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Biomedical subjects

A C Howlett

Publications and source records attributed to A C Howlett.

At least 55 records · Page 3Linked to original sources
PubMed1993

Endogenous ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase that is not regulated by nitric oxide in Dictyostelium discoideum.

A 41,000 M(r) cytosolic protein (p41) in Dictyostelium discoideum was shown to be modified by ADP-ribosylation that was not regulated by nitric oxide (NO). This endogenous ADP-ribosylation was optimal at conditions distinct from those optimal for the NO-stimulated ADP-ribosylation of p41. These two activities were also differentially sensitive to reducing agents and modified different amino acids. The addition of haemoglobin, which sequesters NO, and of NO synthase inhibitors failed to block the endogenous ADP-ribosylation. P41 was purified to homogeneity. The N-terminal sequence of the purified protein was shown to be highly homologous to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both endogenous and NO-stimulated activities ADP-ribosylated three isoforms of the protein, with pI values of 6.6, 6.8 and 7.0. In each case, the isoform with pI 6.8 was preferentially modified. Experiments using purified GAPDH indicate that both the endogenous and NO-stimulated ADP-ribosylation are self-catalysed modifications.

Adenosine Diphosphate↗
PubMed1993

Vasoactive intestinal polypeptide-related peptides modulate tyrosine hydroxylase gene expression in PC12 cells through multiple adenylate cyclase-coupled receptors.

We investigated the receptor mechanisms by which vasoactive intestinal polypeptide (VIP) and related peptides exert their effects on tyrosine hydroxylase (TH) gene expression. VIP, secretin, and peptide histidine isoleucine (PHI) each produced increases in TH gene expression, as measured by increases in TH mRNA levels and TH activity. The concentrations at which the effects of these peptides were maximal differed for TH activity and TH mRNA. Moreover, maximal increases in TH activity were 130-140% of control, whereas maximal increases in TH mRNA were 250% of control. The concentration dependence of the increases in TH mRNA in response to the three peptides was analyzed by fitting the data to nonlinear regression models that assume either one or two components to the response. The data for secretin fit best to a model that assumes a single component to the increase in TH mRNA levels. The data derived for PHI and VIP fit best to models that assumed two components to the TH mRNA response. These data suggested that there may be more than one receptor or signal transduction mechanism involved in the response to the various peptides. We examined whether the peptides exerted their effects through common or multiple second messenger systems. The ability of maximally active concentrations of these peptides to stimulate increases in TH mRNA was not additive, indicating that the peptides work through a common receptor or signal transduction pathway. Each peptide stimulated increases in protein kinase A (PKA) activity. Secretin and VIP were ineffective in increasing TH mRNA levels in a PKA-deficient mutant PC12 cell line (A126-1B2). Moreover, the adenylate cyclase antagonist 2',5'-dideoxyadenosine prevented the increase in TH mRNA produced by each peptide. Thus, each peptide requires an intact cyclic AMP second messenger pathway to produce changes in TH gene expression, suggesting that the complex pattern of response to VIP and PHI revealed by concentration-response analysis was due to the actions of these peptides at multiple receptors. To evaluate this possibility, we examined the effect of several peptide receptor antagonists on the increase in TH gene expression elicited by VIP, PHI, and secretin. The secretin antagonist secretin (5-27) (20 microM) had no significant effect on VIP or PHI stimulation of TH gene expression, but reduced the effect of secretin. The VIP antagonist VIP (10-28) (20 microM) reduced the effect of VIP on increasing TH mRNA, but had no significant effect on the response of TH mRNA to secretin or PHI.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenylyl Cyclases↗
PubMed1993

Structure-activity relationships for cannabinoid receptor-binding and analgesic activity: studies of bicyclic cannabinoid analogs.

Cannabimimetic compounds, such as delta 9-tetrahydrocannabinol (delta 9-THC), evoke analgesia in addition to other behavioral responses in humans and animals. The cannabinoid receptor mediating this response has been characterized by its ability to bind the cannabinoid agonist [3H]CP-55,940 and to inhibit adenylyl cyclase via Gi. An investigation of structural requirements for antinociceptive activity of cannabinoid structures led to the development of a simple bicyclic cannabinoid agonist, CP-47,497, that possessed a spectrum of cannabinoid activities in animals that resembled that of delta 9-THC. The present investigation examines several series of CP-47,497 analogs for their binding affinity at the cannabinoid receptor and their ability to evoke analgesia in rodents. Analogs substituted at the C-3 alkyl side chain exhibited maximal affinity for the cannabinoid receptor with side chains of seven or eight carbons in length. Analgesic potency paralleled the receptor-binding affinity. The cyclohexyl ring was optimized as a six- or seven-membered ring structure for binding as well as analgesic activity. Cyclohexyl alkyl side chain extensions of up to four carbons in length had little influence on the affinity for the receptor or analgesic activity. Hydroxyalkyl side chains exhibited optimal binding affinity and antinociceptive activity at three or four carbon atoms in length; however, polar groups closer to the ring diminished binding to the receptor. The importance of the phenolic and cyclohexyl hydroxyl groups for binding affinity was demonstrated. In general, analgesic activity correlated well with the affinity of these analogs for the cannabinoid receptor. Exceptions could be explained by metabolic transformations likely to occur in vivo.

