Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.6.1.1 (adenylate cyclase)
19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Regulation of intracellular cAMP levels serves as a cellular model for chronic drug action. Since the adenylate cyclase effector system is under dual control of both stimulatory as well as inhibitory receptor systems, a permanent cell line was created in order to allow evaluation of acute and chronic opioid effects on stimulatory receptor function. For this purpose, the cloned rat mu-opioid receptor was stably expressed in human epidermoid carcinoma (A431) cells, which carries high levels of endogenous beta 2-adrenoceptors. Four out of 16 cell clones were found to express considerable amounts of [3H]diprenorphine binding sites and were further characterized. Scatchard analysis of saturation binding data revealed maximal binding capacities (Bmax) between 242.2 +/- 11 and 1,271.8 +/- 221 fmol/mg of membrane protein, whereas drug affinity was found similar among all cell clones tested (Kd = 1.4 +/- 0.2 nM). The expressed mu-receptors also mediated agonist inhibition of adenylate cyclase, indicating that these receptors are functionally coupled to intracellular signalling pathways. Long-term exposure of the cells to morphine (10 microM; 2 days) produced cellular correlates of chronic opioid action as displayed by both a decrease in the maximal degree of adenylate cyclase inhibition (tolerance) as well as an increase in overall effector activity (dependence). Thus, based on these parameters, mu-opioid receptor expressing A431 cells provide a promising tool to investigate cellular mechanisms of chronic drug action.
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PMID:Stable expression and functional characterization of the cloned rat mu-opioid receptor in human epidermoid carcinoma (A431) cells. 876 28

Rat-1 fibroblasts were transfected with a cDNA encoding the mouse delta opioid receptor. Two separate clones, D2 (which expressed some 6 pmol of the receptor/mg of membrane protein) and DOE (which expressed some 0.2 pmol/mg of membrane protein), were examined in detail. With membranes from both clones, the opioid agonist [D-Ala2]leucine enkephalin (DADLE) caused stimulation of high-affinity GTPase activity and of the binding of guanosine 5'-[gamma-[35S]thio]triphosphate, and inhibition of forskolin-amplified adenylate cyclase activity. DADLE also induced phosphorylation and activation of both the p42MAPK (42 kDa isoform) and p44MAPK (44 kDa isoform) members of the mitogen-activated protein kinase (MAP kinase) family. All of these effects of DADLE were prevented in both clones by pretreatment of the cells with pertussis toxin. The maximal response that could be produced by DADLE in direct assays of G-protein activation were substantially greater in clone D2 than in clone DOE, but in both clones essentially full phosphorylation of both p42MAPK and p44MAPK could be achieved. EC50 values for DADLE stimulation of GTPase activity and for activation of p44MAPK were substantially lower in clone D2 than in clone DOE. Moreover, in both clones the EC50 value for DADLE stimulation of p44MAPK was substantially lower than that for stimulation of GTPase activity, and the Hill coefficients for agonist activation of p44MAPK (h > 1) displayed marked co-operativity whereas those for G-protein activation did not (h 0.8-1.0). DADLE activation of p44MAPK showed more sustained kinetics in clone D2 than in clone DOE. By contrast, lysophosphatidic acid, acting at an endogenously expressed G-protein-coupled receptor, also activated p44MAPK in both clones in a pertussis toxinsensitive manner, but both the kinetics and the concentration-response curve for activation of p44MAPK by this ligand were similar. As with other systems, maintained cellular levels of a cAMP analogue prevented the effects of both G-protein-coupled receptors on activation of p44MAPK. These results demonstrate for the first time that an opioid receptor, at least when expressed in Rat-1 fibroblasts, is able to initiate activation of the MAP kinase cascade in a G1-dependent manner, and show that only a very small proportion of the cellular G1 population is required to be activated to result in full phosphorylation of the p42MAPK and p44MAPK MAP kinases.
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PMID:Agonist activation of p42 and p44 mitogen-activated protein kinases following expression of the mouse delta opioid receptor in Rat-1 fibroblasts: effects of receptor expression levels and comparisons with G-protein activation. 894 92

