Two 5-HT receptors linked to adenylate cyclase in guinea pig hippocampus are discriminated by 5-carboxamidotryptamine and spiperone.
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Adenylyl Cyclases↗
Biomedical subjects
S Maayani
Publications and source records attributed to S Maayani.
5-Hydroxytryptamine receptor in isolated rabbit aorta: characterization with tryptamine analogs.
The 5-HT2 receptor in isolated rabbit thoracic aorta was characterized by examining the relationships between structure and activity of nine tryptamine analogs. All assays were conducted after blockade of the alpha adrenergic receptor and inactivation of the neuronal uptake-1 system and monoamine oxidase. Seven of the analogs tested were agonists. 6-Hydroxytryptamine and 7-hydroxytryptamine showed little or no agonist activity in this preparation. The pA2 of spiperone was independent of the agonist assayed and defined the receptor activated by each agonist as the 5-HT2 receptor. The dissociation constant (KA) and relative intrinsic efficacy were determined for each agonist. The KA and relative intrinsic efficacy values for 5-hydroxytryptamine were 0.25 microM and 1, respectively. The KA and relative intrinsic efficacy values for 5-methoxytryptamine were 0.14 microM and 0.86, respectively, and were not significantly different from those for 5-hydroxytryptamine. The other five analogs were partial agonists. N-Methyl-5-hydroxytryptamine and bufotenine had relative intrinsic efficacies of about 0.3 and KA values not statistically different from the KA value for 5-hydroxytryptamine. Tryptamine, 5-methyltryptamine and alpha-methyl-tryptamine had KA values of about 1 microM and relative intrinsic efficacies of 0.6, 0.6 and 0.4, respectively. These results revealed the differential effects of structural changes on drug affinity and intrinsic efficacy. This information will be applicable in the design of selective agonists or antagonists for the classification of less well defined 5-hydroxytryptamine receptors.
Animals↗
Regional differences in binding of [3H]LSD and [3H]5-HT in calf hippocampal slices revealed by radioautography and rapid filtration studies.
Previous radioautographic experiments demonstrated that binding sites labeled by [3H]5-HT and [3H]LSD in rat brain were seen in all layers of CA1, CA4 and the dentate gyrus but not in fields CA2 and CA3 of the hippocampus. In an attempt to confirm this observation we performed binding assays on homogenates from selected areas of calf hippocampus since the small size of the rat hippocampus precluded using preparations from this animal for this purpose. Studies on homogenates from calf hippocampal regions, were done after we determined that the binding to slices in vitro was similar in the calf and rat. Binding of both [3H]5-HT and [3H]LSD by homogenates of CA1 and dentate gyrus, but not of CA3, was saturable. These studies show that the qualitative differences in binding site distribution within the calf hippocampus seen by radioautography reflect quantitative differences in the densities of binding sites revealed by the homogenate studies.
Animals↗
Kinetic characterization of the rabbit aorta contractile response to an alpha adrenergic agonist.
The alpha-1 adrenergic response of the rabbit aorta to phenylephrine (PE) was separated into a phasic and a tonic response by virtue of their different dependence on extracellular [Ca++]. The kinetics of each response was characterized with respect to its dependence on [PE] and [Ca++]. The phasic response is independent of extracellular calcium and has a rapid onset followed by a first order decay. Although its maximal attainable response is saturable with respect to [PE] and [Ca++], its rate constant for onset does not depend on the concentration of calcium in the preincubation buffer. We were unable to show that this rate constant for onset is saturable with respect to [PE]. This suggests that the rate-determining step of the phasic response is the diffusion-controlled formation of the drug-receptor complex. The tonic response depends on extracellular calcium, shows first order kinetics of onset and reaches a steady-state level of contraction that is saturable with respect to [PE] and extracellular [Ca++]. The rate constant for the generation of the tonic response depends on [PE] in a saturable manner and linearly on extracellular [Ca++]. This suggests that the rate-determining step could be the activation of a hypothetical effector by the drug-receptor complex. The activated effector would enable the transport of calcium ions into the cell. The kinetic studies predict that the efficacy of a drug in this system is the maximal rate of activation of the effector by the drug-receptor complex.
Adrenergic alpha-Agonists↗
Discriminant effects of behaviorally active and inactive analogs of phencyclidine on membrane electrical excitability.
