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Biomedical subjects

A C Howlett

Publications and source records attributed to A C Howlett.

At least 73 records · Page 4Linked to original sources
PubMed1990

Involvement of a pertussis toxin-sensitive G protein in the regulation of angiotensinogen production by an angiotensin II analog in HepG2 cells.

The cellular mechanism by which the angiotensin II (AII) agonist, Sar1-AII, inhibits production and release of angiotensinogen in human hepatoma HepG2 cells was examined. Pretreatment of HepG2 cells with pertussis toxin attenuated the ability of Sar1-AII to block angiotensinogen production. This effect could be correlated with the in situ ADP-ribosylation of a protein(s) of apparent molecular weight 39,000-41,000 on SDS-PAGE, and attenuation of the ability of Sar1-AII to inhibit cAMP accumulation. The role of cAMP in angiotensinogen production was examined. A transient increase in cAMP accumulation above basal could be evoked by forskolin (8-fold) or by glucagon (5-fold) using insulin-deficient media. Although neither forskolin nor glucagon had a significant effect on angiotensinogen production agents producing a sustained increase in intracellular cAMP (8-bromo-cAMP, dibutyryl-cAMP, cholera toxin) were able to increase angiotensinogen production. Although these data indicate that intracellular cAMP is a regulatory factor in angiotensinogen production other evidence suggests that modulation of intracellular cAMP is not entirely responsible for the effects of Sar1-AII.

Adenosine Diphosphate Ribose↗
PubMed1990

Cannabinoid receptors and modulation of cyclic AMP accumulation in the rat brain.

The mechanism by which cannabinoid compounds produce their effects in the rat brain was evaluated in this investigation. Cannabinoid receptors, quantitated by [3H]CP-55,940 binding, were found in greatest abundance in the rat cortex, cerebellum, hippocampus, and striatum, with smaller but significant binding also found in the hypothalamus, brainstem, and spinal cord. Using rat brain slice preparations, we evaluated the effect of desacetyllevonantradol on basal and forskolin-stimulated cyclic AMP accumulation in the regions exhibiting the greatest cannabinoid receptor density. Desacetyllevonantradol (10 microM) reduced cyclic AMP levels in the hippocampus, frontal cortex, and striatum. In the cerebellum, however, the response to desacetyllevonantradol was biphasic with cyclic AMP accumulation being decreased at lower and increased at higher concentrations. Desacetyllevonantradol reduced cyclic AMP accumulation in isoproterenol-stimulated slices in the cortex and cerebellum, but not in the hippocampus. Cells that responded to vasoactive intestinal peptide with an increase in cyclic AMP accumulation in the hippocampus and cortex also responded to desacetyllevonantradol. The modulation of cyclic AMP accumulation by desacetyllevonantradol could be attenuated following stereotaxic implantation of pertussis toxin, supporting the involvement of a G protein in the cannabinoid response in the brain. However, other actions of cannabinoid compounds may also affect the cyclic AMP levels in brain slice preparations.

Analgesics↗
PubMed1989

Regulation of adenylate cyclase by cannabinoid drugs. Insights based on thermodynamic studies.

