Inhibition Of Cyclic AMP Production Research Articles

Unique Pharmacological Properties of the Kappa Opioid Receptor Signaling Through Gαz as Shown with Bioluminescence Resonance Energy Tranfer.

Opioid receptors (ORs) convert extracellular messages to signaling events by coupling to the heterotrimeric G proteins, Gα•βγ Classic pharmacological methods, such as [35S]GTPγS binding and inhibition of cyclic AMP production, allow for general opioid characterization, but they are subject to the varying endogenous Gα proteins in a given cell type. Bioluminescence resonance energy transfer (BRET) technology offers new insight by allowing the direct observation of Gα subunit-specific effects on opioid pharmacology. Using a Venus-tagged Gβγ and nanoluciferase-tagged truncated G protein receptor kinase 3, an increase in BRET signal correlated with OR activation mediated by a specific Gα protein. The magnitude of the BRET signal was normalized to the maximum response obtained with 10 µM 2-(3,4-dichlorophenyl)-N-methyl-N-[(1R,2R)-2-pyrrolidin-1-ylcyclohexyl]acetamide (U50,488) for the kappa OR (KOR). Opioids reached equilibrium with the KOR, and concentration-response curves were generated. Although the full agonists U50,488, salvinorin A, nalfurafine, and dynorphin peptides were equally efficacious regardless of the Gα subunit present, the concentration-response curves were leftward shifted when the KOR was signaling through Gαz compared with other Gαi/o subunits. In contrast, the Gα subunit distinctly affected both the efficacy and potency of partial kappa agonists, such as the benzomorphans, and the classic mu opioid antagonists, naloxone, naltrexone, and nalmefene. For example, (-)pentazocine had EC50 values of 7.3 and 110 nM and maximal stimulation values of 79% and 35% when the KOR signaled through Gαz and Gαi1, respectively. Together, these observations suggest KOR pharmacology varies based on the specific Gα subunit coupled to the KOR. SIGNIFICANCE STATEMENT: Opioid receptors couple to various heterotrimeric Gαβγ proteins to convert extracellular cues to precise intracellular events. This paper focuses on how the various inhibitory Gα subunits influence the pharmacology of full and partial agonists at the kappa opioid receptor. Using a bioluminescent assay, the efficacy and potency of kappa opioids was determined. Opioid signaling was more potent through Gαz compared with other Gα proteins. These observations suggest that Gαz may impact opioid pharmacology and cellular physiology more than previously thought.

Lack of effect of the α2C‐adrenoceptor Del322‐325 polymorphism on inhibition of cyclic AMP production in HEK293 cells

The alpha(2C)-adrenoceptor has multiple functions, including inhibiting release of noradrenaline from presynaptic nerve terminals. A human alpha(2C) polymorphism, Del322-325, a potential risk factor for heart failure, has been reported to exhibit reduced signalling in CHO cells. To further understand the role of the Del322-325 polymorphism on receptor signalling, we attempted to replicate and further study the reduced signalling in HEK293 cells. Human alpha(2C) wild-type (WT) and Del322-325 adrenoceptors were stably transfected into HEK293 cells. Radioligand binding was performed to determine affinities for both receptors. In intact cells, inhibition of forskolin-stimulated cyclic AMP production by WT and Del322-325 clones with a range of receptor densities (200-2320 fmol.mg(-1) protein) was measured following agonist treatment. Noradrenaline, brimonidine and clonidine exhibited similar binding affinities for WT and Del322-325. Brimonidine and clonidine also had similar efficacies and potencies for both receptors for the inhibition of cyclic AMP production at all receptor densities tested. A linear regression analysis comparing efficacy and potency with receptor expression levels showed no differences in slopes between WT and Del322-325. The alpha(2C) WT and Del322-325 adrenoceptors exhibited similar binding properties. Additionally, inhibition of cyclic AMP production by Del322-325 was similar to that of WT over a range of receptor densities. Therefore, in intact HEK293 cells, the alpha(2C)-Del322-325 polymorphism does not exhibit reduced signalling to adenylyl cyclase and may not represent a clinically important phenotype.