Chronic opioid regulation of stimulatory beta-2 adrenoceptor (beta-2 AR) signaling was investigated in human mammary epidermoid carcinoma A431 cells stably expressing the cloned rat mu opioid receptor. In the cell clone used (A431/mu 13; Bmax = 302.9 +/- 46 fmol/mg membrane protein), the addition of morphine acutely attenuated basal as well as (-)-isoproterenol-stimulated cAMP accumulation. Prolonged exposure of the cells to morphine (10 microM; 2 d) resulted in homologous desensitization of MOR function as well as heterologous sensitization of adenylate cyclase (AC). Up-regulation of AC in A431/mu 13 cells is characterized by an increased capacity rather than an increased sensitivity of beta-2 AR-stimulated AC. Moreover, opioid withdrawal falls to precipitate a cAMP overshoot in this cell system. Sensitization of stimulatory AC signaling by chronic morphine develops in a time- and dose-dependent manner and is blocked by both naloxone and pertussis toxin. Investigation into the mechanism leading to up-regulation of AC revealed a 40% increase in the number of beta-2 ARs as assessed by [125I]-cyanopindolol binding experiments. No additional quantitative changes were found for stimulatory G proteins and the effector enzyme itself. Sensitization of AC appears to be mediated solely by the increase in beta-2 AR numbers, because (+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3- [(1-methylethyl)amino]-2-butanol hydrochloride, which acts as an "inverse agonist" at the beta-2 AR, completely reversed elevated basal AC activities, and because the ratio between functional active beta-2 ARs and stimulatory G proteins remained unchanged. In conclusion, chronic exposure of clonal A431/ mu13 cells to morphine increases the capacity of stimulatory AC signaling by up-regulating beta-2 AR number. These results demonstrate participation of stimulatory receptor systems in the cellular mechanisms underlying opioid dependence.
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PMID:Chronic morphine treatment increases stimulatory beta-2 adrenoceptor signaling in A431 cells stably expressing the mu opioid receptor. 899 36

The present study evaluates the influence of cholera toxin and its B-subunit on thermic responses to morphine in the rats. The holotoxin (1 microg/rat) and the B-subunit (5 microg) were administered ICV and three days later rats were challenged ICV with morphine and tested for changes of body temperature. Cholera toxin, but not its B-subunit, modified the time course of the hyperthermic response induced by a low dose of morphine (2.5 microg), converted the hypothermia due to a higher dose of morphine (18 microg) to a consistent hyperthermia and only partially reduced the greater hypothermia induced by 36 microg of morphine. Cholera toxin-induced modifications of thermic responses to morphine were paralleled with a decreased Gs(alpha) immunoreactivity and a reduced ability for the toxin to catalyse the "in vitro" ADP-ribosylation of Gs(alpha) in hypothalamic membranes. In contrast, at the same time when morphine-induced effects on body temperature were assessed, no changes in pertussis toxin-mediated ADP-ribosylation of Gi(alpha)/Go(alpha), or basal adenylate cyclase activity, or binding of mu-opioid receptor selective ligand [3H]-DAMGO were observed in hypothalamic areas from rats treated with cholera toxin. These findings suggest that adaptative events secondary to prolonged activation of Gs(alpha) play a role in the modifications of thermic responses to morphine induced by CTX.
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PMID:Cholera toxin effects on body temperature changes induced by morphine. 907 89

