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Biomedical subjects

C D Unsworth

Publications and source records attributed to C D Unsworth.

15 recordsLinked to original sources
PubMed1994

Regulation of mRNA encoding 5-HT2A receptors in P11 cells through a post-transcriptional mechanism requiring activation of protein kinase C.

Exposure of P11 cells to serotonin (5-HT) resulted in a transient increase in levels of 5-HT2A receptor mRNA. Exposure to 5-HT for as short a time as 1 min was sufficient to trigger a delayed increase in receptor mRNA. 5-HT-induced increases in receptor mRNA levels were not antagonized by the protein synthesis inhibitor cycloheximide. The increase in receptor mRNA levels was accompanied by a transient increase in the half-life of receptor mRNA; the rate of transcription of receptor mRNA was unchanged. Submaximal stimulation of phosphinositide hydrolysis by partial agonists or 6-fluoronorepinephrine, an alpha 1-adrenergic receptor agonist, also increased receptor mRNA levels. Exposure to phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, mimicked these effects, whereas the protein kinase C inhibitor bisindolylmaleimide antagonized the effects of both 5-HT and PMA. When agonist-promoted increases in receptor mRNA were prevented, the rate of agonist-induced down-regulation was accelerated. These data suggest that levels of 5-HT2A receptor mRNA are regulated by phospholipase C-coupled receptors via a protein kinase C-dependent, post-transcriptional mechanism and indicate that agonist-promoted increases in levels of 5-HT2A receptor mRNA modulate receptor expression.

Amino Acid Isomerases↗
PubMed1994

Characterization of a 5-hydroxytryptamine receptor in mouse neuroblastoma N18TG2 cells.

Cyclic AMP formation was found to increase in mouse neuroblastoma N18TG2 cells exposed to 5-hydroxytryptamine (5-HT). This response was concentration-dependent with an EC50 value of 0.22 microM. Tryptamine and other tryptamine-related compounds were also agonists in this assay with a rank order of potency of 5-methoxytryptamine > 5-HT > tryptamine > 2-methyl-5-HT > 5-carboxamidotryptamine >> alpha-methyl-5-HT. (D)-Lysergic acid diethylamide and 2-bromo-lysergic acid diethylamide were partial agonists in this system with maximal responses of 44 and 34%, respectively, compared to 5-HT. 5-HT-stimulated cyclic AMP formation was inhibited by clozapine, mianserin and methiothepin with pA2 values of 6.6, 6.5 and 6.4, respectively. Radioligand binding studies using [125I]iodolysergic acid diethylamide revealed a binding site present at a density of 10.4 fmol/mg of protein, with an affinity for the ligand of 1.18 nM. In competition studies this binding site displayed a pharmacology similar to that defined in studies of cyclic AMP formation. The pharmacological profile of this receptor, characterized by both radioligand binding and functional coupling to adenylyl cyclase, does not correspond to that of any of the currently classified subtypes of 5-HT receptor, but is similar to the 5-HT receptor cloned recently from rat striatum and referred to as the 5-HT6 receptor. The N18TG2 cell line represents a useful model system in which this novel 5-HT receptor may be characterized fully.

Adenylyl Cyclases↗
PubMed1993

18F-labeled benzamides for studying the dopamine D2 receptor with positron emission tomography.

Two series of (N-benzylpiperidin-4-yl)- and (9-azabicyclo[3.3.1]nonan- 3 beta-yl)benzamides were prepared, and in vitro binding assays were used to measure the affinity of these compounds for dopamine D2, dopamine D3, serotonin 5-HT2, and alpha 2-adrenergic receptors. The results of these studies indicated compounds 23, 26b, and 34 have the selectivity needed for in vivo studies of the D2 (and possibly D3) receptors. 18F-Labeled analogues of 23, 26b and 34 were prepared by N-alkylation of the corresponding desbenzyl precursors with [18F]-4-fluorobenzyl iodide. Preliminary in vivo studies demonstrated that [18F]-23 and [18F]-26b are suitable candidates for further evaluation in positron emission tomography imaging studies. The slow rate of washout of [18F]-34 from nondopaminergic regions and its comparatively high lipophilicity indicates that this compound may not be suitable for imaging studies because of a high level of nonspecific binding.

Animals↗
PubMed1993

The use of [18F]4-fluorobenzyl iodide (FBI) in PET radiotracer synthesis: model alkylation studies and its application in the design of dopamine D1 and D2 receptor-based imaging agents.