P.1.c.047 Hippocampal nociceptin receptors and memory: possible implications for "anti-alcohol" effects of nociceptin ligands

The opioid system comprises four receptor subtypes: μ (MOP), κ (KOP), δ (DOP), now called the ‘classical’ opioid receptors, and the ‘non-classical’ nociceptin/orphanin FQ peptide (N/OFQ) receptor (NOP). Selective endogenous peptides, cleaved from larger precursor proteins, have been identified for all subtypes. Both classical and non-classical opioid receptors couple to inhibitory, pertussis toxin-sensitive G-proteins. Opioid receptors activate the same major intracellular pathways, which include: closing of voltage-sensitive calcium channels; opening of potassium channels and subsequent cellular hyperpolarization; and inhibition of cyclic AMP production through inhibition of the enzyme adenylate cyclase. All current, clinically used opioids work through activation of the MOP receptor. In an experimental setting, co-administration of MOP and DOP agonists has been shown to have a synergistic analgesic action. Administration of DOP-receptor antagonists has also been shown to reduce tolerance, physical dependence and other side effects of MOP-receptor agonists, without detriment to their analgesic action. In animal models NOP agonists are analgesic when administered spinally and have a pronociceptive/anti-analgesic (or anti-opioid) effect supraspinally. NOP knockout mice show a partial loss of tolerance to morphine and there is an up-regulation of N/OFQ production in chronic morphine tolerant mice. Analgesic tolerance that develops from repeated exposure to morphine is markedly attenuated in NOP knockout mice. The development of ligands with mixed action at MOP, DOP and NOP receptors offer new opportunities for opioid pharmacology.

Distinct roles of the DRY motif in rat melanin-concentrating hormone receptor 1 in signaling control

Rhodopsin family (class A) G protein-coupled receptors possess common key residues or motifs that appear to be important for receptor function. To clarify the roles of the highly conserved amino acid triplet Asp 3.49–Arg 3.50–Tyr 3.51 (DRY motif), we examined how single-substitution mutations of the amino acids in the motif influenced specific features of rat melanin-concentrating hormone receptor 1 (MCH1R) activity. Substitution of either Asp140 3.49 or Tyr142 3.51 to Ala resulted in nonfunctional receptors, despite the retention of apparent potencies for agonist binding. These loss-of-function phenotypes may be caused by the lack of stimulation for GDP-GTP exchange observed in GTPγS-binding assays. On the other hand, substitution of Arg141 3.50 to Ala caused a 4-fold reduction in the agonist binding affinity and, concomitantly, a rightward shift of the dose-dependency curve for calcium mobilization and inhibition of cyclic AMP production. Although many experimental studies have suggested that the DRY motif is involved in maintaining the receptor in its ground state, none of the DRY motif substitutions to Ala in MCH1R led to constitutive activation, in terms of the basal signaling level for ERK1/2 activation or GTPγS binding. These data suggest that the major contribution of the DRY motif in MCH1R is to govern receptor conformation and G protein coupling/recognition.

Inhibition of cyclic AMP production is not reduced in HEK293 cells expressing the alpha‐2C adrenergic receptor Del322–325 polymorphism

The human alpha‐2C adrenergic receptor inhibits norepinephrine release in the sympathetic nervous system. A four residue deletion in the third intracellular loop of this receptor, Del322–325, has been reported to exhibit reduced signaling in vitro (Small, et al., 2000) and be a potential risk factor for heart failure, presumably due to excess norepinephrine release and action. We attempted to replicate the reduced signaling, with the hypothesis that decreased signaling is due to decreased coupling of Del322–325 to Gi proteins. Our objective was to compare cyclic AMP inhibition of alpha‐2C wild type and Del322–325 receptors stably expressed in HEK293 cells. Cyclic AMP production was stimulated by 30 μM forskolin, and alpha‐2 agonists were used to inhibit this production in clones with high or low receptor expression levels. WT and Del322–325 clones with high expression levels (Bmax = 1500 and 2300 fmol/mg) did not differ in their percent inhibition (94 and 98%) or in their potencies (0.5 and 0.4 nM) for UK‐14,304. Clonidine also showed no difference between WT and Del322–325 clones with high Bmax (93 and 97%, 2.2 and 1.7 nM EC50s). Similar results were obtained in clones with low Bmax (20 and 20 fmol/mg). Therefore, in HEK293 cells, Del322–325 does not have a reduced ability to inhibit cyclic AMP production in comparison to WT.Supported by a donation from Procter and Gamble.