Nociceptin, also known as orphanin FQ, was recently identified as the naturally occurring agonist of orphan opioid receptor-like ORL1 receptor (Meunier et al., 1995, Nature 377, 532; Reinscheid et al., 1995, Science 270, 792). Nociceptin is a heptadecapeptide which, although it resembles dynorphin A, the endogenous agonist of the kappa-opioid receptor, displays very low potency in competing with binding of [3H]diprenorphine to or inhibiting adenylate cyclase via mu-, delta- and kappa-opioid receptors. Tritium-labeled nociceptin ([3H]nociceptin) was used here to establish a pharmacological profile in vitro of the ORL 1 receptor. In membranes from recombinant Chinese hamster ovary (CHO) cells expressing the ORL 1 receptor, equilibrium binding of [3H]nociceptin is highly specific, saturable (Bmax in the range 1.3-1.8 pmol/mg protein) and of high affinity (Kd approximately equal to 0.1 nM). It is selectively decreased in the presence of Na+ ions and/or of the GTP analog 5'-guanylylimido-diphosphate, an allosteric regulation that is analogous to that of opiate binding to opioid receptors. A few opiates, namely lofentanil, a 4-anilinopiperidine derivative and etorphine, a 6,14-endo-ethenotetrahydrothebaine derivative, were found to be quite potent not only in competing with binding of [3H]nociceptin at the ORL 1 receptor but also in inhibiting forskolin-induced accumulation of cyclic AMP in intact recombinant CHO cells. In a preliminary attempt to delineate active parts of the neuropeptide, nociceptin analogs were also tested, including N- and C-terminal truncation products. Our results suggest that the highly basic, internal core of nociceptin might be essential in conferring on the peptide both affinity for and activity at the ORL 1 receptor. In this respect, the message and address division of dynorphin A, nociceptin's closest structural analog, do not seem to apply to nociceptin.
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PMID:Recognition and activation of the opioid receptor-like ORL 1 receptor by nociceptin, nociceptin analogs and opioids. 908 91

The existence of mu, delta and kappa opioid receptors in the central nervous system is well documented. The present review focuses on the relationships between opioid receptor types and physical and psychic dependences. Mu and delta, but not kappa opioid receptor agonists produce physical dependence. From behavioral, biochemical and molecular biological studies, it is suggested so far that development of physical dependence on morphine results predominantly from an activation of mu 1 and mu 2 opioid receptors which causes functional changes in Gi/o, adenylate cyclase, protein kinases A and C, beta-adrenoceptor and NMDA receptor in the locus coeruleus. Recently, there have been significant advances in studies on psychic dependence. Mu and delta opioid receptor agonists produce psychic dependence, but kappa opioid receptor agonists rather produce an aversive effect. Activation of the mesolimbic dopamine system may lead to psychic dependence on opioids. Mu and delta 1 opioid receptor agonists activate the mesolimbic dopamine system to induce a rewarding effect, whereas the rewarding effect of delta 2 opioid receptor agonists may be produced through a non-dopaminergic system. There are complicated interactions among opioid receptor types. The activation of kappa opioid receptor suppresses physical and psychic dependences on mu and delta opioid receptor agonists, but the activation of delta opioid receptor potentiates the dependence on mu opioid receptor agonists. The clinical use of morphine in patients with cancer pain won't develop dependence probably due to the balance of the opioid system coming from these interactions.
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PMID:[Opioid receptor types and dependence]. 916 Mar 46

The highly potent and efficacious mu-opioid agonist fentanyl was SC infused into rats with submaximal analgesic doses (0-1.14 mumol/kg/day) continuously for 8 days, checked by the constant daily urinary recovery of intact drug (0.43 +/- 0.031% of the daily dose). Tail-flick latencies measured at 24 (day 1) and 48 h (day 2) after starting the infusion were increased in a dose-dependent fashion compared with those before the infusion (day 0). However, at day 8, the latencies were increased only weakly, not significantly, revealing tolerance to the antinociceptive activity of fentanyl. Fentanyl at all doses showed no significant effect on the capacity (Bmax) and affinity (Kd) of the mu-opioid receptor binding of DAMGO to whole brain (Bmax 126.2 +/- 3.00 fmol/mg protein, Kd 1.00 +/- 0.04 nM) and spinal cord (Bmax 48.24 +/- 2.71 fmol/mg protein, Kd 1.93 +/- 0.13 nM) membranes gained from the rats after killing them at day 8. Gpp(NH)p increased the Kd for brain and spinal cord sites by 3.09 and 2.65, respectively, independent of the fentanyl dose. The infusion with fentanyl did not after the basal and forskolin-stimulated adenylate cyclase activity in the whole brain membranes, nor did it change the inhibition of the forskolin-stimulated activity by DAMGO. It is concluded that, in rats, constant long-term body levels of highly potent mu-agonists result in a tolerant state that, however, does not produce overall changes in the parameters of their specific receptor sites in the CNS, i.e., receptor capacity and affinity, and in the events closely related to them, i.e., their regulation by GTP and of adenylate cyclase. This does not exclude such possible changes to be restricted to specific regions in the CNS.
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PMID:Influence of continuous levels of fentanyl in rats on the mu-opioid receptor in the central nervous system. 926 90

