Cyclic AMP Accumulation In Slices Research Articles

Study of adenosine A2 receptors in membrane preparations from optic tectum of chicks.

Binding properties of the subtypes of adenosine A2 receptors in membrane preparations and the effects of adenosine receptor ligands on cAMP accumulation in slices from the optic tectum of neonatal chicks have been investigated. [3H]2-[4-(2-p-carboxyethyl)phenylamino]-5'-N-ethylcarboxaminoadenosin e (CGS 21680), a selective ligand for adenosine A2a receptors, did not bind to optic tectal membranes, as observed with rat striatal membranes. CGS 21680 also did not induce cyclic AMP accumulation in optic tectum slices. However, 5'-N-ethylcarboxamidoadenosine (NECA), 2-chloro-adenosine or adenosine induced a 2.5- to 3-fold increase on cyclic AMP accumulation in this preparation. [3H]NECA binds to fresh non-washed-membranes obtained from optic tectum of chicks, displaying one population of binding sites, which can be displaced by NECA, 8-phenyltheophylline, 2-chloro-adenosine, but is not affected by CGS 21680. The estimated K(D) value was 400.90 +/- 80.50 nM and the Bmax was estimated to be 2.51 +/- 0.54 pmol/mg protein. Guanine nucleotides, which modulate G-proteins activity intracellularly, are also involved in the inhibition of glutamate responses by acting extracellularly. Moreover, we have previously reported that guanine nucleotides potentiate, while glutamate inhibits, adenosine-induced cyclic AMP accumulation in slices from optic tectum of chicks. However, the guanine nucleotides, GMP or GppNHp and the metabotropic glutamate receptors agonist, 1S,3R-ACPD did not alter the [3H]NECA binding observed in fresh non-washed-membranes. Therefore, the adenosine A2 receptor found in the optic tectum must be the adenosine A2b receptor which is available only in fresh membrane preparations, and its not modulated by guanine nucleotides or glutamate analogs.

Evidence for altered alpha 2-adrenoceptor function following isolation-rearing in the rat.

This study investigated central alpha 2-adrenoceptor function in differentially reared rats. Rats reared from weaning were either housed singly or in groups of five. Measurements of spontaneous ambulatory activity at 4 weeks postweaning showed that isolates were more hyperactive on exposure to a novel environment than grouped rats. alpha 2-Adrenoceptors were investigated using alpha 2-adrenoceptor agonist-induced behaviours, [3H]-idazoxan binding and measurement of forskolin-stimulated cyclic AMP accumulation. Clonidine (0.001-1.0 mg/kg IP) induced mydriasis in both groups with no difference observed in the response between the isolation and group-reared animals. Clonidine (0.01-0.5 mg/kg IP) induced hypoactivity in both groups, with the effect significantly greater in the isolation-reared rats. Idazoxan markedly attenuated both responses, confirming their induction by alpha 2-adrenoceptor stimulation. Clonidine-induced hypoactivity and mydriasis are mediated by pre- and postsynaptic alpha 2-adrenoceptors, respectively; therefore the results suggest rats reared in isolation have enhanced presynaptic but unchanged postsynaptic alpha 2-adrenoceptor function. Saturation binding experiments using [3H]-idazoxan were undertaken to determine alpha 2-adrenoceptor number (Bmax) and affinity (Kd) in membranes prepared from the frontal cortex and hippocampus. Analysis of binding data revealed an increase in receptor number in the hippocampus of isolates. Cyclic AMP accumulation was measured in hippocampal slices from differentially reared rats. Isolation-rearing did not affect cyclic AMP accumulation in response to stimulation by forskolin (30 microM). However, the selective alpha 2-adrenoceptor agonist, UK14304, produced a significantly greater inhibition of cyclic AMP accumulation in slices from isolated rats, confirming changes in alpha 2-adrenoceptor function following isolation rearing.

Dissociation of the striatal D-2 dopamine receptor from adenylyl cyclase following 6-hydroxydopamine-induced denervation

Intracellular cyclic AMP accumulation following exposure to dopamine (DA) agonists and and antagonists was measured in striatal slices from rats with a unilateral 6-hydroxydopamine (6-OHDA) lesion of the nigrostriatal pathway and which showed contralateral circling to apomorphine. Both DA (10–320 μM) and the D-1 agonist SKF 38393 (0.1–32 μM) increased cyclic AMP accumulation in striatal slices from the lesioned and intact hemispheres. The EC 50 for DA to increase cyclic AMP accumulation in slices was greater in the 6-OHDA-lesioned striata compared to the intact striatum, but the EC 50 for SKF 38393 was not affected. The D-1 antagonist SCH 23390 (10 μM) completely inhibited the ability of DA and SKF 38393 to increase cyclic AMP accumulation in striatal slices from both denervated and intact sides of the brain. In slices from the intact hemisphere the increase in DA-induced cyclic AMP accumulation was enhanced by the D-2 antagonist (±)-sulpiride (50 μM) but (±)-sulpiride had no effect on the DA response in slices from the lesioned side. Similarly, the ability of SKF 38393 to enhance cyclic AMP accumulation was blocked by the D-2 agonist quinpirole (10 μM) in striatal slices from the intact hemisphere but not in tissue from the lesioned side. The density of striatal D-1 and D-2 receptors assessed by [ 3H]SCH 23390 and [ 3H]spiperone binding did not differ between the hemispheres although there was an increase in the affinity of D-1 receptors for [ 3H]SCH 23390 in the lesioned striatum. After striatal deafferentiation there appears to be an uncoupling of the “inhibitory” D-2 receptor from the D-1 receptor-associated adenylyl cyclase.

