Evidence for a cyclic GMP mechanism in the mediation of hippocampal post-tetanic potentiation.

Correlative electrophysiological and biochemical techniques were used to study hippocampal post-tetanic potentiation in acutely prepared rabbits following stimulation of the medial septal region and contralateral hippocampal field CA3. The results indicate that calcium ions, guanosine-3':5'-monophosphate, and phosphodiesterase inhibitors selectively enhanced the duration of post-tetanic potentiation. Potassium ions selectively enhanced tetanic potentiation. Adenosine-3':5'-cyclic monophosphate suppressed both tetanic and post-tetanic potentiation. The electrophysiological findings were supported by biochemical observations that guanosine-3':5'-monophosphate levels show marked increases following tetanic stimulation of either the medial septal region or contralateral hippocampal field CA3 pathways. The data suggest that a calcium-dependent process in the presence of a guanosine-3':5'-monophosphate mechanism promotes periods of hippocampal pyramidal cell hyperexcitability. The mechanism by which the cyclic nucleotide alters potentiation does not appear to be coupled to a single receptor variety.

Selective effects of an essential sulfhydryl group on the activation of dopamine- and guanine nucleotide-sensitive adenylate cyclase.

Treatment of membranes from the dog caudate nucleus with sulfhydryl alkylating agents, N-ethylmaleimide and p-chloromercuribenzoate, results in selective inhibition of dopamine-sensitive adenylate cyclase activity that can be distinguished from effects on basal enzyme activity. Fifty per cent inhibition of dopamine-sensitive adenylate cyclase activity was observed in the presence of 10(-5) M N-ethylmaleimide and 3 X 10(-6) M p-chloromercuribenzoate. N-Ethylmaleimide (10(-5) M or less) also inhibited GTP- and NaF-stimulated adenylate cyclase activity, but had no effect on basal adenylate cyclase activity (assayed in the presence of magnesium) and on enzyme activity assayed in the presence of manganese. The reducing agents dithiothreitol, 2-mercaptoethanol, glutathione, and cysteine had no inhibitory effect on dopamine-sensitive adenylate cyclase activity. Pretreatment of membranes with guanyl-5'-yl imidodiphosphate or guanosine 5'-O-(3-thio)triphosphate prior to incubation with N-ethylmaleimide prevented the inhibitory effect of N-ethylmaleimide on adenylate cyclase activity. The results suggest that a reactive sulfhydryl group in the domain of the GTP-binding protein is important for the coupling of the components of the dopamine-sensitive adenylate cyclase complex in brain.

Detection of dopamine receptors in the area postrema.

[3H]Spiroperidol labeled dog area postrema membranes with high affinity (KD, 0.1 nM) and stereospecificity. The displacement of the radioligand by different dopamine agonists and antagonists indicated that the labeled sites were dopaminergic in nature. Guanine nucleotides decreased agonist affinity for the labeled sites without affecting the affinity for antagonists. Adenylate cyclase in the area postrema was stimulated by guanine nucleotides and sodium fluoride but not by dopamine. The results suggest that the dopamine receptor in the area postrema can be classified as D2-type receptors.

Sodium-dependent interaction of benzamides with dopamine receptors in rat and dog anterior pituitary glands.

Sulpiride and other benzamides' displacement of [3H]-spiroperidol binding to rat and dog anterior pituitary dopaminergic receptors was found to be selectively sodium dependent, while typical neuroleptic activity was not influenced by NaCl. These results indicate the existence in the anterior pituitary of a subpopulation of dopaminergic receptors with which benzamides interact.

Regulation of adenylate cyclase activity in GH1 cells by chlorpromazine and a heat-stable factor.

An adenylate cyclase present in a PRL-producing tumor cell line, GH1, which is selectively stimulated by chlorpromazine, has been characterized with respect to several biochemical properties. The parameters studied include divalent metal ion specificity, guanyl nucleotide interaction, and sensitivity to sodium fluoride. The effects of calcium and the calcium chelator, EGTA, were also tested on the chlorpromazine response. The data reported herein establish the optimal conditions for the activation of adenylate cyclase by chlorpromazine in homogenates of GH1 cells. In addition, the results from this study identify a heat-stable protein in these cells which regulates cyclase activity. This protein, which can be released into the supernatant by the pretreatment of GH1 cells with EGTA, is an absolute requirement for chlorpromazine stimulation of adenylate cyclase activity in these cells. The activation of adenylate cyclase by chlorpromazine in homogenates of normal rat pituitaries was demonstrated as well as the presence of a protein factor which regulates this activity.

[3H]2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalen (ADTN). Regional distribution and in vivo binding after acute and chronic drug treatment.