The discriminant effects of several behaviorally active and inactive analogs of phencyclidine [PCP; 1-(phenylcyclohexyl) piperidine] and the actions of PCP and three Ca-channel antagonists were examined on electrical excitability in frog and crayfish skeletal muscles. In frog sartorius muscle, 1-[1-(2-thienylcyclohexyl)piperidine (TCP; 100 microM), a behaviorally active analog of PCP, increased action potential duration nearly 9-fold, blocked delayed rectification and at 0.5 to 1 microM also increased the quantal release of transmitter. A partial blockade of delayed rectification and slight prolongation of the action potential occurred with 1-(p-fluorophenylcyclohexyl)piperidine (p-F-PCP; 100 microM), which possesses about 25% of the behavioral activity of PCP. Of the remaining p-phenyl- substituted analogs which never exhibited more than 10% of the behavioral potency of PCP, only 1-(1-p-nitrophenylcyclohexyl)piperidine (p-NO2-PCP; 100 microM) produced a frequency-dependent prolongation of the action potential but, like the p-methoxy-, p-chloro- and p-methyl- analog, it did not block delayed rectification. The order of potencies of these analogs in blocking delayed rectification, prolonging the muscle action potential and in affecting alternation impairment and response rate depression is therefore: PCP much greater than TCP greater than p-F-PCP much greater than p-CH3-PCP = p-CH3O-PCP = p-Cl-PCP = p-NO2-PCP. Like PCP and its behaviorally active analogs, verapamil (50 microM) and bepridil (50 microM), two Ca-channel blockers, also blocked delayed rectification in frog sartorius muscles whereas nifedipine (50 microM), another Ca-channel blocker, did not.(ABSTRACT TRUNCATED AT 250 WORDS)
Action Potentials↗
5-Hydroxytryptamine receptor in rabbit aorta: characterization by butyrophenone analogs.
The contractile response of isolated rabbit aorta rings to 5-hydroxytryptamine (5-HT) was antagonized by spiperone and four other butyrophenone analogs in a competitive manner. The Kb values were (nanomolar):spiperone, 0.8; spirilene , 2.1; benperidol , 4.4; azaperone, 16.6; and haloperidol, 96.6. The Kd values for four of these drugs, whose affinities for [3H]ketanserin and [3H]spiperone binding sites in rat brain membranes have been measured, are almost indistinguishable from the Kb values in inhibiting 5-HT-induced contraction of the rabbit aorta. This suggests a congruence between the aortic "D" receptors and 5-HT2 type binding sites in rat brain. Among the drugs we tested, one portion of the molecule is almost identical; the other portion of the molecule differs in four of the five compounds. It is suggested that their rank order as antagonists of the 5-HT receptor in the aorta depends on the degree of recognition of the nonbutyrophenone part of the molecules by the receptor.
Animals↗
Differences in d-[3H]lysergic acid diethylamide binding in mouse cortex and hippocampus in vivo and in vitro revealed by radioautography and rapid filtration studies.
The localization of d-[3H]lysergic acid diethylamide ([3H]LSD) binding sites in mouse brain was compared in vivo and in vitro. Radioautography of brain sections incubated with 6 nM [3H]LSD in vitro revealed substantial specific binding in cortex (CTX), especially in layers III to IV and anterior cingulate gyrus, and in areas CA1 and dentate gyrus of hippocampus (HIP). In sections of brains from mice that received 100 nmol of [3H]LSD per kg and were killed 10, 15 or 30 min later, specific [3H]LSD binding in CTX had a pattern of distribution similar to that observed in vitro. In contrast, the pattern of specific [3H]LSD binding in HIP in vivo differed from the results obtained in vitro, in that it was sparse and lacked differential subregional distribution. The low specific [3H]LSD binding in vivo in HIP but not in CTX was confirmed by homogenate filtration studies of brain areas from mice that received 100 nmol of [3H]LSD per kg. The levels of free [3H]LSD, obtained after correction for time-dependent metabolism of [3H]LSD, did not vary among regions, but [3H]LSD specifically bound in HIP was 30 to 50% of that in CTX. In contrast, steady-state binding studies in vitro in membrane preparations from CTX and HIP demonstrated a similar density and affinity of [3H]LSD binding sites in the two regions. Comparison of [3H]LSD binding characteristics in vivo and in vitro suggests possible mechanisms causing the lower specific binding in HIP in vivo, including modulation of the binding sites that differ in CTX and HIP.
Animals↗
Enhanced serotonin-stimulated adenylate cyclase activity in membranes from adult guinea pig hippocampus.