Viral envelope protein gp64 transgenic mouse facilitates the generation of monoclonal antibodies against exogenous membrane proteins displayed on baculovirus

We have been investigating the functional display of multipass membrane protein such as transporter or G-protein coupled receptor on the budded baculovirus (BV). We tested the use of a viral envelope protein gp64 transgenic mouse for the direct immunization of these membrane proteins displayed on BVs. The gp64 transgenic mice showed only a weak response to virus compared to wild type BALB/c mice. Immunizing gp64 transgenic mice with the BV expressing peptide transporter PepT1, we obtained 47 monoclonal antibodies (mAbs). These mAbs were specific to the PepT1 on the pancreatic cancer cells AsPC-1 by fluorocytometric analysis and exhibited antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity to AsPC-1. We also generated 7 mAbs by immunizing gp64 transgenic mice on a CCR2-deficient background with the BV expressing chemokine receptor CCR2 together with partially purified CCR2. These mAbs possessed specific binding to CCR2 in CHO cells on fluorocytometric analysis, and exhibited neutralizing activities for ligand-dependent inhibition of cyclic AMP production. This method provides a powerful tool for the generation of therapeutic/diagnostic mAbs against membrane proteins.

C-Reactive Protein Decreases Interleukin-10 Secretion in Activated Human Monocyte-Derived Macrophages via Inhibition of Cyclic AMP Production

C-Reactive protein (CRP), a cardiovascular risk marker, could also participate in atherosclerosis. Atherosclerotic plaques express CRP and interleukin (IL)-10, a major antiinflammatory cytokine. IL-10 deficiency results in increased lesion formation, whereas IL-10 delivery attenuates lesions. We tested the effect of CRP on lipopolysaccharide (LPS)-induced IL-10 secretion in human monocyte-derived macrophages (HMDMs). Incubation of HMDMs with CRP significantly decreased LPS-induced IL-10 mRNA and intracellular and secreted IL-10 protein and destabilized IL-10 mRNA. Also, CRP alone increased secretion of IL-8, IL-6, and tumor necrosis factor from HMDMs and did not inhibit LPS-induced secretion of these cytokines. Fc gamma receptor I antibodies significantly reversed CRP-mediated IL-10 inhibition. CRP significantly decreased intracellular cAMP, phospho-cAMP response element binding protein (pCREB), and adenyl cyclase activity. cAMP agonists reversed CRP-mediated IL-10 inhibition. Overexpression of wild-type and constitutively active CREB in THP-1 cells revealed attenuation of the inhibitory effect of CRP on LPS-induced IL-10 levels. CRP also inhibited hemoglobin:haptoglobin-induced IL-10 and heme oxygenase-1. Furthermore, administration of human CRP to rats significantly decreased IL-10 levels. This study provides novel evidence that CRP, by decreasing IL-10 alters the antiinflammatory/proinflammatory balance, accentuating inflammation, which is pivotal in atherothrombosis.

Cloning and characterization of a Caenorhabditis elegans D2-like dopamine receptor.

The neurotransmitter dopamine plays an important role in the regulation of behavior in both vertebrates and invertebrates. In mammals, dopamine binds and activates two classes of dopamine receptors, D1-like and D2-like receptors. However, D2-like dopamine receptors in Caenorhabditis elegans have not yet been characterized. We have cloned a cDNA encoding a putative C. elegans D2-like dopamine receptor. The deduced amino acid sequence of the cloned cDNA shows higher sequence similarities to vertebrate D2-like dopamine receptors than to D1-like receptors. Two splice variants that differ in the length of their predicted third intracellular loops were identified. The receptor heterologously expressed in cultured cells showed high affinity binding to [125I]iodo-lysergic acid diethylamide. Dopamine showed the highest affinity for this receptor among several amine neurotransmitters tested. Activation of the heterologously expressed receptor led to the inhibition of cyclic AMP production, confirming that this receptor has the functional property of a D2-like receptor. We have also analyzed the expression pattern of this receptor and found that the receptor is expressed in several neurons including all the dopaminergic neurons in C. elegans.

Evidence for cocaine and methylecgonidine stimulation of M(2) muscarinic receptors in cultured human embryonic lung cells.