The abilities of lipophilic cannabinoid drugs to regulate adenylate cyclase activity in neuroblastoma cell membranes were analyzed by thermodynamic studies. Arrhenius plots of hormone-stimulated adenylate cyclase activity exhibited a break point at 20 degrees. The break point was reduced to 14 degrees by benzyl alcohol, consistent with results from other laboratories that have correlated this response with the increase in membrane fluidity induced by benzyl alcohol. Because cannabinoid drugs partition into membrane lipids and alter membrane fluidity parameters in a number of model systems, it was of interest to examine the influence of delta 9-tetrahydrocannabinol and cannabidiol on enzyme activity analyzed by the Arrhenius plot. delta 9-Tetrahydrocannabinol, known to inhibit adenylate cyclase, failed to decrease the transition temperature either at 1 microM or at concentrations exceeding its aqueous solubility (30 microM), suggesting that delta 9-tetrahydrocannabinol could not mimic the effects observed with benzyl alcohol. In contrast, 30 microM cannabidiol, which stimulated enzyme activity slightly, decreased the Arrhenius plot break point to 17.5 degrees. The decrease in the transition temperature in response to benzyl alcohol or cannabidiol was not accompanied by a change in activation energies above or below the transition temperature. delta 9-Tetrahydrocannabinol inhibits adenylate cyclase activity via Gi as does the muscarinic agonist carbachol (Howlett et al., Mol Pharmacol 29: 307-313, 1986). Both carbachol and delta 9-tetrahydrocannabinol decreased the enthalpy and entropy of activation. The net free energy of activation at 37 degrees was increased in the presence of both of these inhibitory agonists. These data suggest that, for the entropy-driven hormone-stimulated adenylate cyclase enzyme, less disorder of the system occurs in the presence of regulators that inhibit the enzyme via Gi. In summary, thermodynamic data suggest that cannabidiol can influence adenylate cyclase by increasing membrane fluidity, but that the inhibition of adenylate cyclase by delta 9-tetrahydrocannabinol is not related to membrane fluidization.

Adenylyl Cyclase Inhibitors↗
PubMed1988

Determination and characterization of a cannabinoid receptor in rat brain.

The determination and characterization of a cannabinoid receptor from brain are reported. A biologically active bicyclic cannabinoid analgetic CP-55,940 was tritium-labeled to high specific activity. Conditions for binding to rat brain P2 membranes and synaptosomes were established. The pH optimum was between 7 and 8, and specific binding could be eliminated by heating the membranes to 60 degrees. Binding to the P2 membranes was linear within the range of 10 to 50 micrograms of protein/ml. Specific binding (defined as total binding displaced by 1 microM delta 9-tetrahydrocannabinol (delta 9-THC) or 100 nM desacetyllevonantradol) was saturable. The Kd determined from Scatchard analysis was 133 pM, and the Bmax for rat cortical P2 membranes was 1.85 pmol/mg of protein. The Hill coefficient for [3H]CP-55,940 approximated 1, indicating that, under the conditions of assay, a single class of binding sites was determined that did not exhibit cooperativity. The binding was rapid (kon approximately 2.6 x 10(-4) pM-1 min-1) and reversible (Koff approximately 0.016 min-1) and (koff' greater than 0.06 min-1). The two Kd values estimated from the kinetic constants approximately 55 pM and exceeded 200 pM, respectively. The binding of the agonist ligand [3H]CP-55,940 was decreased by the nonhydrolyzable GTP analog guanylylimidodiphosphate. The guanine nucleotide induced a more rapid dissociation of the ligand from the binding site, consistent with an allosteric regulation of the putative receptor by a G protein. The binding was also sensitive to MgCl2 and CaCl2. Binding of [3H]CP-55,940 was displaced by cannabinoid drugs in the following order of potency: CP-55,940 greater than or equal to desacetyllevonantradol greater than 11-OH-delta 9-THC = delta 9-THC greater than cannabinol. Cannabidiol and cannabigerol displaced [3H]CP-55,940 by less than 50% at 1 microM concentrations. The (-)-isomer of CP-55,940 displaced with 50-fold greater potency than the (+)-isomer. This pharmacology is comparable to both the inhibition of adenylate cyclase in vitro and the analgetic activity of these compounds in vivo. The criteria for a high affinity, stereoselective, pharmacologically distinct cannabinoid receptor in brain tissue have been fulfilled.

Allosteric Regulation↗
PubMed1988

Regulation of adenylate cyclase by chronic exposure to cannabimimetic drugs.