We have developed an assay for serotonin (5-HT) stimulation of adenylate cyclase activity in membranes from adult guinea pig hippocampus. The response to 5-HT is concentration-dependent, with an EC50 of 0.01 microM, a shallow slope, and mean maximal stimulation of 90% over basal activity. The response to 5-HT is GTP-dependent and additive to the maximal stimulation by histamine. Micromolar concentrations of the known 5-HT receptor agonists, tryptamine and 5-methoxytryptamine, also stimulate cAMP production in this system, and their effect is not additive to that elicited by a maximal concentration of 5-HT. These results are consistent with the hypothesis that the response to 5-HT is elicited through a distinct receptor coupled to adenylate cyclase; the magnitude and the reproducibility of the 5-HT response in this system should make it useful for receptor classification. To examine the effect of prior exposure to endogenous 5-HT on the responsiveness of the system, we assayed 5-HT stimulation of enzyme activity in membranes prepared from animals 25-27 hrs after treatment with a single injection of reserpine (5 mg/kg, i.p.). The mean maximal stimulation of adenylate cyclase by 5-HT was increased to 150% over basal activity with no effect on the EC50 or slope of the 5-HT concentration-response curve. Reserpine pretreatment did not affect basal activity or histamine-stimulated adenylate cyclase activity. These results are discussed in the context of a hypothesis that endogenous 5-HT normally exerts a desensitizing effect on its receptors in situ.
5-Methoxytryptamine↗
Effects of phencyclidine on cardiac action potential: pH dependence and structure-activity relationships.
The effects of phencyclidine [1-(1-phenylcyclohexyl)-piperidine; PCP] on cardiac action potential duration (APD) were compared to those of some of its derivatives, in strips of isolated frog ventricular muscle perfused with normal Ringer solution. We studied compounds with PCP-like behavioral actions (N-ethyl-1-phenyl-cyclohexylamine: PCE; and m-amino-PCP) as well as behaviorally inactive analogs (m-nitro-PCP; the quaternary derivative PCP-methyl iodide; and various fragments of the PCP molecule). Exposure to PCP, 3 microM to 1 mM, produced reversible, dose- and pH-dependent prolongations, of the APD to over 100% above control. The observed effects of the drugs are compatible with a mechanism of blockade of potassium conductance. An intracellular site for this action is suggested by: (i) the inactivity of the quaternary analog; (ii) the marked increase in the potency of the compounds when the external pH is changed in the region of their respective pKa values to increase the concentration of the unionized species; and (iii) the pronounced acceleration of the termination of the PCP effect by washout with a series of buffer solutions with decreasing pH values. The rank order of potency of the compounds in lengthening APD (PCE greater than m-amino PCP greater than PCP much much greater than m-nitro-PCP) is the same as reported from other pharmacological studies of specific PCP actions, and matches the rank of behavioral activity of the drugs.
Action Potentials↗
Multiple actions of phencyclidine: discriminant structure-activity relationships from molecular conformations and assay conditions.
A major approach to the elucidation of the mechanisms by which phencyclidine (PCP) elicits the varied and complex responses observed in biological systems consists of comparisons of the pharmacological profiles of PCP derivatives with those of drugs for which the mechanism of action is better understood. Such studies depend on the definition of discriminant structure-activity relationships and on comparisons of rank orders of potency for the actions of PCP derivatives. Using the muscarinic cholinergic system in brain and the potassium ion channels in cardiac muscle as targets for PCP derivatives, we review the elucidation of the structural determinants for PCP recognition at muscarinic receptors, and the dramatic effect of assay conditions on the rank order of potency for the action of PCP derivatives at intracellular sites. The structural and physicochemical considerations illustrated here must be considered basic to the establishment of any assay aimed at the elucidation of the mechanism of action of PCP.
Animals↗
Response of the histamine h2-receptor in brain to antidepressant drugs.
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Adenylyl Cyclases↗
Site of action of phencyclidine. IV. Interaction of phencyclidine and its analogues on ionic channels of the electrically excitable membrane and nicotinic receptor: implications for behavioral effects.
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Animals↗
[3H]dihydroergocryptine binding to bovine striatal membranes defined by a low d-butaclamol concentration: antagonism by substituted benzamides.
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Animals↗
The behavioral effects of phencyclidines may be due to their blockade of potassium channels.