Caffeine inhibits forskolin-stimulated cyclic AMP accumulation in rat brain

Caffeine potently inhibited forskolin-stimulated cyclic AMP accumulation in slices of rat cerebral cortex. The IC 50 for this inhibition was 21 ± 2.9 μM, and 200 μM caffeine almost completely blocked the forskolin response. Theophylline which is a behavioural stimulant mimicked the effect of caffeine. However, there was only a slight inhibition of the forskolin response by theobromine and IBMX (3-isobutyl-1-methylxanthine), both of which lack behavioral stimulant effects.

Effects of histamine H 3-receptor ligands on various biochemical indices of histaminergic neuron activity in rat brain

The interaction of the potent histamine H 3-receptor ligands i.e. (R)α-methylhistamine, an agonist, and thioperamide, an antagonist, with the three classes of cerebral histamine receptors was studied in vitro and in vivo. The histamine-induced stimulation of 3',5'-cyclic AMP accumulation in slices of the guinea-pig hippocampus was not modified by thioperamide (up to 0.1 mM) and (R)α-methylhiistamine stimulated cyclic AMP accumulation only at millimolar concentrations. Hence, both (R)α-methylhistamine and thioperamide were at least 100 000-fold more potent at H 3- than at H 1- or H 2-receptors in brain. In vivo, the turnover of histamine in rat cerebral cortex, as determined from its depletion elicited by α-fluoromethylhistidine in a synaptosomal fraction was not nmodified by mepyramine and zolantidine but was markiedly enhanced by thioperamide at a (ED 50 = 2 mg/kg). Thiopermide also elicited a long-lasting decrease in synaptosomal histamine and increase radioimmunoassayable N τ-methylhistamine. In contrast, (R)α-methylhistamine markedley reduced cortical [ 3H]histamine synthesis (ED 50 = 5 mg/kg). This long-lasting action was accompanied by an increase in synaptosomal histamine and a decrease in N τ-methylahistamine levels. These changes were compared with those in plasma drug levels. Hence the two H 3-receptor ligands appear to modify the activity of cerebral histamine neurons markedly and in a long-lasting and opposite manner.

Inhibitory effects of neuropeptide Y on cyclic AMP accumulation in slices of the nucleus tractus solitarius region of the rat

The effects of neuropeptide Y (NPY) on cyclic AMP (cAMP) accumulation in slices of the dorsal midline area of the caudal part of the medulla oblongata containing the nucleus tractus solitarius (nTS) have been studied. Neuropeptide Y (30 and 300 nM) significantly reduced the [ 3H]cAMP accumulation induced by forskolin and phorboldibutyrate. Similar results were obtained after incubation with the α-adrenoceptor agonist clonidine (1 μM). These results indicate that stimulation of the NPY receptors and α 2-adrenoceptors in the nTS region may cause inhibition of the adenylate cyclase. Such a mechanism may at least partly underlie the centrally mediated hypotensive effects of the costored transmitters adrenaline and NPY.

Specific Inhibition of Dopamine D‐1‐Mediated Cyclic AMP Formation by Dopamine D‐2, Muscarinic Cholinergic, and Opiate Receptor Stimulation in Rat Striatal Slices

The ability of different receptors to mediate inhibition of cyclic AMP accumulation due to a variety of agonists was examined in rat striatal slices. In the presence of 1 mM 3-isobutyl-1-methylxanthine, dopamine D-2, muscarinic cholinergic, and opiate receptor stimulation by RU 24926, carbachol, and morphine (all at 10(-8)-10(-5) M), respectively, inhibited the increase in cyclic AMP accumulation in slices of rat striatum due to dopamine D-1 receptor stimulation by 1 microM SKF 38393. In contrast, these inhibitory agents were unable to reduce the ability of a number of other agonists, including isoprenaline, prostaglandin E1, 2-chloroadenosine, vasoactive intestinal polypeptide, and cholera toxin, to increase cyclic AMP levels in striatal slices. These results suggest that in rat striatum either dopamine D-2, muscarinic cholinergic, and opiate receptors are only functionally linked to dopamine D-1 receptors or that the D-1 and D-2 receptors linked to adenylate cyclase lie on the cells, distinct from other receptors capable of elevating striatal cyclic AMP levels.