1. Muscarinic cholinoceptor stimulation leads to an increase in guanylyl cyclase activity and to a decrease in adenylyl cyclase activity. This study examined the effects of cocaine and methylecgonidine (MEG) on muscarinic receptors by measurement of cyclic GMP and cyclic AMP content in cultured human embryonic lung (HEL299) cells which specifically express M(2) muscarinic receptors. 2. A concentration-dependent increase in cyclic GMP production was observed in HEL299 cells incubated with carbachol, cocaine, or MEG for 24 h. The increase in cyclic GMP content was 3.6 fold for 1 microM carbachol (P < 0.01), 3.1 fold for 1 microM cocaine (P < 0.01), and 7.8 fold for 1 microM MEG (P < 0.001), respectively. This increase in cyclic GMP content was significantly attenuated or abolished by the muscarinic receptor antagonist atropine or the M(2) blocker methoctramine. 3. In contrast, cocaine, MEG, and carbachol produced a significant inhibition of cyclic AMP production in HEL299 cells. Compared to the control, HEL299 cells treated with 1 microM cocaine decreased cyclic AMP production by 30%. MEG and carbachol at 1 microM decreased cyclic AMP production by 37 and 38%, respectively. Atropine or methoctramine at 1 or 10 microM significantly attenuated or abolished the cocaine-induced decrease in cyclic AMP production. However, the antagonists alone had neither an effect on cyclic GMP nor cyclic AMP production. Pretreatment of HEL299 cells with pertussis toxin prevented the cocaine-induced reduction of cyclic AMP production. 4. Western blot analysis showed that HEL299 cells specifically express M(2) muscarinic receptors without detectable M(1) and M(3). Incubation of HEL299 cells with cocaine, carbachol, and atropine did not alter the expression of M(2) protein levels. However, the inducible isoform of nitric oxide synthase (iNOS) was induced in the presence of cocaine or carbachol and this induction was significantly attenuated after addition of atropine or methoctramine. 5. The present data show that cocaine and MEG significantly affect cyclic GMP and cyclic AMP production in cultured HEL299 cells. Our results also show that these effects result from the drug-induced stimulation of M(2) muscarinic receptors accompanied with no alterations of receptor expression. However, the induction of iNOS by cocaine may result in the increase in cyclic GMP production.

Involvement of the cyclic AMP system in the switch from tolerance into supersensitivity to the antinociceptive effect of the opioid sufentanil.

1. We have previously demonstrated that chronic and simultaneous treatment of rats with the mu-opioid receptor agonist sufentanil and the Ca(2+) channel blocker nimodipine, not only prevented tolerance development, but the animals became supersensitive to the antinociceptive effect of the opioid. The focus of the present work was to determine the possible involvement of cross interactions between the adenylyl cyclase pathway and L-type voltage-sensitive Ca(2+)-channels, in modulating the switch from opioid tolerance into supersensitivity. 2. The modulatory effect of sufentanil on adenylyl cyclase activity was determined by measuring cyclic AMP production in slices from the cortex of rats rendered tolerant or supersensitive to the antinociceptive effect of the opioid. Tolerance was induced by chronic infusion of sufentanil, at a rate of 2 microg h(-1), for 7 days. Supersensitivity was induced by concurrent infusion of sufentanil (2 microg h(-1)) and nimodipine (1 microg h(-1)) for 7 days. Antinociception was evaluated by the tail-flick test. 3. Tolerance to the analgesic effect of sufentanil was associated with a significant reduction in the response of adenylyl cyclase to forskolin. Furthermore, the effect of the opioid on forskolin-induced cyclic AMP accumulation was abolished. On the other hand, supersensitivity to the analgesic effect of the opioid was associated with an increase in both, the adenylyl cyclase response to forskolin, and the opioid inhibition of cyclic AMP production. 4. We suggest that sustained L-type Ca(2+) channel blockade may result in changes in the adenylyl cyclase effector system triggered by mu-opioid receptor activation, leading to the switch from opioid tolerance into supersensitivity.

Pharmacological characterization of human recombinant melatonin mt(1) and MT(2) receptors.

We have pharmacologically characterized recombinant human mt(1) and MT(2) receptors, stably expressed in Chinese hamster ovary cells (CHO-mt(1) and CHO-MT(2)), by measurement of [(3)H]-melatonin binding and forskolin-stimulated cyclic AMP (cAMP) production. [3H]-melatonin bound to mt(1) and MT(2) receptors with pK(D) values of 9.89 and 9.56 and B(max) values of 1.20 and 0.82 pmol mg(-1) protein, respectively. Whilst most melatonin receptor agonists had similar affinities for mt(1) and MT(2) receptors, a number of putative antagonists had substantially higher affinities for MT(2) receptors, including luzindole (11 fold), GR128107 (23 fold) and 4-P-PDOT (61 fold). In both CHO-mt(1) and CHO-MT(2) cells, melatonin inhibited forskolin-stimulated accumulation of cyclic AMP in a concentration-dependent manner (pIC(50) 9.53 and 9.74, respectively) causing 83 and 64% inhibition of cyclic AMP production at 100 nM, respectively. The potencies of a range of melatonin receptor agonists were determined. At MT(2) receptors, melatonin, 2-iodomelatonin and 6-chloromelatonin were essentially equipotent, whilst at the mt(1) receptor these agonists gave the rank order of potency of 2-iodomelatonin>melatonin>6-chloromelatonin. In both CHO-mt(1) and CHO-MT(2) cells, melatonin-induced inhibition of forskolin-stimulated cyclic AMP production was antagonized in a concentration-dependent manner by the melatonin receptor antagonist luzindole, with pA(2) values of 5.75 and 7.64, respectively. Melatonin-mediated responses were abolished by pre-treatment of cells with pertussis toxin, consistent with activation of G(i)/G(o) G-proteins. This is the first report of the use of [(3)H]-melatonin for the characterization of recombinant mt(1) and MT(2) receptors. Our results demonstrate that these receptor subtypes have distinct pharmacological profiles.