Previous studies in this laboratory have demonstrated that a cloned neuroblastoma cell line (N18TG2) responds to delta 9-tetrahydrocannabinol (THC), the major psychoactive product of marihuana, with an attenuation of cyclic AMP accumulation that results from an inhibition of adenylate cyclase. The requirement for the Gi regulatory protein, stereoselectivity, pharmacologic specificity and cell selectivity of this response suggest that a receptor for cannabimimetic compounds may be associated with adenylate cyclase in the neuroblastoma cell. Presented here is a comprehensive investigation of cellular effects of chronic exposure to cannabimimetic agents. Short-term exposure to either delta 9-THC or the more potent nantradol analog, desacetyllevonantradol (DALN), at doses up to 100 microM did not compromise the plating efficiency of the cells. Cells that were exposed to 1 microM delta 9-THC (maximally effective for inhibiting cyclic AMP production) for 24 hr in a serum-free medium were shown to accumulate the drug but not to metabolize it. Exposure to 10 microM delta 9-THC or DALN for up to 48 hr failed to significantly affect cell growth rate or protein content per cell. The gross morphology of cannabinoid-treated cells was not altered at the light or the electron microscope level. The cellular organelles and membranes appeared intact, with no remarkable differences from control cells. The inhibition of cyclic AMP accumulation in response to cannabimimetic drugs was diminished in cells treated with delta 9-THC or DALN for 24 hr. This desensitization was homologous because both delta 9-THC and DALN responses were attenuated after exposure to either cannabimimetic drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1988

Nonclassical cannabinoid analgetics inhibit adenylate cyclase: development of a cannabinoid receptor model.

Extensive structure-activity relationship studies have demonstrated that specific requirements within the cannabinoid structure are necessary to produce potent analgesia. A three-point association between the agonist and the receptor mediating analgesia consists of: 1) the C ring hydroxyl, 2) the phenolic A ring hydroxyl, and 3) the A ring alkyl hydrophobic side chain. Potent tricyclic and bicyclic structures were synthesized as "nonclassical" cannabinoid analgetics that conform to this agonist-receptor three-point interaction model. At the cellular level, centrally active cannabinoid drugs inhibit adenylate cyclase activity in a neuroblastoma cell line. The structure-activity relationship profile for inhibition of adenylate cyclase in vitro was consistent with this same three-point association of agonists with the receptor. A correlation exists between the potency of drugs to produce analgesia in vivo and to inhibit adenylate cyclase in vitro. Enantio- and stereoselectivity were exhibited by the nonclassical cannabinoid compounds for both the analgetic response and the ability to inhibit adenylate cyclase. The magnitude of the enantioselective response was equal for both the biochemical and physiological endpoints. Based on the parallels in structure-activity relationships and the enantioselective effects, it is postulated that the receptor that is associated with the regulation of adenylate cyclase in vitro may be the same receptor as that mediating analgesia in vivo. A conceptualization of the cannabinoid analgetic receptor is presented.

Adenylyl Cyclase Inhibitors↗
PubMed1987

Cyclic nucleotide phosphodiesterase isozymes in neuroblastoma cells.

Adenosine 3',5'-cyclic monophosphate (cAMP) content of neurons is determined not only by the rate of synthesis but also by the rate of hydrolysis by cyclic nucleotide phosphodiesterases. Multiple forms of cyclic nucleotide phosphodiesterase exist in brain and other tissues, and these may be regulated by various hormones and neuromodulators. The present study examines this regulation in a cloned line of neuroblastoma cells (N18TG2). A biphasic Lineweaver-Burk plot of cAMP hydrolysis revealed two Kms approximating 5 and 25 microM. Lineweaver-Burk plots of cGMP hydrolysis were linear over a range of 1 microM to 1 mM and exhibited a Km of 37 microM. Neither cAMP nor cGMP competed for hydrolysis of the alternative cyclic nucleotide. No evidence for an allosteric activation of cAMP phosphodiesterase by cGMP was found. Calcium regulation of phosphodiesterase was not found in spite of preparation of the cell extract with several protease inhibitors, and addition of exogenous calmodulin. No effect of calmodulin antagonists (calmidazolium, W7, or trifluoperazine) was observed in vitro or in situ. Growth of the cells in the presence of 200 nM 3,5,3'-triiodothyronine (T3) resulted in an increased hydrolysis of cAMP but of cGMP. This increase was attributed to an increase in Vmax with no change in either high or low Km. This response was blocked by cycloheximide, suggesting that the thyroid hormone effect requires protein synthesis. The thyroid hormone response in neuroblastoma cells is compared with the results of other studies of thyroid hormone effects on phosphodiesterase in other tissues in vivo.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1987