Behavioural sensitivity to pia in selectively bred mice is related to a number of A 1 adenosine receptors but not to cyclic AMP accumulation in brain slices

In a dose of 0.1 mg/kg, PIA had marked behavioural effects in long-sleep mice (which show a high sensitivity to ethanol, while no significant effect was observed in short sleep mice (low sensitivity to ethanol). The number of [ 3H]PIA binding sites in cortex and subcortical brain regions was significantly higher in long-sleep than in short-sleep mice. The K D value was higher in cortex and cerebellum in the short-sleep mice, but there were no differences in the number of hippocampal β-adrenoceptors or in the adenosine analogue-induced increase in cyclic AMP accumulation in slices of mouse hippocampus.

Stimulation by forskolin of the thyroid adenylate cyclase, cyclic amp accumulation and iodine metabolism

Forskolin, a diterpene hypotensive drug, activates adenylate cyclase in brain and in some other tissues (Seamon et al., 1981). Forskolin activated adenylate cyclase in particulate preparations and enhanced cyclic AMP accumulation in slices of dog thyroid. These effects were maximal within minutes and remained constant afterwards. The action of forskolin on intact cells disappeared rapidly after washing. It reproduced two known cyclic AMP-mediated TSH effects: the activation of secretion and of protein iodination. Forskolin thus provides a very convenient tool for the study of the action of defined elevations of cyclic AMP level in thyroid cells. The activation by forskolin of adenylate cyclase was not reduced by Mn 2+ which uncouples TSH and PGE 1 action. This suggests that in the thyroid also, forskolin acts beyond the receptor level. The effect of forskolin on cyclic AMP accumulation was inhibited by the known negative regulators of this system in the thyroid, acetylcholine, iodide, norepinephrine, PGF 1α and adenosine. On the other hand, forskolin potentiated the effects of TSH, PGE, and cholera toxin. These data show that, though it does not require the receptors for its action, forskolin does not uncouple them from the catalytic unit of adenylate cyclase.

Depolarizing agent-induced cyclic AMP accumulation in isolated rat spinal cord

The stimulation of cyclic adenosine 3′, 5′ -monophosphate (cyclic AMP) accumulation by the depolarizing agents K +, ouabain and veratridine, was studied in rat and guinea pig spinal cord tissue slices. Significantly increased accumulation of cyclic AMP was produced by each of the agents in a concentration-dependent manner. Veratridine and ouabain were equipotent (EC 50 = 5 × 10 −5M) and approximately 500 fold more potent than K + (EC 50 = 10 −2M). Depolarizing agent-induced cyclic AMP accumulation in slices from guinea pig spinal cord was approximately double the response in rat spinal cord. Maximum stimulation occurred within 2.5 min of incubation with these agents and lasted for at least 30 min. Regional studies demonstrated that the maximal accumulation of cyclic AMP occurred to a greater degree in tissue slices from the dorsal section of spinal cord from both rat and guinea pig. Whereas the ouabain and veratridine stimulatory responses are completely dependent on extracellular Ca ++, the K + response is only partially dependent. Stimulation due to ouabain and veratridine is dependent, and K + is independent, of release of neurohumoral substances such as norepinephrine or adenosine from spinal neurons. These experiments indicate the possible modulatory role of depolarization-linked events in regulating the spinal cord cyclic AMP system.

Possible involvement of cyclic AMP and frontal cortex in amitriptyline mediated suppression of the hypotensive effect of clonidine

Administration of amitriptyline (10mg/kg/day IM) to conscious Wistar rats for 14 days inhibited the hypotensive and bradycardiac response to chronic administration of clonidine (0.3mg/kg/day IM), when this antihypertensive drug was administered on the eighth through fourteenth day of treatment with the tricyclic antidepressant. This effect was not due to physiologic antagonism since amitriptyline did not affect the blood pressure or heart rate of conscious Wistar rats. Amitriptyline did not alter the plasma levels or cerebral cortical and brain stem accumulation of N-2-C 14-clonidine but suppressed norepinephrine-sensitive cyclic AMP accumulation in slices of frontal cortex. Norepinephrine-stimulated cyclic AMP accumulation was restored to normal values when tested in slices of frontal cortex obtained from Wistar rats refractory to the cardiovascular actions of clonidine. These effects did not occur in slices of medulla-pons. The data strongly support the postulate that amitriptyline mediated suppression of the cardiovascular action of clonidine may occur at higher centers of the brain (frontal cortex) and not in the medulla.

Histamine receptors mediating cyclic AMP accumulation in slices from guinea-pig hippocampus: Characterization and localization

Histamine receptors mediating cyclic AMP accumulation in slices from guinea-pig hippocampus: Characterization and localization