Adenylyl Cyclase Inhibitors↗
PubMed1993

Solubilization of the cannabinoid receptor from rat brain and its functional interaction with guanine nucleotide-binding proteins.

The present investigation was undertaken to characterize cannabinoid receptor binding in the absence of the membrane environment, inasmuch as cannabinoid drugs have been noted to influence the behavior of integral membrane proteins. The zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) was able to solubilize the cannabinoid receptor from rat brain membranes, with the greatest yield and specific activity being obtained at a detergent/protein ratio of 0.5:1. [3H]CP-55940 bound to a single class of binding sites in the CHAPS extract, which exhibited a Kd of 0.94 nM as determined by nonlinear regression analysis of equilibrium binding data. The order of potency for cannabinoid agonists in heterologous equilibrium binding studies was CP-55244 > or = desacetyllevonantradol > delta 9-tetrahydrocannabinol > cannabinol >> cannabidiol, consistent with the relative affinities for these agonists in brain membrane preparations. CP-55243, the biologically inactive enantiomer of CP-55244, competed for binding of [3H]CP-55940 by < 50% at 1 microM, similar to its poor affinity for the receptor in membranes. The CHAPS-solubilized cannabinoid receptor exhibited functional interactions with guanine nucleotide-binding proteins (G proteins). GTP and nonhydrolyzable analogs decreased [3H]CP-55940 binding by 75%. The concentration-effect curves for guanine nucleotides exhibited a potency order similar to that observed for other G protein-linked receptors. Kinetic analyses indicated that GTP analogs increased the rate of agonist dissociation, decreasing the t1/2 from 60 min at 0-4 degrees to a multiphasic dissociation that exhibited a component having a t1/2 of < 1 min. The cannabinoid agonist desacetyllevonantradol was able to reduce pertussis toxin-catalyzed ADP-ribosylation of G proteins by 50%, demonstrating a receptor effect on G protein functions. These studies demonstrate that the membrane environment is not necessary for agonist binding to the cannabinoid receptor. Furthermore, the cannabinoid receptor maintains its functional interactions with pertussis toxin-sensitive G proteins in detergent solution.

Animals↗
PubMed1992

Ca(2+)-dependent release from rat brain of cannabinoid receptor binding activity.

As a result of the identification, pharmacological characterization, and localization of the cannabinoid receptor in the CNS, the existence of an endogenous ligand for this receptor can be hypothesized. Following the premise that such a substance could have the properties of a neuromodulator being stored in intracellular vesicles, we tested the ability of increased intracellular Ca2+ levels to stimulate release. We demonstrate here that the Ca2+ ionophore A23187 can induce release of cannabinoid-like binding activity in the presence but not in the absence of Ca2+. The effect of A23187 was maximal at 1.2 microM, consistent with vesicular release. It was necessary to increase the concentration of extracellular free Ca2+ to greater than 60 nM to evoke release. The released cannabinoid-like binding activity displaced [3H]CP-55940 binding to cannabinoid receptors in rat synaptosomal membranes in a concentration-dependent manner. This is the first report of a substance present endogenously in brain that can be released in a Ca(2+)-dependent manner and that binds to the cannabinoid receptor.

Animals↗
PubMed1991

Inhibition of adenylate cyclase by p-bromophenacyl bromide.

p-Bromophenacyl bromide (BPB) is an alkylating agent which has been used in biochemical studies as an inhibitor of phospholipase A2 activity. We report here that BPB irreversibly inhibited adenylate cyclase activity stimulated by hormones, forskolin, GppNHp, NaF, and cholera toxin. The action of BPB in S49 lymphoma cell membranes (wild type and cyc-) indicates that it can inhibit adenylate cyclase function in the absence of Gs. In the presence of Gs, however, inhibition of adenylate cyclase by BPB was enhanced, suggesting that BPB may covalently modify the catalytic protein on a site involved in activated catalytic functioning or critical to its interaction with Gs and/or additionally on the alpha s protein.