Cannabinoid inhibition of adenylate cyclase: relative activity of constituents and metabolites of marihuana.

delta 9Tetrahydrocannabinol (THC) has been shown to inhibit the activity of adenylate cyclase in the N18TG2 clone of murine neuroblastoma cells. The concentration of delta 9THC exhibiting half-maximal inhibition was 500 nM. delta 8Tetrahydrocannabinol was less active, and cannabinol was only partially active. Cannabidiol, cannabigerol, cannabichromene, olivetol and compounds having a reduced length of the C3 alkyl side chain were inactive. The metabolites of delta 8THC and delta 9THC hydroxylated at the C11 position were more potent than the parent drugs. However, hydroxylation at the C8 position of the terpenoid ring resulted in loss of activity. Compounds hydroxylated along the C3 alkyl side chain were equally efficacious but less potent than delta 9THC. These findings are compared to the pharmacology of cannabinoids reported for psychological effects in humans and behavioral effects in a variety of animal models.

Adenylyl Cyclase Inhibitors↗
PubMed1987

Thyroid effects on adenosine 3',5'-monophosphate levels and adenylate cyclase in cultured neuroblastoma cells.

Using neuroblastoma cells as a model of developing neurons, we have tested the hypothesis that thyroid hormones alter cAMP metabolism. Neuroblastoma cells were grown in serum-free defined medium for 48 h with or without thyroid hormones. Treatment with 20-200 nM 3,5,3'-triiodo-L-thyronine (T3) increased the accumulation of cAMP by intact cells without altering growth, gross morphology, or DNA or protein content. The increase in cAMP accumulation could be detected 5 h after the addition of T3 and was abolished by the addition of cycloheximide. The maximum stimulation produced by prostaglandin E1 was increased in T3 cells without a significant alteration of the half-maximal concentration. T4 and D-T3 in concentrations up to 20 microM did not increase cAMP accumulation. Adenylate cyclase activity in response to forskolin, guanine nucleotides, and stimulatory hormones was increased in purified membranes from cells grown in T3, suggesting that increased adenylate cyclase is probably the major mechanism of the observed response to thyroid hormone.

Adenylyl Cyclases↗
PubMed1986

An assessment of the role of opioid receptors in the response to cannabimimetic drugs.

Cannabimimetic drugs have been shown to inhibit adenylate cyclase activity in N18TG2 neuroblastoma cells. This investigation examines the possible role of opioid receptors in the cannabimimetic response. Opioid receptors of the delta subtype were found on N18TG2 membranes using [3H]D-Ala2-D-Leu5-enkephalin. No mu or kappa receptors were detected using selective ligands for these sites. The delta binding affinity and capacity were unaltered by cannabimimetic drugs. To test if cannabimimetic drugs may modulate opioid effector mechanisms, cyclic AMP metabolism was determined in intact cells and in membranes. N18TG2 adenylate cyclase was inhibited by the cannabimimetic drugs delta 9-tetrahydrocannabinol and desacetyllevonantradol, and by the opioid agents morphine, etorphine, and D-Ala2-Met5-enkephalinamide. The opioid inhibition was reversed by naloxone and naltrexone; however, the cannabimimetic response was unaffected. Both cannabimimetic and opioid drugs decreased cyclic AMP accumulation in intact cells, but opioid antagonists blocked the response only to the latter. Thus, cannabimimetic effects are observed even though opioid receptors are blocked by antagonist drugs. The interaction between desacetyllevonantradol and etorphine was neither synergistic nor additive at maximal concentrations, suggesting that these two drugs operate via the same effector mechanism. Other neuronal cell lines having an opioid response were also examined. The cannabimimetic inhibition of cyclic AMP accumulation in NG108-15 neuroblastoma X glioma cells was not as great as the response in N18TG2. N4TG1 neuroblastoma cells did not respond to cannabimimetic drugs under any conditions tested. Thus, the cannabimimetic inhibition of adenylate cyclase is not universally observed, and the efficacy of the cannabimimetic response does not correlate with the efficacy of the opioid response.