The rat mu-opioid receptor (rMOR1), expressed in human embryonic kidney 293 (HEK293) cells, shows a desensitization to the inhibitory effect of the mu agonist DAMGO on adenylate cyclase activity within 4 h of DAMGO preincubation. To investigate the role of calcium/calmodulin-dependent protein kinase II (CaM kinase II) on mu-opioid receptor desensitization, we coexpressed rMOR1 and constitutively active CaM kinase II in HEK293 cells. This coexpression led to a faster time course of agonist-induced desensitization of the mu-opioid receptor. The increase of desensitization could not be observed with a mu-opioid receptor mutant (S261A/S266A) that lacks two putative CaM kinase II phosphorylation sites in the third intracellular loop. In addition, injection of CaM kinase II in Xenopus oocytes led only to desensitization of expressed rMOR1, but not of an S261A/S266A receptor mutant. These results suggest that phosphorylation of Ser261 and Ser266 by CaM kinase II is involved in the desensitization of the mu-opioid receptor.
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PMID:Site mutation in the rat mu-opioid receptor demonstrates the involvement of calcium/calmodulin-dependent protein kinase II in agonist-mediated desensitization. 932 7

The effect of intrathecal injection of dynorphin A (1-17) on second messenger systems of spinal cord relative to behavioral change in rats was studied. Dynorphin A (1-17) 5, 10 (20 nmol) caused dose-dependent flaccid paralysis of hindlimbs. Dynorphin A (1-17) 10, 20 nmol dose-dependently decreased spinal adenylate cyclase (AC) activity, cyclic AMP production, calmodulin (CaM) level and cyclic-nucleotide phosphodiesterase (PDE) activity 10 min after intrathecal injection. They recovered to a varying extent two hours later. Pretreatment with selective kappa-opioid receptor antagonist nor-BNI 30 nmol 10 min before dynorphin A (1-17) markedly antagonized the effects of dynorphin A (1-17) at 20 nmol on hindlimb paralysis and inhibition of intracellular second messengers. The L-type calcium channel blocker verapamil (100 nmol) also played a role in blocking dynorphin neurotoxicity. The NMDA receptor antagonist APV could partially or completely block dynorphin inhibition of CaM level and PDE activity without affecting paralysis and decrease of AC-cAMP level induced by dynorphin A (1-17) 10 min after intrathecal injection.
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PMID:Effects of dynorphin A (1-17) on motor function and spinal intracellular messenger systems in rat. 938 10

We have recently shown that the cytoplasmic tail of the rat mu-opioid receptor undergoes alternative splicing giving rise to two isoforms, rMOR1 and rMOR1B. These isoforms exhibit similar pharmacological profiles, however, differ in agonist-induced desensitization of coupling to adenylate cyclase. In the present study, we have raised polyclonal antibodies that specifically detect either rMOR1 or rMOR1B and used these antisera for immunocytochemical localization of the receptor proteins in the rat central nervous system. Prominent MOR1B-like immunoreactivity was found in the external plexiform layer of the main olfactory bulb localized to a dense plexus of dendrites mostly originating from mitral cells and extending into the glomerular layer. MOR1-like immunoreactivity was restricted to the perikarya of mitral cells and to distinct juxtaglomerular cells as well as their processes. While MOR1-, DOR1- and KOR1-like immunoreactivity was absent from the external plexiform layer, high densities of opioid peptides were found in this layer suggesting that MOR1B may be a targeted receptor of these peptides. MOR1-like immunoreactivity was observed in many pain-controlling brain areas including the spinal cord dorsal horn, sensory trigeminal complex, raphe nuclei and periaqueductal gray while MOR1B-like immunoreactivity was not detectable in these regions. Taken together, we provide evidence that the mu receptor isoforms, MOR1 and MOR1B, exhibit a strikingly different distribution in that MOR1 appears to be the major isoform widely distributed throughout the central nervous system and MOR1B being predominantly localized to the olfactory bulb.
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PMID:Immunolocalization of two mu-opioid receptor isoforms (MOR1 and MOR1B) in the rat central nervous system. 946 65


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