Structural determinants of the partial agonist-inverse agonist properties of 6'-azidohex-2'-yne-delta8-tetrahydrocannabinol at cannabinoid receptors.

1. We have extended previous investigations of four analogues of Delta8-tetrahydrocannabinol (Delta8-THC): 6'-azidohex-2'-yne-Delta8-THC (O-1184), 6'-azidohex-cis-2'-ene-Delta8-THC (O-1238) and octyl-2'-yne-Delta8-THC (O-584) and its 1-deoxy-analogue (O-1315). 2. O-1184, O-1238 and O-584 displaced [3H]-CP55940 from specific binding sites on Chinese hamster ovary (CHO) cell membranes expressing CB1 or CB2 cannabinoid receptors, with pKi values of 8.28 to 8.45 (CB1) and 8.03 to 8.13 (CB2). The pKi values of O-1315 were significantly less, 7.63 (CB1) and 7.01 (CB2). 3. All the analogues inhibited forskolin-stimulated cyclic AMP production by CB1-transfected CHO cells (pEC50=9.16 to 9.72). Only O-1238 behaved as a full agonist in this cell line. 4. In mouse vasa deferentia, O-1238 inhibited electrically-evoked contractions (pEC50=10.18 and Emax=70.5%). Corresponding values for O-1184 were 9.08 and 21.1% respectively. At 1 nM, O-1184 produced surmountable antagonism of the cannabinoid receptor agonist, CP55940. However, at 0.1 nM, O-1184 did not attenuate CP55940-induced inhibition of cyclic AMP production by CB1-transfected CHO cells. 5. In CB2-transfected CHO cells, cyclic AMP production was inhibited by CP55940 (pEC50=8.59), enhanced by O-1184 and O-584 (pEC50=8.20 and 6.86 respectively) and not significantly affected by O-1238 or O-1315. 6. At 100 nM, O-1184 and O-1238 produced surmountable antagonism of CP55940 in CB2 cells, decreasing the pEC50 of CP55940 from 8.61 to 7.42 (O-1184) or from 8. 54 to 7.44 (O-1238). 7. These data support the hypothesis that increasing the degree of unsaturation of the aliphatic side-chain of Delta8-THC analogues has little effect on CB1 or CB2 receptor affinity but can reduce CB1 receptor efficacy and reverse the direction of responses elicited at CB2 receptors.

The effect of site-directed mutagenesis of two transmembrane serine residues on agonist-specific coupling of a cloned human alpha2A-adrenoceptor to adenylyl cyclase.

1. The effects of substitution of the Ser200 and Ser204 residues with alanine on the signalling properties of the cloned human alpha2A-adrenoceptor, stably expressed in Chinese hamster ovary (CHO) cell lines, have been investigated using noradrenaline and the structural isomers of octopamine. 2. The Ser-->Ala200 or the Ser-->Ala204 mutant forms of the alpha2A-adrenoceptor, when expressed in cells in the absence of pertussis toxin pretreatment, are two orders of magnitude more sensitive to inhibition of cyclic AMP production by (+/-)-para-octopamine and (+/-)-meta-octopamine, respectively, than cells expressing the wild-type receptor. Binding studies indicate that the effects are not due to an increased agonist affinity for the mutant receptors and that they are likely to be due to agonist-mediated conformational changes in receptor structure. 3. After incubation with pertussis toxin, (+/-)-meta-octopamine (100 microM and above) produced a stimulation of cyclic AMP levels in cells expressing the Ser-->Ala204 mutant form of the alpha2A-adrenoceptor but showed no stimulation in cells expressing the Ser-->Ala200 mutant receptor. Under these conditions (+/-)-para-octopamine did not produce any increases in cyclic AMP production in cells expressing either of the mutant receptor forms or the wild-type receptor. 4. The results emphasise the importance of the Ser200 and Ser204 residues of the alpha2A-adrenoceptor in exerting an inhibitory influence on the ability of (+/-)-para-octopamine and (+/-)-meta-octopamine respectively, to induce a receptor-agonist conformation capable of inhibiting forskolin-stimulation of cyclic AMP levels. 5. It is clear that Ser204 also prevents meta-octopamine from generating a receptor-agonist conformation that can increase cyclic AMP levels, emphasising the importance of this residue in the agonist-specific coupling of this receptor to different second messenger systems.