Acetophenones↗
PubMed1991

Muscarinic receptor binding is inhibited by quinacrine.

Quinacrine altered the equilibrium binding of the muscarinic antagonist quinuclidinyl benzilate (QNB) to the muscarinic receptor in membranes from N18TG2 neuroblastoma cells. At 1 microM quinacrine, the apparent Kd for [3H]QNB binding was shifted from 48 to 210 pM without a decrease in the maximum binding. The competition binding profile of quinacrine indicates that it displaced [3H]QNB binding with an IC50 of 460 nM in a manner consistent with competitive inhibition. Quinacrine blocked the muscarinic inhibition of cyclic AMP accumulation by carbachol in cultured N18TG2 neuroblastoma cells without affecting arachidonic acid release or Gi coupling to the adenylate cyclase. The disruption of muscarinic receptor function by quinacrine may provide an explanation for its blockade of the muscarinic response in cells.

Binding, Competitive↗
PubMed1991

Vasoactive intestinal polypeptide induces tyrosine hydroxylase in PC12 cells.

Physiological stress induces tyrosine hydroxylase, the rate-limiting enzyme for catecholamine biosynthesis, via trans-synaptic mechanisms within the adrenal medulla. Previous studies have implicated cAMP as a second messenger capable of inducing tyrosine hydroxylase; however, it is unclear whether any receptor coupled to adenylate cyclase mediates tyrosine hydroxylase induction. Recently, vasoactive intestinal polypeptide, whose receptor is coupled to adenylate cyclase in many tissues, has been shown to meet many of the criteria for a neuromodulator within the adrenal medulla. We therefore undertook a series of studies to determine whether vasoactive intestinal polypeptide may induce tyrosine hydroxylase in PC12 cells, a cell line derived from rat adrenal medulla. Here we report that vasoactive intestinal polypeptide produces a transient, time- and concentration-dependent increase in tyrosine hydroxylase mRNA levels which is followed by a stable increase in tyrosine hydroxylase protein. The increase in tyrosine hydroxylase mRNA does not occur in a mutant PC12 cell line deficient in cAMP-dependent protein kinase activity, indicating that the effect of vasoactive intestinal polypeptide is mediated through the cAMP second messenger pathway. This is the first report demonstrating that a neuromodulator which acts on an adenylate cyclase-coupled receptor can induce tyrosine hydroxylase.

Animals↗
PubMed1991

Chronic exposure to delta 9-tetrahydrocannabinol fails to irreversibly alter brain cannabinoid receptors.

The effects of chronic delta 9-tetrahydrocannabinol (delta 9-THC) and marijuana administration on the properties of brain cannabinoid receptor populations of the rat and monkey, respectively, were examined in this study. It was determined that the properties of the cannabinoid receptors in the striatum, cerebral cortex, cerebellum, hippocampus, and brainstem/spinal cord of the rat do not appear to be irreversibly altered by chronic exposure to delta 9-THC. Similarly, the cannabinoid receptors in the caudate, prefrontal cortex, and cerebellum of the monkey do not appear to be irreversibly altered by chronic exposure to marijuana smoke.

Analgesics↗
PubMed1991

Dopaminergic regulation of dopamine release from PC12 cells via a pertussis toxin-sensitive G protein.

Regulation of dopamine (DA) release from PC12 cells was investigated. Apomorphine and quinpirole, a selective D2 agonist, significantly reduced K(+)-evoked DA release, and this reduction was reversed by haloperidol. Furthermore, spiroperidol, a selective D2 antagonist, and haloperidol, a nonselective DA antagonist, enhanced the K(+)-evoked DA release. Pertussis toxin treatment of the cells abolished the quinpirole-induced reduction of K(+)-evoked DA release. Also, the haloperidol-induced enhancement of K(+)-evoked DA release was not seen in pertussis toxin treated cells. These results, therefore, suggest the presence of D2 receptors on PC12 cells which result in the modulation of K(+)-evoked DA release via a pertussis toxin-sensitive G protein.

Adrenal Gland Neoplasms↗
PubMed1991

Regulation of nicotine-evoked dopamine release from PC12 cells.

The nature of second messengers involved in the nicotine-evoked release of dopamine from PC12 cells was examined. Calmidazolium, a calmodulin inhibitor, abolished the nicotine-evoked release. A23187, a Ca2+ ionophore, enhanced dopamine release, and this was inhibited by calmidazolium. Further, 2', 5'-dideoxyadenosine abolished both the nicotine- and A23187-evoked release. Forskolin, dibutyryl-cyclic AMP, and rolipram (a cyclic AMP phosphodiesterase inhibitor) all enhanced dopamine release. 1, 9-Dideoxyforskolin, a forskolin analog which does not activate adenylate cyclase, did not alter dopamine release. These results suggest an obligatory role for Ca2+ and calmodulin-sensitive adenylate cyclase in the nicotine-evoked release process.