[18F]4-Fluorobenzyl iodide ([18F]FBI) was prepared, and a series of model alkylation studies were conducted to determine its chemical reactivity toward nitrogen and sulfur nucleophiles of varying nucleophilicities. [18F]FBI was found to react rapidly with secondary amines and anilines to give the corresponding N-[18F]4-fluorobenzyl analogue in high yield. Amides and thiol groups required the use of a base catalyst. The utility of [18F]FBI was documented by investigation of dopamine D1 and D2 receptor-based radiotracers.

Alkylation↗
PubMed1993

Effects of agonists, partial agonists, and antagonists on the regulation of 5-hydroxytryptamine2 receptors in P11 cells.

Studies of the regulation of 5-hydroxytryptamine (5-HT2) receptors in vivo have generated anomalous and sometimes contradictory results. In particular, administration of antagonists unexpectedly results in a reduction in the density of 5-HT2 receptors. P11 cells, which express a high density of 5-HT2 receptors coupled to phosphoinositide hydrolysis, were used to investigate the regulation of receptors in vitro by agonists, partial agonists, and antagonists. (+/-)-2,5-Dimethoxy-4-iodophenylisopropylamine (DOI) and (+)-lysergic acid diethylamide (LSD) caused marked reductions in the density of 5-HT2 receptors as has been observed in vivo. Down-regulation was prevented by coincubation with ketanserin. The decrease in the density of 5-HT2 receptors after exposure to 5-HT, LSD, or DOI was time dependent and was not a consequence of residual drug in binding assays or irreversibly bound drug. The ability of 5-HT, DOI, and LSD to down-regulate 5-HT2 receptors was not proportional to the ability of these compounds to stimulate phosphoinositide hydrolysis. Ketanserin and mianserin, antagonists which cause paradoxical decreases in the density of 5-HT2 receptors in vivo, did not alter the density of 5-HT2 receptors on P11 cells, even after prolonged incubation with drug. Results of the current studies, which demonstrate agonist- but not antagonist-induced down-regulation of 5-HT2 receptors, lead to the conclusion that the ability of ketanserin and mianserin to down-regulate receptors in vivo is the result of indirect actions of these drugs and is unlikely to be a direct consequence of receptor occupancy by antagonists.

Amphetamines↗
PubMed1992

Regulation of the 5-hydroxytryptamine1B receptor in opossum kidney cells after exposure to agonists.

The density of 5-hydroxytryptamine (5-HT)1B receptors and their coupling to the inhibition of cAMP accumulation were investigated in opossum kidney cells maintained in culture. The density and properties of the receptor were determined using [125I] iodocyanopindolol as the radioligand. The pharmacological specificity of the binding site was consistent with that expected for a 5-HT1B receptor. Serotonin inhibited forskolin-stimulated cAMP accumulation with an EC50 of 4-8 nM. Compounds known to show selectivity at the 5-HT1B receptor, such as trifluoromethyl-phenylpiperazine and CGS-12066B, also inhibited forskolin-stimulated cAMP accumulation, acting as full agonists with efficacies comparable to that of serotonin. Other beta-adrenergic receptor antagonists, including (-)-pindolol and (-)-alprenolol, bound to the receptor with high affinity and acted as partial agonists capable of inhibiting forskolin-stimulated cAMP accumulation. Exposure of cells to 5-HT resulted in a time- and dose-dependent decrease in the density of 5-HT1B receptors that was not accompanied by a change in the Kd of the binding site for [125I] iodocyanopindolol. A maximum decrease of 60% in the number of 5-HT1B receptors was evident after a 16-hr treatment with 1 microM 5-HT. Concomitant with the observed decrease in the density of receptors was a marked increase in the EC50 for 5-HT-mediated inhibition of forskolin-stimulated cAMP accumulation. The EC50 was increased 4-5-fold after a 16-hr exposure to 1 microM 5-HT, and the maximal level of inhibition was markedly decreased. Whereas pretreatment with moderate concentrations of 5-HT (100-300 nM) for 16 hr produced significant decreases in the density of 5-HT1B receptors and increases in the EC50 for inhibition of forskolin-stimulated cAMP formation, there was little change in the maximal level of inhibition that could be attained. Such a combination of changes could be explained by the presence of "spare" 5-HT1B receptors on these cells.

Animals↗
PubMed1990

Acetylcholine and ATP are coreleased from the electromotor nerve terminals of Narcine brasiliensis by an exocytotic mechanism.

