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D A Haycock

Publications and source records attributed to D A Haycock.

11 recordsLinked to original sources
PubMed1992

Distribution of cannabinoid receptors in rat brain determined with aminoalkylindoles.

Extensive mapping of the cannabinoid receptor in rat brain has been reported recently using synthetic cannabinoids. Another class of compounds, the aminoalkylindoles (AAIs), does not resemble the cannabinoids structurally. Ligand binding data on isolated membranes, however, indicate that AAIs bind to the cannabinoid receptor. The present experiments compared the binding of AAIs and synthetic cannabinoids in vitro and by receptor autoradiography. The AAIs bound to a receptor in rat cerebellum with high affinity (Kd = 15 nM), and synthetic cannabinoids were potent competitors for AAI binding sites. In the autoradiographic studies in rat brain, an AAI and a synthetic cannabinoid were used to compete for the binding of a radiolabeled AAI to compare regionally and quantitatively the inhibition of AAI binding by the two classes of compounds. The distribution of the AAI binding was very similar to that reported for synthetic cannabinoid binding. These data add further evidence that the aminoalkylindoles bind to the cannabinoid receptor. Furthermore, the autoradiographic data for AAI binding, in addition to the autoradiographic data for the synthetic cannabinoid, provide a high degree of confidence in the localization of the cannabinoid receptor in the rat brain.

Animals↗
PubMed1992

Conformationally restrained analogues of pravadoline: nanomolar potent, enantioselective, (aminoalkyl)indole agonists of the cannabinoid receptor.

Pravadoline (1) is an (aminoalkyl)indole analgesic agent which is an inhibitor of cyclooxygenase and, in contrast to other NSAIDs, inhibits neuronally stimulated contractions in mouse vas deferens (MVD) preparations (IC50 = 0.45 microM). A number of conformationally restrained heterocyclic analogues of pravadoline were synthesized in which the morpholinoethyl side chain was tethered to the indole nucleus. Restraining the morpholine diminished the ability of these pravadoline analogues to inhibit prostaglandin synthesis in vitro. In contrast, mouse vas deferens inhibitory activity was enhanced in [2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl] pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-(4-methoxyphenyl)methano ne (20). Only the R enantiomer of 20 was active (IC50 = 0.044 microM). An optimal orientation of the morpholine nitrogen for MVD inhibitory activity within the analogues studied was in the lower right quadrant, below the plane defined by the indole ring. A subseries of analogues of 20 and a radioligand of the most potent analogue, (R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo [1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone (21) were prepared. Inhibition of radioligand binding in rat cerebellar membranes was observed to correlate with functional activity in mouse vas deferens preparations. Binding studies with this ligand (Win 55212-2) have helped demonstrate that the (aminoalkyl)indole binding site is functionally equivalent with the CP-55,940 cannabinoid binding site. These compounds represent a new class of cannabinoid receptor agonists.

Analgesics↗
PubMed1991

Tyrosine hydroxylase in rat brain dopaminergic nerve terminals. Multiple-site phosphorylation in vivo and in synaptosomes.

Tyrosine hydroxylase, which catalyzes the initial step in catecholamine biosynthesis, is phosphorylated at serines 8, 19, 31, and 40 in intact pheochromocytoma (PC12) cells (Haycock, J.W. (1990) J. Biol. Chem. 265, 11682-11691). After 32Pi labeling of rat corpus striata in vivo or rat corpus striatal synaptosomes, 32P incorporation into tyrosine hydroxylase occurred predominantly at serines 19, 31, and 40. Electrical stimulation (30 Hz, 20 min) of the medial forebrain bundle (containing the afferent dopaminergic fibers) increased 32P incorporation into each of the three sites. Brief depolarization of the synaptosomes with elevated [K+]o (20-60 mM, 5-30 s) or veratridine (50 microM, 2 min) produced a selective increase in 32P incorporation into Ser19. Phorbol 12,13-dibutyrate (1 microM, 5 min) increased 32P incorporation into Ser31, and cAMP-acting agents such as forskolin (10 microM, 5 min) increased 32P incorporation into Ser40. In contrast, 32P incorporation into Ser8, which was usually detectable but very low, was not regulated either in vivo or in situ by any of the activators of signal transduction pathways. In synaptosomes, the only treatment found to increase Ser8 phosphorylation was okadaic acid (a protein phosphatase inhibitor), which increased 32P incorporation into all four phosphorylation sites. Thus, three different signal transduction systems appear to mediate the physiological regulation of tyrosine hydroxylase phosphorylation at three different sites.

Amino Acid Sequence↗
PubMed1984

Differential effects of calcium on catecholamine synthesis regulation in olfactory tubercle and hypothalamic synaptosomes.

We have compared catecholamine synthesis regulation in rat brain olfactory tubercle and hypothalamic synaptosomes with regard to: calcium-dependency of stimulant drug-induced synthesis activation; tyrosine-dependency of amphetamine-induced synthesis stimulation, and the effects of the calcium chelator, ethylene glycol-bis-(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA). Amphetamine-induced synthesis stimulation was markedly calcium-dependent in the olfactory tubercle, but was completely calcium-independent in the hypothalamus. The effect of amphetamine on catecholamine synthesis in the olfactory tubercle was dependent upon the concentration of tyrosine in the incubation buffer; amphetamine produced a biphasic response, inhibiting synthesis at low tyrosine concentrations and activating synthesis at higher tyrosine concentrations. In contrast to the olfactory tubercle, amphetamine-induced stimulation of synthesis in the hypothalamus was observed at both low and high tyrosine concentrations. Distinct tissue differences in drug responsiveness were also observed following exposure to EGTA; synthesis was stimulated by EGTA in the olfactory tubercle but not in the hypothalamus. These data provide evidence for regional differences in catecholamine regulation in the brain, and suggest that some of these differences may be related to differential effects of calcium on catecholamine formation.

