellum (molecular layer) and the stratum oriens and stratum radiatum of the hippocampus (CAI region). Displacement curves obtained using N-ethylcarboxamidoadenosine (NECA) and the R- and S-diastereoisomers of phenylisopropyl adenosine (PIA) showed the [3H]CHA binding sites to have the pharmacological properties of A1-adenosine receptors, i.e. the order of potency for these derivatives was R-PIA greater than NECA greater than S-PIA, in all regions tested. Further, [3H]CHA binding was in all cases attenuated by the guanosine triphosphate derivative, beta, gamma-imidoguanosine triphosphate. These results indicate that [3H]CHA binding sites throughout the central nervous system have the properties of A1-adenosine receptors and that these are in all regions associated with guanine nucleotide regulatory proteins.

Parallel investigations of the effects of adenosine on evoked potentials and cyclic AMP accumulation in hippocampus slices of the rat.

In slices of rat hippocampus, adenosine and its receptor agonist, 2-chloro-adenosine, were both found to (a) enhance the intracellular accumulation of cyclic adenosine monophosphate (cAMP) and (b) depress neuronal evoked responses to electrical stimulation. The differences in the dose/effect curves for the biochemical and electrophysiological changes cast doubt on a causal relationship between the two effects.

Effect of carbamazepine on stimulus-evoked Ca2+ fluxes in rat hippocampal slices and its interaction with A1-adenosine receptors.

In rat hippocampal slices superfused with a medium lacking Mg2+ ions, CA1 neurons generated burst discharges which were sensitive to blockade by 2-amino-5-phosphonovaleric acid (APV) and antagonized by 20-50 microM carbamazepine (CBZ). Decreases of [Ca2+]o (delta Ca), evoked by repetitive synaptic activation, were reduced in the presence of CBZ by 20-50%, associated with a reduced membrane depolarization. CBZ also abolished an APV-sensitive increase of delta Ca in the synaptic area elicited by theophylline. CBZ inhibited binding of the A1-adenosine receptor antagonist, [3H]8-cyclopentyl-1,3-dipropylxanthine [( 3H]DPCPX), in a dose-dependent manner. The displacement curve was shifted to the left in the presence of guanine nucleotide, suggesting that CBZ acts as an antagonist at A1-receptors. It is concluded that CBZ exerts its electrophysiological actions in the hippocampus at a site beyond the ligand recognition moiety.

An A1-adenosine receptor, characterized by [3H] cyclohexyladenosine binding, mediates the depression of evoked potentials in a rat hippocampal slice preparation.

In slices of rat hippocampus, adenosine and several adenosine derivatives depressed evoked neuronal responses to afferent stimulation. The nanomolar potency of adenosine derivatives and their relative effectiveness indicate that the depression of evoked potentials is mediated via an A1-adenosine receptor. A remarkable similarity was found between the relative potencies of nucleoside derivatives with respect to their electrophysiological effects and to their inhibition of high affinity [3H] cyclohexyladenosine ([3H]CHA) binding to rat brain membranes. We conclude that the [3H] CHA binding site in rat brain membranes represents a physiological receptor of the A1-type.

Modulation of A1 adenosine receptor function in rat brain by the polyamine, spermine.

Studies on factors modulating the binding of agonist ligands to A1 adenosine receptors in rat forebrain membranes revealed that the reduction of [3H]cyclohexyladenosine [( 3H]CHA) binding, observed after removing Mg2+ by pretreatment with ethylene-dinitrilo-tetraacetic acid (EDTA), was restored by the polyamine, spermine (1mM). Parallel electrophysiological experiments performed on rat hippocampal slices in Mg(2+)-free medium indicated that spermine also led to a recovery of the depressive effect of 1 microM adenosine on stimulus train-evoked neuronal Ca2+ influx. These observations suggest that the polyamine, spermine is, like Mg2+, able to control the physiological adenosine-mediated modulation of synaptic transmission by changing the affinity state of A1 receptors.

Adenosine receptor density and the depression of evoked neuronal activity in the rat hippocampus in vitro.