Although the exocytotic mechanism for quantal acetylcholine (ACh) release has been widely accepted for many years, it has repeatedly been challenged by reports that ACh released upon stimulation originates from the cytosol rather than synaptic vesicles. In this report, two independent experimental approaches were taken to establish the source of ACh released from the electromotor system of Narcine brasiliensis. Since ATP is colocalized with ACh in the cholinergic vesicle, the exocytotic theory predicts the corelease of these two components with a stoichiometry identical to that of the vesicle contents. The stimulated release of ATP from isolated synaptosomes could be accurately quantitated in the presence of the ATPase inhibitor adenosine 5'-[alpha, beta-methylene]triphosphate (500 microM), which prevented degradation of the released ATP. Various concentrations of elevated extracellular potassium (25-75 mM), veratridine (100 microM), and the calcium ionophore ionomycin (5 microM) all induced the corelease of ACh and ATP in a constant molar ratio of 5-6:1 (ACh/ATP), a stoichiometry consistent with that established for the vesicle content. In parallel to these stoichiometry studies, the compound 2-(4-phenylpiperidino)cyclohexanol (AH5183) was used to inhibit specifically the vesicular accumulation of newly synthesized (radiolabeled) ACh without affecting cytosolic levels of newly synthesized ACh in cholinergic nerve terminals. Treatment with AH5183 (10 microM) was shown to inhibit the release of newly synthesized ACh without markedly affecting total ACh release; thus, the entry of newly synthesized ACh into the synaptic vesicle is essential for its release. We conclude that ACh released upon stimulation originates exclusively from the vesicular pool and is coreleased stoichiometrically with other soluble vesicle contents.

Acetylcholine↗
PubMed1986

Reflex splanchnic nerve stimulation increases levels of proenkephalin A mRNA and proenkephalin A-related peptides in the rat adrenal medulla.

The effect of reflex splanchnic nerve stimulation on proenkephalin A biosynthesis was investigated in the rat adrenal medulla. Tissue levels of native [Met5]enkephalin-like immunoreactivity (IR) (measured by direct RIA of tissue extracts), cryptic [Met5]enkephalin-like IR (calculated as the increase in [Met5]enkephalin-like IR detected in tissue extracts after sequential digestion with trypsin and carboxypeptidase B), and proenkephalin A mRNA were determined in adrenal medulla from rats sacrificed at various times after a period of insulin-induced hypoglycemia. Two hours of insulin hypoglycemia, which produced intense reflex stimulation of the splanchnic nerves as evidenced by a 55% decrease in the adrenal medulla catecholamine levels, resulted in a 3-fold increase in proenkephalin A mRNA levels in this tissue. The proenkephalin A mRNA levels reached a maximum 15-fold increase over control values 24 hr after this period of hypoglycemic stress and then gradually declined with an approximate half-life of 4 days. Native and cryptic [Met5]enkephalin-like IR had increased 9-fold and 12-fold, respectively, 24 hr after this period of hypoglycemia, and both demonstrated maximum increases of 130-fold and 50-fold, respectively, after 96 hr. Combined pretreatment (i.p. administration) with the ganglionic and muscarinic blocking agents chlorisondamine (5 mg/kg of body weight) and atropine (1 mg/kg) blocked the increase in levels of proenkephalin A mRNA seen in the rat adrenal medulla following insulin hypoglycemia. These data indicate that reflex splanchnic nerve discharge stimulates proenkephalin biosynthesis, probably at the level of gene expression.

Adrenal Medulla↗
PubMed1984

Regulation of opioid peptide synthesis and processing in adrenal chromaffin cells by catecholamines and cyclic adenosine 3':5'-monophosphate.

Primary cultures of bovine adrenal medullary chromaffin cells were used to study the regulation of opioid peptide (OP) synthesis. Chromaffin cells continuously exposed to tetrabenazine, a drug that depletes cellular catecholamine stores, increase their OP contents between 32 hr and 6 days of treatment. At no time following tetrabenazine addition were increases in opiate receptor-inactive enkephalin-containing peptides (IECPs) observed. Because IECPs may serve as precursors to OPs, these results suggest increased processing of OP precursors following treatment with catecholamine-depleting drugs in addition to an increased rate of OP precursor synthesis. The increases in cellular OP levels induced by tetrabenazine were approximately proportional to the depletion in cellular catecholamines produced by this drug. Also, the effects of tetrabenazine on chromaffin cell OP and IECP contents were mimicked by inhibitors of catecholamine biosynthesis and other agents that decreased catecholamine stores, but not by supplementing the culture medium with catecholamines or catecholamine receptor agonists. Addition of 8-bromo-cAMP or forskolin, an activator of adenylate cyclase, to chromaffin cell cultures increased both OP and IECP stores. Inhibitors of cyclic nucleotide phosphodiesterase also increase chromaffin cell OP and IECP contents, although it is unclear whether these increases result from increased cyclic nucleotide levels. Hence, both alterations in some intracellular catecholamine pool and elevations of cAMP levels may trigger increases in the synthesis and processing of OPs and IECPs in the adrenal medullary chromaffin cell.

Adrenal Medulla↗