Amphetamine↗
PubMed1984

The stability of 6-hydroxydopamine under minipump conditions.

A number of investigators have recently used osmotic minipumps to continuously deliver the neurotoxin 6-hydroxydopamine (6-OHDA) to kitten cerebral cortex for periods up to 7 days. Because this compound is known to be particularly labile, we studied the stability of 6-OHDA stored under conditions similar to those found in an osmotic minipump. In 0.4% ascorbic acid, 4 mM 6-OHDA-HBr was found to be stable for at least one week as determined by (1) assay of the drug by high performance liquid chromatography and electrochemical detection and (2) test of the drug's ability to deplete mouse heart norepinephrine.

Animals↗
PubMed1984

Dihydroxyphenylalanine production in rat brain striatal synaptosomes: stimulation by a calcium chelator.

By inhibiting aromatic L-amino-acid decarboxylase (EC 4.1.1.28) in rat brain striatal synaptosomes, we have been able to measure dihydroxyphenylalanine production via high performance liquid chromatography-electrochemical oxidation. This dihydroxyphenylalanine assay was compared to a standard radioisotopic assay of catecholamine synthesis (14CO2 production from L-[1-14C]tyrosine) in terms of (1) units of activity, (2) effects of known inhibitory and stimulatory agents, and (3) effects of the calcium chelator, EGTA. The units of activity in the dihydroxyphenylalanine assay were 40% greater than the units in the radioisotopic assay, indicating a mixing of labeled and endogenous tyrosine pools before conversion of the labeled tyrosine to labeled dihydroxyphenylalanine. The inhibition of synthesis produced by either 3-iodotyrosine or 3,4-dihydroxyphenylethylamine was similar in the two assays, as was the stimulation produced by 8-bromo cyclic AMP. The calcium chelator, EGTA, also activated synthesis to the same extent in the two assays, indicating that the increase observed in the radioisotopic assay is not an artifact of altered precursor specific activity. These data thus indicate the general utility of the synaptosomal dihydroxyphenylalanine synthesis assay, and also demonstrate the specific advantages of this assay for analyzing the effects of agents such as EGTA, which can alter tissue catecholamine precursor levels.

Animals↗
PubMed1983

Chromatographic and histochemical identification of dopamine within an identified neuron in the leech nervous system.

Each segmental ganglion of the leech nervous system has two pairs of lateral roots extending to the body wall and viscera. A cluster of about eight neuron cell bodies is located proximal to the first major branch of each anterior root and is termed the anterior root ganglion (ARG). Only one of these eight cells is vitally-stained by Neutral Red dye and fluoresces an intense blue-green following the glyoxylic acid histochemical condensation. The emission spectrum of this anterior root cell (AR) is stable under continuous UV illumination and is bimodal, with peaks at about 480 and 515 nm. This spectrum is indistinguishable from that of millimolar solutions of dopamine (DA) in gelatin droplets following glyoxylic acid histochemistry. We utilized high performance liquid chromatography and an amperometric detector to measure DA within the AR neurosomata at 1.01 pmol/cell. The AR cells in this study had an average diameter of 23 micron and therefore, the minimum intrasomatal concentration of DA is 160 mM, an unusually high level for any neurotransmitter. We measured DA in anterior axons at 0.83 pmol, in segmental ganglia at 1.07 pmol, and in longitudinal connectives at 0.16 pmol. Control neurosomata (Retzius cells) and axonal tracts which lack blue-green fluorescence (posterior and distal anterior roots), had no detectable DA (less than 0.06 pmol/sample). These data establish that the catecholamine DA is responsible for the fluorescence of the AR cell.

Animals↗
PubMed1981

Catecholamine synthesis regulation in hypothalamic synaptosomes.

In order to characterize the properties of synaptosomal catecholamine formation in a predominantly noradrenergic preparation, we have studied catecholamine synthesis in rat brain hypothalamic synaptosomes. Kinetic analysis revealed an apparent Km for tyrosine of 2.5 microM and an apparent Vmax of 2.1 nmol/h/g. In the hypothalamus, norepinephrine-induced synthesis inhibition was completely reversed by preincubation with desipramine, a blocker of catecholamine uptake into noradrenergic tissue. In contrast, desipramine was relatively ineffective in the predominantly dopaminergic striatum, indicating that most of the catecholamine synthesis observed in the hypothalamic synaptosomes was taking place in noradrenergic, as opposed to dopaminergic, terminals. Synthesis was stimulated approximately 30% in the hypothalamus by elevated (55 mM) potassium. This stimulation was markedly antagonized in a calcium-free buffer and by the addition of tetraethylammonium chloride. Phenylethylamine compounds could produce either stimulation (amphetamine) or inhibition (tyramine). The most effective synthesis stimulator was dibutyryl cyclic AMP (80% stimulation at 2 mM). Lowering the pH of the incubation buffer from 7.2 to 6.2 increased the basal rate but decreased the stimulatory response to elevated potassium. These data suggest that synaptosomal preparations from the hypothalamus offer a convenient system for studying drug effects on catecholamine synthesis in noradrenergic terminals.

Animals↗