Ligand binding and modulation of cyclic AMP levels depend on the chemical nature of residue 192 of the human cannabinoid receptor 1.

The human cannabinoid receptor associated with the CNS (CB1) binds delta9-tetrahydrocannabinol, the psychoactive component of marijuana, and other cannabimimetic compounds. This receptor is a member of the seven transmembrane domain G protein-coupled receptor family and mediates its effects through inhibition of adenylyl cyclase. An understanding of the molecular mechanisms involved in ligand binding and receptor activation requires identification of the active site residues and their role. Lys192 of the third transmembrane domain of the receptor is noteworthy because it is the only nonconserved, charged residue in the transmembrane region. To investigate the properties of this residue, which are important for both ligand binding and receptor activation, we generated mutant receptors in which this amino acid was changed to either Arg (K192R), Gln (K192Q), or Glu (K192E). Wild-type and mutant receptors were stably expressed in Chinese hamster ovary cells and were evaluated in binding assays with the bicyclic cannabinoid CP-55,940 and the aminoalkylindole WIN 55,212-2. We found that only the most conservative change of Lys to Arg allowed retention of binding affinity to CP-55,940, whereas WIN 55,212-2 bound to all of the mutant receptors in the same range as it bound the wild type. Analysis of the ligand-induced inhibition of cyclic AMP production in cells expressing each of the receptors gave an EC50 value for each agonist that was comparable to its binding affinity, with one exception. Although the mutant K192E receptor displayed similar binding affinity as the wild type with WIN 55,212-2, an order of magnitude difference was observed for the EC50 for cyclic AMP inhibition with this compound. The results of this study indicate that binding of CP-55,940 is highly sensitive to the chemical nature of residue 192. In contrast, although this residue is not critical for WIN 55,212-2 binding, the data suggest a role for Lys192 in WIN 55,212-2-induced receptor activation.

Modulation of GH4 cell cycle via A1 adenosine receptors.

Identification and characterization of A1 adenosine receptors (A1Rs) in a tumor cell line derived from rat pituitary (GH4 cells) was performed by ligand binding and immunocytochemistry. Subsequently, the involvement of A1Rs in the regulation of cell proliferation was studied in these cells. The agonist N6-(R)-phenylisopropyladenosine (R-PIA) did not modify the number of cultured cells, but it regulated the kinetics of the cell cycle. By means of experiments of pulse and of pulse and chase with bromodeoxyuridine and further labeling with Hoechst 33258, propidium iodide, and/or fluorescein-conjugated antibodies against bromodeoxyuridine, it was demonstrated that R-PIA, via A1Rs, accelerated progression from G0/G1 to S phase and from S to G2/M phase of the cell cycle, whereas the initiation of a new cycle occurred at the same time in treated and untreated cells. As a consequence, R-PIA did not change the total length of the cycle. This is the first description of cell cycle regulation without modification of cell proliferation. Although pertussis toxin blocked the R-PIA-induced inhibition of cyclic AMP production in these cells, it did not affect the R-PIA action on the cell cycle. In contrast, cholera toxin mimicked the action of R-PIA. Thus, it is likely that regulation of the cell cycle via A1Rs is mediated by heterotrimeric G proteins different from those that mediate inhibition of adenylate cyclase. Due to the fact that cells in G0/G1 phase were less susceptible to secretory signals, adenosine, in an autocrine manner and by regulating the cell cycle kinetics, may contribute to the modulation of the secretory capacity of pituitary cells.

Adenosine A3 receptor agonist-induced neurotoxicity in rat cerebellar granule neurons