Adenylyl Cyclases↗
PubMed1991

The cannabinoid receptor: biochemical and cellular properties in neuroblastoma cells.

The cannabinoid receptor that has been pharmacologically characterized for hypothermia, spontaneous activity, analgesia and catalepsy in rodents is the same pharmacological receptor that inhibits adenylate cyclase in vitro. The inhibition of adenylate cyclase by the cannabinoid receptor results from an interaction with Gi, based on the biochemical kinetic properties of the response, the sensitivity to pertussis toxin ADP-ribosylation, and the thermodynamic characteristics of the response. From precedents based on studies of the well-characterized G protein coupled receptors, rhodopsin and the beta-adrenergic receptor, we can predict the tertiary structure of the cannabinoid receptor. Three sites of potential glycosylation are present on the receptor. However, treatment of N18TG2 neuroblastoma cells with tunicamycin to prevent glycosylation of newly synthesized receptors failed to alter cannabinoid-induced inhibition of cyclic AMP accumulation. The cannabinoid response was rapidly desensitized (within 1/2 h). Treatment of cells with tunicamycin failed to alter agonist-induced desensitization processes. These findings can be more veraciously interpreted as we gain a better understanding of the cellular dynamics of the cannabinoid receptor.

Animals↗
PubMed1991

Cannabinoid receptor-regulated cyclic AMP accumulation in the rat striatum.

The present study demonstrates that desacetyllevonantradol, a synthetic cannabinoid analog, reduces cyclic AMP levels in rat striatal slices stimulated with vasoactive intestinal peptide or SKF 38393, a D1-dopamine agonist. Desacetyllevonantradol and the D2 agonist LY 171555 both inhibited D1-stimulated cyclic AMP accumulation in the striatum. Spiperone, a specific D2-dopamine antagonist, fully reversed the inhibitory effect of LY 171555 but not that of desacetyllevonantradol, indicating that this cannabinoid response is not occurring through a D2-dopaminergic mechanism. Morphine also inhibited cyclic AMP accumulation in striatal slices stimulated with either SKF 38393 or vasoactive intestinal peptide. Naloxone, an opioid antagonist, fully reversed the effect of morphine but not that of desacetyllevonantradol, indicating that cannabinoid drugs are not acting via a mechanism involving opioid receptors. The response to maximally effective concentrations of desacetyllevonantradol was not additive to that of maximally effective concentrations of either morphine or LY 171555, suggesting that dopaminergic, opioid, and cannabinoid receptors may be present on the same populations of cells.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1990

Stereochemical effects of 11-OH-delta 8-tetrahydrocannabinol-dimethylheptyl to inhibit adenylate cyclase and bind to the cannabinoid receptor.

The recent preparation of the enantiomers of 11-OH-delta 8-tetrahydrocannabinol-dimethylheptyl (THC-DMH), recrystallized to absolute enantiomeric purity, has made it possible to examine the requirement for stereospecificity for the interaction of this component with the cannabinoid receptor, defined by the binding of [3H]CP-55,940 and the adenylate cyclase enzyme. The enantiomer (-)11-OH-delta 8-THC-DMH exhibited a fully efficacious and potent (IC50 = 1.8 nM) inhibition of the accumulation of cAMP in intact N18TG2 cells. The (-)enantiomer was as efficacious and potent (Kinh = 7.2 nM) as desacetyllevonantradol in inhibiting adenylate cyclase activity in membrane preparations. The (-)enantiomer was able to compete fully for the specific binding of [3H]CP-55,940 to membranes from the brain of the rat in homologous displacement studies (Ki = 234 pM). The potency ratios exhibited by the (-) to (+)enantiomers of 11-OH-delta 8-THC-DMH exceeded 1000 for each of these activities.

Adenylyl Cyclase Inhibitors↗
PubMed1990

The cannabinoid receptor: biochemical, anatomical and behavioral characterization.

The actions of the active principle of marihuana, delta 9-tetrahydrocannabinol, are mimicked by synthetic cannabinoid agonists showing high potency and enantio-selectivity in behavioral assays. These drugs have been used to characterize cannabinoid receptor binding, biochemistry and pharmacology, leading to a better understanding of the effects of cannabinoids in the CNS of humans and experimental animals.

Animals↗