Adenylyl Cyclase Inhibitors↗
PubMed1986

Involvement of Gi in the inhibition of adenylate cyclase by cannabimimetic drugs.

The cellular mechanism of action of the cannabimimetic drugs is examined using cultured cells. In membranes from N18TG2 neuroblastoma cells and the neuroblastoma X glioma hybrid cells, NG108-15, the psychoactive cannabinoid drugs and their nantradol analogs could inhibit adenylate cyclase activity. This response was not observed in either the soluble adenylate cyclase from rat sperm or membrane-bound adenylate cyclases from C6 glioma or S49 lymphoma cells. This cellular selectivity provides further evidence for the existence of specific receptors for the cannabimimetic compounds. Receptor-mediated inhibition of adenylate cyclase requires the presence of a guanine nucleotide-binding protein complex, Gi. Gi can be functionally inactivated as a result of an ADP-ribosylation modification catalyzed by pertussis toxin. The present study demonstrates that pertussis toxin treatment of cells abolished the cannabimimetic response in intact cells and in membranes derived therefrom. The action of pertussis toxin required NAD+ as substrate for in vitro modification of neuroblastoma membranes. Furthermore, pertussis toxin was able to catalyze the labeling of a neuroblastoma membrane protein in vitro using [32P] NAD+ under conditions similar to those by which attenuation of the cannabimimetic inhibition of adenylate cyclase could be demonstrated. This evidence demonstrates the requirement for a functional Gi in the action of cannabimimetic drugs.

Adenosine Diphosphate Ribose↗
PubMed1985

Cannabinoid inhibition of adenylate cyclase. Biochemistry of the response in neuroblastoma cell membranes.

The inhibition of adenylate cyclase activity by cannabimimetic compounds in a membrane fraction from cultured neuroblastoma cells has been examined. The inhibition was shown to be concentration-dependent over a nanomolar range for both delta 9-tetrahydrocannabinol and its synthetic analog, desacetyllevonantradol. Inhibition was rapid and reversible. The cannabimimetic compounds caused a decrease in Vmax of the enzyme, with no alteration in the Km for substrate. The effects of these compounds were related to the ability of the enzyme to be regulated by divalent cations and guanine nucleotides. The inhibition was greatest at micromolar Mg2+ or Mn2+ concentrations and was abolished at less than 1 mM MnCl2. In the hormone-stimulated state, the enzyme appeared to be regulated by one Mg2+ site. The addition of cannabimimetic or muscarinic cholinergic agents transformed the enzyme into one in which more complex regulation by divalent cations was observed. Half-maximal inhibition of adenylate cyclase was observed at 800 nM GTP for both cannabimimetic and muscarinic cholinergic agents. The substitution for GTP of a nonhydrolyzable analog resulted in activation of the enzyme and failure to respond to either class of inhibitory agents. If the Mg2+ concentration was reduced and exposure to the GTP analog was of short duration, inhibition by both cannabimimetic and muscarinic agents could be observed in the presence of forskolin. This study points to the similarities between the enzyme inhibition by cannabimimetic compounds and by muscarinic cholinergic compounds. It is inferred that the cannabimimetic compounds must act via regulatory mechanisms similar to those operating for receptor-mediated inhibition of adenylate cyclase.