Acute stimulation of A3 adenosine receptors has been shown to intensify damage in an in vivo model of global cerebral ischemia and to induce cell death in cell cultures. To investigate the mechanism for these cytotoxic phenomena, adenosine analogs of varied receptor subtype selectivities were applied directly to rat cerebellar granule neurons in culture. Agonists of A1, A2A, and/or A2B receptors (CPA, NECA and CGS 21680) had no effect on neuronal survival during a 16 h incubation period. The highly selective adenosine A3 receptor agonist 2-chloro-N6–(3-iodobenzyl)adenosine-5′-N-methyluronamide (Cl-IB-MECA) at ≥10 μM induced cell death in a concentration-dependent fashion. A structurally related nucleoside, cladribine, which has cytotoxic properties via a non-adenosine receptor related mechanism, had no effect on cell survival. Adding dbcAMP to the culture to activate cyclic AMP-dependent protein kinases attenuated Cl-IB-MECA-induced neurotoxicity, suggesting the Cl-IB-MECA-induced neuronal cell death is mediated by an inhibition of cyclic AMP production. Glutamate (50 μM) induced cell death to a degree comparable to that induced by 10 μM Cl-IB-MECA. Furthermore, a subcytotoxic concentration of Cl-IB-MECA (1 μM) significantly augmented glutamate neurotoxicity. In contrast, the adenosine A1/A2 agonists, the adenosine A1/A2 antagonist XAC, and the A2A antagonist SCH 58261 at micromolar concentrations had no effect on neuronal viability or on glutamate neurotoxicity. These results suggest that activation of adenosine A3 receptors may play an important role in regulating neuronal survival and death in cerebellar neurons. Drug Dev. Res. 40:267–273, 1997. © 1997 Wiley-Liss, Inc.

Deregulation of hearing ability in mice lacking nociceptin/orphanin FQ receptor

The opioid system comprises four receptor subtypes: μ (MOP), κ (KOP), δ (DOP), now called the ‘classical’ opioid receptors, and the ‘non-classical’ nociceptin/orphanin FQ peptide (N/OFQ) receptor (NOP). Selective endogenous peptides, cleaved from larger precursor proteins, have been identified for all subtypes. Both classical and non-classical opioid receptors couple to inhibitory, pertussis toxin-sensitive G-proteins. Opioid receptors activate the same major intracellular pathways, which include: closing of voltage-sensitive calcium channels; opening of potassium channels and subsequent cellular hyperpolarization; and inhibition of cyclic AMP production through inhibition of the enzyme adenylate cyclase. All current, clinically used opioids work through activation of the MOP receptor. In an experimental setting, co-administration of MOP and DOP agonists has been shown to have a synergistic analgesic action. Administration of DOP-receptor antagonists has also been shown to reduce tolerance, physical dependence and other side effects of MOP-receptor agonists, without detriment to their analgesic action. In animal models NOP agonists are analgesic when administered spinally and have a pronociceptive/anti-analgesic (or anti-opioid) effect supraspinally. NOP knockout mice show a partial loss of tolerance to morphine and there is an up-regulation of N/OFQ production in chronic morphine tolerant mice. Analgesic tolerance that develops from repeated exposure to morphine is markedly attenuated in NOP knockout mice. The development of ligands with mixed action at MOP, DOP and NOP receptors offer new opportunities for opioid pharmacology.

Further evidence for the presence of cannabinoid CB 1 receptors in mouse vas deferens

Our results provide further evidence for the hypothesis that the mouse vas deferens contains cannabinoid CB 1 receptors. Thus we found that in the presence of forskolin, the cannabinoid receptor agonist, CP 55,940 ((−)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-l-ol) produced a concentration related inhibition of cyclic AMP production by the vas deferens (EC 50 = 6.0 nM). At 100 nM, SR141716A ( N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H-pyrazole-3-carboxamide hydrochloride) attenuated this effect of CP 55,940, producing a parallel rightward shift in its log concentration-response curve ( K d = 4.3 nM). We also found that cyclic AMP production was inhibited by (−)-11-hydroxy-1′,1′-dimethylheptyl- Δ 8-tetrahydrocannabinol but not by the (+) enantiomer.

Investigation of the inhibitory effects of PGE2 and selective EP agonists on chemotaxis of human neutrophils.