The regional distribution and in vivo binding of the dopamine analog 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalen (ADTN) was studied in the brain. The highest density of binding sites was in the striatum, with virtually no binding in the cerebellum. The binding of [3H]ADTN reflects an occupation of specific dopamine sites because the binding was diminished by the simultaneous administration of the dopamine antagonist haloperidol or the dopamine precursor L-3,4-dihydroxyphenylalanine (L-dopa). Chronic administration of haloperidol or L-dopa prior to assaying for in vivo binding resulted in an increase in the number of sites for [3H]ADTN which correlates to the increase observed in in vitro assays following long-term treatment with these agents. The subcellular distribution of in vivo labeled ADTN sites in the caudate nucleus indicate a high density of specific binding sites in the microsomal fraction, P3. Overall, these data demonstrate that the aminotetralins, such as ADTN, which bind with high affinity to the dopamine receptor in the caudate nucleus in vitro and in vivo, can provide precise information on the topography of this receptor.

Identification and partial purification of a hydrophobic protein component associated with [3H]spiroperidol-binding activity.

The binding activity of radiolabelled neuroleptic drugs has been used to biochemically and pharmacologically characterize the dopamine receptor in brain. An extract which binds [3H]spiroperidol and exhibits stereoselectivity for (+)- and (-)-butaclamol, has been isolated from the calf striatal microsomal fraction. Specific binding activity in the chloroform-methanol extract of this preparation is enhanced over that of the crude homogenate. The highest specific binding of the chloroform methanol extract is associated with the crude phospholipid component which is enriched in hydrophobic proteins and acidic phospholipids. Subfractionation of the crude phospholipid extract by gel filtration (Sephadex LH-20) yields multiple peaks of [3H]spiroperidol binding activity, however four major zones of specific binding activity were detected. These results demonstrating a close association of phospholipids with a dopamine binding site suggest a functional role for proteolipid in receptor recognition and regulation.

Inhibition of cyclic nucleotide accumulation following hippocampal tetanic potentiation: effects of diazepam.

Biochemical studies on the hippocampus of acutely prepared rabbits revealed more than twofold increases in cyclic GMP levels following tetanic potentiation of the pathway from the medial septal region to CA1 pyramidal cells. Diazepam, administered intravenously, prevented the elevation in cyclic GMP levels in this region and also attenuated the post-tetanic potentiation seen following the presentation of trains at frequencies within theta rhythm. The results imply a modulatory role for cyclic nucleotides in the enhancement of pyramidal cell excitability and suggest that the biochemical mechanism for the psychoactive benzodiazepines may well include the suppression of cyclic GMP levels.

Biochemical alterations of dopamine receptor responses following chronic L-dopa therapy.

The effects of chronic l-dopa treatment on rat striatal adenylate cyclase and dopaminereceptor binding activities were studied using an oral dose of Sinemet (250 mg l-dopa/25 mg carbidopa). The calculated average daily oral intake of l-dopa was 150 mg/rat. A 4-fold increase in the ec(50) for dopamine on adenylate cyclase activity in homogenstes of the caudate nucleus was observed in the oral drug-treated group with no change in the maximal level of enzyme activity. Binding studies using [(3)H]spiroperidol, a dopamine-receptor antagonist, revealed a decrease in the dissociation constant from 0.26 nM in the control group to 0.069 nM in the oral drug-treated group. In addition, the B(max) for [(3)H]spiroperidol specific binding in the animals receiving oral l-dopa increased by 300 fmoles/mg over that observed in the control group. Dopamine-sensitive adenylate cyclase and binding activities in animals receiving a lower dose of l-dopa alone, given intraperitoneally (50 mg/kg) twice daily, were determined to be similar to control values. Analysis of the cerebrospinal fluid biogenic amine metabolites, homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA) and 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG), by gas chromatography/mass spectrometry revealed a 13-fold increase in HVA following oral l-dopa and a 44 per cent increase in MHPG levels. These data, which demonstrate an increased number of dopamine binding sites following long-term l-dopa therapy, are consistent with demonstrations of behavioral hypersensitivity in animals undergoing this particular drug treatment. The results also suggest that the subsensitivity of the adenylate cyclase system may reflect a side effect of this drug, due to prolonged administration.

GABA and dopamine receptor binding in retinal synaptosomal fractions.

Binding sites for a variety of agonists and anatagonists of GABA and dopamine were demonstrated in retina subcellular fractions. These sites exhibited the binding kinetics and pharmacologic specificity consistent with known properties of the physiological receptors for GABA and for dopamine. Important differences noted between brain and retinal receptors will be discussed.