Adenosine Triphosphate↗
PubMed1984

Inhibition of neuroblastoma adenylate cyclase by cannabinoid and nantradol compounds.

This study was undertaken to ascertain the effects of cannabinoid drugs on prostanoid-stimulated adenylate cyclase in neuroblastoma cells. This report demonstrates that delta 9-tetrahydrocannabinol (THC) and levonantradol could decrease initial rate cyclic AMP accumulation in response to prostacyclin in intact cells. Basal accumulation was also diminished. Prostanoid-stimulated adenylate cyclase in a membrane preparation from these cells was inhibited by cannabinoid and nantradol compounds. However, this inhibition was not competitive with prostaglandin E1 or prostacyclin. Further, inhibition was also observed when the enzyme was stimulated by peptide hormones at the secretin receptor. In contrast, enzyme activated by NaF was not inhibited by cannabinoid compounds. Cyclic AMP phosphodiesterase activity in subcellular fractions was unaltered by these agents. These data demonstrate that cannabinoid and nantradol compounds decrease cyclic AMP accumulation in neuronally derived cells, and that this results from an inhibition of basal and hormone-stimulated adenylate cyclase activity.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1984

Secretin receptors on neuroblastoma cell membranes: characterization of 125I-labeled secretin binding and association with adenylate cyclase.

Secretin, a gut-brain peptide, elicited cyclic AMP production in a clone of neuroblastoma cells derived from the C1300 mouse tumor. Adenylate cyclase (EC 4.6.1.1) in plasma membranes from these cells was stimulated by secretin greater than vasoactive intestinal peptide greater than peptide histidine isoleucine amide, but not by the related peptides glucagon, gastric inhibitory polypeptide, or human growth hormone releasing factor. Hill coefficients for stimulation approximated one and the response to submaximal peptide concentrations was additive, as expected for hormones competing for a single receptor associated with the enzyme. Binding of 125I-labeled secretin to the neuroblastoma plasma membranes was saturable, time-dependent, and reversible. The KD determined from kinetic and equilibrium binding studies approximated 1 nM. The binding site displayed marked ligand specificity that paralleled that for stimulation of adenylate cyclase. The secretin receptor was regulated by guanine nucleotides, with guanosine 5'-(beta, gamma-imino)-triphosphate being the most potent to accelerate the rate of dissociation of bound secretin. These findings demonstrate the functional association of the secretin receptor with adenylate cyclase in neuronally derived cells.

Adenylyl Cyclases↗
PubMed1984

Cannabinoid inhibition of adenylate cyclase. Pharmacology of the response in neuroblastoma cell membranes.

Adenylate cyclase in plasma membranes was inhibited by micromolar concentrations of delta 8-tetrahydrocannabinol and delta 9-tetrahydrocannabinol and by levonantradol and desacetyllevonantradol. This inhibition was noncompetitive for stimulation of the enzyme at the prostanoid receptor by prostaglandin E1 or prostacyclin, or at the peptide receptor by secretin or vasoactive intestinal peptide. Forskolin-activated adenylate cyclase was also inhibited by cannabimimetic agents. Inhibition by cannabinoid compounds was neither synergistic nor additive with muscarinic or alpha-adrenergic agents when each was present at maximally inhibitory concentrations. Cannabinoid inhibition was not blocked by atropine, yohimbine, or naloxone, suggesting that muscarinic, alpha 2-adrenergic and certain opiate receptors may not be required for the response. The inhibition of adenylate cyclase was specific for psychoactive cannabinoids, since cannabinol and cannabidiol produced minimal or no response. Inhibition was also stereoselective, since dextronantradol did not produce the response. A biphasic log dose-response curve was observed for each of the cannabinoid drugs, such that reversal of the inhibition occurred at 3-10 microM. Possible mechanisms for the effects of cannabinoid drugs on adenylate cyclase activity are discussed.

Adenylyl Cyclase Inhibitors↗