1. The aims of this study were to investigate the inhibitory effects of prostaglandin E2 (PGE2) on chemotaxis of N-formyl-methionyl-leucine-phenylalanine (FMLP)-stimulated human neutrophils, and to test the hypothesis that cyclic AMP is the second messenger involved. For this purpose, the inhibitory effect of selective EP agonists, and the modulatory effects of the adenylate cyclase inhibitor, SQ 22536, the protein kinase A (PKA) inhibitors H-89 and Rp-cAMPs, and the type IV phosphodiesterase (PDE) inhibitors, rolipram and Ro20-1724 have been examined. 2. Chemotaxis has been measured using blindwell chambers. When human neutrophils were stimulated with FMLP (100 nM), PGE2 inhibited chemotaxis in a concentration-dependent manner (0.01-10 microM), with an EC50 of 90 +/- 24.5 nM, a maximum effect ranging from 45-75% and a mean inhibition of 64.5 +/- 2.4%. 3. The EP2-receptor agonists, 11-deoxy PGE1, butaprost and AH 13205 also inhibited chemotaxis. The order of potency of these agonists was PGE2 > butaprost (EC50 = 106.4 +/- 63 nM) > 11-deoxy PGE1 (EC50 = 140.9 +/- 64.7 nM) > AH 13205 (EC50 = 1.58 +/- 0.73 microM). Correlation of the ability of EP2 agonists to increase cyclic AMP and to inhibit chemotaxis was poor (r = 0.38). 4. The IP agonist, cicaprost gave similar increases in cyclic AMP to those achieved with PGE2, yet produced 50% of the maximum inhibition of chemotaxis observed with PGE2. 5. Slight potentiation of the inhibitory effects of PGE2 after type IV PDE block was observed with rolipram (EC50 for PGE2 = 57.2 +/- 5.9; 35.2 +/- 6.8 nM) but not Ro20-1724 (EC50 for PGE2 = 216.0 +/- 59.7; 97.8 +/- 50.6 nM). Type IV PDE inhibitors are themselves potent inhibitors of chemotaxis with EC50 values of 23.0 +/- 2.3 and 73.6 +/- 10.3 nM for rolipram and Ro20-1724, respectively. 6. Inhibition of cyclic AMP production with the adenylate cyclase inhibitor SQ 22,536 (0.1 mM) failed to antagonize inhibition of chemotaxis by PGE2 (EC50s for PGE2 of 57.2 +/- 5.9 and 56.8 +/- 27.3 nM, in the absence and presence of SQ 22,536, respectively) despite a reduction in the increase in cyclic AMP induced by PGE2. 7. Inhibition of PKA with either H-89 (10 microM) or Rp cyclic AMPS (10 microM) similarly failed to antagonize inhibition of chemotaxis by PGE2; EC50 for PGE2 of 90 +/- 40 and PGE2 + H-89 60 +/- 17 nM; PGE2 216.0 +/- 58.7 and PGE2 + Rp cyclic AMP 76.9 +/- 14.7 nM. 8. Of the two PKA inhibitors tested, H-89 (10 microM) and Rp cyclic AMPS (10 microM), the more effective inhibitor of PGE2-induced inhibition of neutrophil superoxide anion generation was H-89 (EC50s for PGE2 were 0.36 +/- 0.1 and > 10 microM, respectively). We have previously shown this to be a cyclic AMP-dependent effect of PGE2. 9. Confirmation of block of PKA by H-89 was suggested by the finding that H-89 blocked inhibition of superoxide anion generation observed with the type IV PDE inhibitors rolipram and Ro20-1724; EC50s of 12.9 +/- 8.9 nM for rolipram alone and rolipram+H-89 > 1 microM; Ro20-1724 alone 59.5 +/- 28.1 nM and Ro20-1724 + H-89 > 1 microM. 10. The results suggest that inhibition of chemotaxis by PGE2 and EP2 agonists is not mediated by increased neutrophil cyclic AMP levels.

Pharmacological study of dihydroetorphine in cloned μ-, δ- and κ-opioid receptors

We investigated the binding characteristics of dihydroetorphine, 7,8-dihydro-7α-[1-(R)-hydroxyl-1-methylbutyl]-6,14-endoethano-tetrahydro-oripavine, and its effect on the inhibitory system of cyclic AMP production using cloned μ-, δ- and κ-opioid receptors expressed on Chinese hamster ovary cells. The K i values of dihydroetrophine for the μ-, δ- and κ-opioid receptor were 4.5 × 10 −10, 1.8 × 10 −9 and 5.7 × 10 −10 M, respectively. On the hand, those of morphine were 1.9 × 10 −9, 1.4 × 10 −6 and 1.3 × 10 −7 M, respectively. Through all of these three types of opioid receptors, dihydroetorphine inhibited forskolin (10 μM)-stimulated cyclic AMP production via pertussis toxin-sensitive G protein(s), and the inhibitory effects were antagonized by co-application with opioid receptor antagonists. The IC 50 values of dihydroetorphine for the inhibition of cyclic AMP production through the μ-, δ- and κ-opioid receptors were 4.2 × 10 −11, 8.6 × 10 −10 and 4.3 × 10 −9 M, respectively. On the other hand, those of morphine were 2.6 × 10 −8, 2.6 × 10 −6 and 1.9 × 10 −6 M, respectively. These results indicate that dihydroetorphine, unlike morphine which preferentially binds the μ-opioid receptor, binds not only μ- but also δ- and κ-opioid receptors with high affinity and acts as a more potent agonist than morphine for all of the three types of receptors.