The action of phencyclidine [1-(1-phenylcyclohexyl)piperidine; PCP] and its behaviorally active analog (m-amino-PCP) and of two behaviorally inactive analogs [m-nitro-PCP and 1-piperidinocyclohexanecarbonitrile (PCC)] were examined in this study. In a test of spatial alternation performance in rats, PCP and m-amino-PCP were much more potent behavior modifiers than were PCC and m-nitro-PCP. We studied the effects of the drugs on the ionic channels of the electrically excitable membrane and of the nicotinic acetylcholine (AcCho) receptors at the neuromuscular junction of frog skeletal muscle. All four compounds blocked the indirectly elicited muscle twitch and depressed the amplitude and rate of rise of directly elicited muscle action potentials. They also caused a voltage- and concentration-dependent decrease in the peak amplitude of the endplate current but did not react with the nicotinic AcCho receptor. These observations indicate that the four compounds have comparable blocking effects on the ionic channels associated with the nicotinic AcCho receptor. In contrast, the behaviorally active agents could be distinguished from behaviorally inactive ones by their effects on K+ conductance. PCP and m-amino-PCP blocked delayed rectification in frog sartorius muscles, prolonged the muscle action potential more than 2-fold, and markedly potentiated the directly elicited muscle twitch. The behaviorally active compound also blocked depolarization-induced 86Rb+ efflux from rat brain synaptosomes (presumably a measure of K+ conductance) and increased quantal content at the frog neuromuscular junction. In these actions, m-nitro-PCP was much less effective, and PCC was relatively ineffective. Because PCP and m-amino-PCP are much more potent behavior modifiers than PCC and m-nitro-PCP, we suggest that the behavioral effects of PCP and m-amino-PCP, may be due to a block of K+ conductance and enhancement of transmitter release at central neurons.
Action Potentials↗
Localization of naloxone-sensitive [3H]dihydromorphine binding sites within the hippocampus of the rat.
In vitro radioautographic experiments were performed on coronal rat brain slices to determine the precise localization of [3H]dihydromorphine binding sites sensitive to naloxone, within the hippocampus. Binding was observed in all cell fields and in dentate gyrus (DG) in the order CA2 greater than CA1 greater than CA3 greater than DG. Within each cell field the density of receptors was greatest in the stratum pyramidale, which in field CA2 was 77% as dense as the striatum. The existence of a dense population of opiate binding sites within the hippocampus is consistent with the view that this structure is involved in opiate actions.
Animals↗
Characterization and radioautography of [3H]LSD binding by rat brain slices in vitro: the effect of 5-hydroxytryptamine.
Binding of D-[3H]lysergic acid diethylamide (LSD) to rat coronal brain slices and its blockade by 5-hydroxytryptamine (5-HT) had characteristics similar to those of brain homogenates in respect of KD, kinetics and reversibility of binding. Radioautography was done on slices that had been incubated in 6 nM [3H] LSD and on adjacent slices incubated in the same concentration of tritiated LSD plus 10(-5) M of 5-HT. Choroid plexus showed densest labeling of [3H] LSD. In neuropil, dense labeling occurred within parts of the hippocampal formation except for fields CA2 and CA3 which were sparsely labeled. All layers of the cortex except the posterior cingulate gyrus were labeled by LSD. 5-HT blocked labeling of choroid plexus, hippocampal formation, septum, pons, medulla and parts of cortex but only reduced labeling of most other structures. LSD binding sites may relate to some of its pharmacological effects.
Animals↗
Phencyclidine-induced rotation and hippocampal modulation of nigrostriatal asymmetry.
Phencyclidine (PCP) elicited dose-related rotation in naive rats. The effect of PCP was consistent in direction and magnitude from one week to the next but was dissimilar to the rotatory effects of dopaminergic (D-amphetamine, apomorphine) or anticholinergic (scopolamine) drugs. Study of the effects of PCP on regional brain uptake of labeled 2-deoxy-D-glucose suggested that PCP-induced rotation is at least in part mediated by an action in the hippocampus. PCP elicited ipsilateral rotation following unilateral hippocampal lesions whereas such lesions did not alter the direction of either nocturnal or D-amphetamine-induced rotation. PCP appears to activate a hippocampal mechanism that normally only modulates the intensity of rotation.
Animals↗
Intraperitoneal administration and other modifications of the 2-deoxy-D-glucose technique.
Experiments were conducted to determine the optimal conditions necessary for implementing modifications of the 2-deoxyglucose (2-DDG) technique. Substitution of tritium-labeled 2-DDG with subsequent microdissection of selected brain regions and liquid scintillation counting produced results that were highly correlated with both [14C]radioautograms and glucose utilization values as obtained by Sokoloff et al. The route of administration of isotope was also varied. Whole brain uptake at maximal levels of incorporation was the same for both intravenously and intraperitoneally injected animals. Radioautograms from i.p. and i.v. injected animals were indistinguishable. Densitometric analyses of the i.p. radioautograms were highly correlated with glucose utilization values. Thus, relative indices of functional activity may be obtained when experimental circumstances preclude arteriovenous cannulations and restraint. The use of naive, unrestrained animals, therefore, makes the 2-DDG technique applicable to a broader range of studies.
Animals↗