The relationship between the heterogeneous distribution of A-1 adenosine receptors and the capacity of adenosine to depress neuronal activity was examined in the rat hippocampus. Utilizing autoradiographic techniques, the distribution of A-1 adenosine receptors was assessed by the binding of [3H]cyclohexyladenosine ([3H]CHA) to cryostat sections of rat brain. The apical dendritic region of CA1 showed a differential distribution of adenosine receptors between the stratum radiatum and stratum lacunosum/moleculare. The physiological relevance of this binding difference was studied in the hippocampal slice by examining the capacity of adenosine to depress evoked potentials in these two strata. It was observed that the receptor differences correlated with differential sensitivities to adenosine modulation of the evoked potentials. These data suggest that receptor density, as shown by binding techniques, may provide not only a qualitative but also a quantitative map of the sites of adenosine action.

The action of calcitonin gene-related peptide on astrocyte morphology and cyclic AMP accumulation in astrocyte cultures from neonatal rat brain.

The actions of calcitonin gene-related peptide (CGRP) were investigated on 9- to 14-day-old astrocyte cultures obtained from the cerebra of 1- to 2-day-old rats. The morphological appearance of the cells was monitored by immunofluorescent staining of glial fibrillary acidic protein (GFAP), and the concentration of adenosine 3',5'-monophosphate (cyclic AMP) was measured in the cultures using radioimmunoassay. CGRP induced a morphological change in cultured astrocytes from rat brain. Flat, polygonal cells positive for GFAP became multipolar with many processes. In addition, CGRP stimulated the accumulation of cyclic AMP by up to 30-fold, half maximal stimulation being obtained at CGRP concentrations in the range 100-300 nM. These data provide evidence for the presence of functional CGRP receptors on astrocytes and suggest a role for CGRP as an intercellular messenger controlling the state of differentiation and activation of astroglial cells.

8-Cyclopentyl-1,3-dipropylxanthine (DPCPX)--a selective high affinity antagonist radioligand for A1 adenosine receptors.

The properties of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) as an antagonist ligand for A1 adenosine receptors were examined and compared with other radioligands for this receptor. DPCPX competitively antagonized both the inhibition of adenylate cyclase activity via A1 adenosine receptors and the stimulation via A2 adenosine receptors. The Ki-values of this antagonism were 0.45 nM at the A1 receptor of rat fat cells, and 330 nM at the A2 receptor of human platelets, giving a more than 700-fold A1-selectivity. A similar A1-selectivity was determined in radioligand binding studies. Even at high concentrations, DPCPX did not significantly inhibit the soluble cAMP-phosphodiesterase activity of human platelets. [3H]DPCPX (105 Ci/mmol) bound in a saturable manner with high affinity to A1 receptors in membranes of bovine brain and heart, and rat brain and fat cells (KD-values 50-190 pM). Its nonspecific binding was about 1% of total at KD, except in bovine myocardial membranes (about 10%). Binding studies with bovine myocardial membranes allowed the analysis of both the high and low agonist affinity states of this receptor in a tissue with low receptor density. The binding properties of [3H]DPCPX appear superior to those of other agonist and antagonist radioligands for the A1 receptor.

Red cell flexibility and platelet aggregation in patients with chronic obstructive vascular disease (COAD) and study of therapeutic approaches.

The red blood cell flexibility was studied in the blood of twenty patients with severe peripheral vascular disease and twenty matched controls. Patients exhibited significantly less red cell flexibility than controls. In both groups there was an inverse relationship between age and red cell flexibility. No correlation was found between red cell flexibility and sex or smoking history. Pentoxifylline, a xanthine derivative which inhibits phosphodiesterases and platelet aggregation, was found to increase red cell flexibility. This effect of the drug was greater on red cells with imparied flexibility than on normal cells. Various prostaglandins by contrast were found to decrease red cell flexibility, this could be compensated for by pentoxifylline. Forty patients with COAD were treated intravenously with PgE1. Significant inhibition of platelet aggregation and clinical improvement was noticed. It is suggested that combinations of PgE1 and pentoxifylline should be explored in clinical studies.