Studies on the coupling of opioid and cannabinoid receptors to adenylyl cyclase
thesisposted on 15.12.2014, 10:30 by Robert Anthony. Hirst
This thesis represents a detailed study of the regulation of adenylyl cyclase (AC) by opioid and cannabinoid ligands which act at transmembrane spanning Gi-protein coupled receptors.;In SH-SY5Y human neuroblastoma cells carbachol (CCh) and K+ caused a time and dose dependent increase in cAMP formation and also increased [Ca2+]i. Ni2+ essentially abolished CCh stimulated [Ca2+]i plateau phase and blocked K+ stimulated [Ca2+]i. Preincubation with Ni2+, did not affect the initial CCh mediated rise in cAMP but significantly reduced the 5 and l0min levels. CCh in Ca2+ free buffer increased cAMP and when Ca2+ was re-introduced to the buffer there was a further increase in cAMP. These studies suggest that SH-SY5Y cells express type 1 AC.;The previously characterised SH-SY5Y ([3H]Diprenorphine (DPN) Bmax (an estimate of opioid receptor density)=98fmol/mg protein) and NG108-15 ([3H]DPN Bmax=280fmol/mg protein) cells were used as a model system for the study of endogenous mu- and delta- opioid receptors respectively. In SH-SY5Y membranes, fentanyl, DAMGO and in NG108-15 membranes, DPDPE, DADLE displaced [3H]DPN revealing high affinity binding sites. Fentanyl, DAMGO (SH-SY5Y) and DPDPE, DADLE (NG108-15) dose dependency inhibited forskolin stimulated cAMP formation with IC50 values of 18, 202nM and 0.8, 0.5nM respectively. Maximal inhibition (Imax) in both cell lines with all agonists were relatively constant, 47-65%. The ORL-1 receptor agonist, nociceptin also inhibited forskolin stimulated cAMP formation in SHSY5Y cells with IC50 and Imax values of 72nM and 47% respectively. These studies in SH-SY5Y cells, report the regulation by opioid and OR1-1 receptors of type 1 AC.;Endogenous opioid receptors in SH-SY5Y and NG108-15 cells were then compared with recombinant receptors expressed in CHO cells. Opioid receptor subtypes (mu-, delta-, and kappa-) bound [3H]DPN in a dose dependent and saturable manner, (Bmax, range=35- 561fmol/mc protein). Fentanyl and [D-Ala 2,Me-Phe4,Gly-ol]enkephalin (DAMGO) displaced [ H]DPN from CHOmu membranes with high affinity. Spiradoline displaced [ 3H]DPN from CHOkappa cells. [D-Pen2,D-pen 5]enkephalin (DPDPE) and [D-Ala2,DLeu5] enkephalin (DADLE) displaced [3H]DPN from CHOdelta cell membranes revealing the presence of a high affinity binding site. All opioid agonists used in displacement studies inhibited forskolin stimulated cAMP formation in there respective CHOmu, delta, or kappa cells.;The cannabinoid receptor is also an example of a transmembrane spanning G-protein coupled receptor. In order to study the cannabinoid receptor in detail, an assay was developed using the first central cannabinoid receptor (CB1) selective antagonist, SR141716A. In rat cerebella membranes, [ 3H]SR141716A bound with a Kd of around 0.7nM. A range of cannabinoid agonists displaced [3H]SR141716A from cerebella membranes. In addition, basal cAMP levels were inhibited by WIN55212-2 in rat cerebella membranes in an SR141716A reversible manner.;CB1 cDNA was expressed in CHO cells and the resulting CHOCB1 cells bound [3H]S R M H lbA , which was displaced dose dependently by unlabeled SR141716A yielding a Ki of 2.2nM. These receptors were functionally coupled to adenylyl cyclase. Collectively, these findings add significantly to our current understanding of opioid and cannabinoid receptor regulation of AC.