Activation of the elastin-laminin receptor （S-Gal） induces preconditioning in isolated rat heart submitted to ischemia and reperfusion

AIM: Elastin-laminin receptor (S-Gal), was described to belong to G-protein-coupled receptors (GPCRs). Using an isolated nonworking rat heart model, we investigated whether S-Gal stimulation was able to mimic ischemic preconditioning as observed with some other GPCRs. METHODS: Hearts, after 6-hydroxydopamine pretreatment and a 20-min stabilization period,     

Mechanisms of rapid opioid receptor desensitization, resensitization and tolerance in brain neurons

Agonists acting on μ-opioid receptors (MOR) are very effective analgesics but cause tolerance during long-term or repeated exposure. Intensive efforts have been made to find novel opioid agonists that are efficacious analgesics but can elude the signalling events that cause tolerance. μ-Opioid agonists differentially couple to downstream signalling mechanisms. Some agonists, such as enkephalins, D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO), methadone and sufentanyl are efficacious at mediating G-protein and effector coupling, as well as triggering MOR regulatory events that include MOR phosphorylation, β-arrestin binding, receptor endocytosis and recycling. By contrast, morphine and closely related alkaloids can mediate efficacious MOR-effector coupling but poorly trigger receptor regulation. Several models have been proposed to relate differential MOR regulation by different opioids with their propensity to cause tolerance. Most are based on dogma that β-arrestin-2 (βarr-2) binding causes MOR desensitization and/or that MOR endocytosis and recycling are required for receptor resensitization. This review will examine some of these notions in light of recent evidence establishing that MOR dephosphorylation and resensitization do not require endocytosis. Recent evidence from opioid-treated animals also suggests that impaired MOR-effector coupling is driven, at least in part, by enhanced desensitization, as well as impaired resensitization that appears to be βarr-2 dependent. Better understanding of how chronic exposure to opioids alters receptor regulatory mechanisms may facilitate the development of effective analgesics that produce limited tolerance.     

Response of rat globus pallidus neurons to microiontophoretically applied μ and κ opioid receptor agonists

The effects of the microiontophoretic application of dynorphin A-(1–13) (DYN 13) and the benzomorphans ethylketocyclazocine (EKC), bremazocine and MRZ 2549, (κ) opioid agonists, and of morphine and morphiceptin, (μ) opioid agonists, were compared on spontaneous or glutamate-evoked discharge of globus pallidus (GP) neurons in rat. Our results demonstrate that μ and κ opioid agonists are able to depress the excitability of pallidal neurons, possibly by interacting with μ and κ opioid receptor subtypes, respectively. In addition, the μ agonists and dynorphin A-(1–13), but not the benzomorphans, enhanced the excitability of a number of pallidal neurons. We have proposed a presynaptic site as the basis for this opioid-induced excitation, possibly also mediated by a μ opioid receptor. The selectivity of dynorphin A-(1–13) for benzomorphan κ opioid receptors in the rat GP appears to be low and dynorphin A-(1–13) may elicit effects that are different from those produced by the benzomorphan κ agonists by virtue of its ability to interact with other opioid receptor subtypes, for example μ opioid receptors.     

Analgesic conotoxins: block and G protein-coupled receptor modulation of N-type (Ca(V) 2.2) calcium channels

Conotoxins (conopeptides) are small disulfide bonded peptides from the venom of marine cone snails. These peptides target a wide variety of membrane receptors, ion channels and transporters, and have enormous potential for a range of pharmaceutical applications. Structurally related ω-conotoxins bind directly to and selectively inhibit neuronal (N)-type voltage-gated calcium channels (VGCCs) of nociceptive primary afferent neurones. Among these, ω-conotoxin MVIIA (Prialt) is approved by the Food and Drug Administration (FDA) as an alternative intrathecal analgesic for the management of chronic intractable pain, particularly in patients refractory to opioids. A series of newly discovered ω-conotoxins from Conus catus, including CVID-F, are potent and selective antagonists of N-type VGCCs. In spinal cord slices, these peptides reversibly inhibit excitatory synaptic transmission between primary afferents and dorsal horn superficial lamina neurones, and in the rat partial sciatic nerve ligation model of neuropathic pain, significantly reduce allodynic behaviour. Another family of conotoxins, the α-conotoxins, are competitive antagonists of mammalian nicotinic acetylcholine receptors (nAChRs). α-Conotoxins Vc1.1 and RgIA possess two disulfide bonds and are currently in development as a treatment for neuropathic pain. It was initially proposed that the primary target of these peptides is the α9α10 neuronal nAChR. Surprisingly, however, α-conotoxins Vc1.1, RgIA and PeIA more potently inhibit N-type VGCC currents via a GABA(B) GPCR mechanism in rat sensory neurones. This inhibition is largely voltage-independent and involves complex intracellular signalling. Understanding the molecular mechanisms of conotoxin action will lead to new ways to regulate VGCC block and modulation in normal and diseased states of the nervous system.     

Inhibitory effect of salvinorin A, from Salvia divinorum, on ileitis-induced hypermotility: cross-talk between kappa-opioid and cannabinoid CB(1) receptors

Background and purpose:

Salvinorin A, the active component of the hallucinogenic herb Salvia divinorum, inhibits intestinal motility through activation of κ-opioid receptors (KORs). However, this compound may have target(s) other than the KORs in the inflamed gut. Because intestinal inflammation upregulates cannabinoid receptors and endogenous cannabinoids, in the present study we investigated the possible involvement of the endogenous cannabinoid system in salvinorin A-induced delay in motility in the inflamed gut.

Experimental approach:

Motility in vivo was measured by evaluating the distribution of a fluorescent marker along the small intestine; intestinal inflammation was induced by the irritant croton oil; direct or indirect activity at cannabinoid receptors was evaluated by means of binding, enzymic and cellular uptake assays.

Key results:

Salvinorin A as well as the KOR agonist U-50488 reduced motility in croton oil treated mice. The inhibitory effect of both salvinorin A and U-50488 was counteracted by the KOR antagonist nor-binaltorphimine and by the cannabinoid CB₁ receptor antagonist rimonabant. Rimonabant, however, did not counteract the inhibitory effect of salvinorin A on motility in control mice. Binding experiments showed very weak affinity of salvinorin A for cannabinoid CB₁ and CB₂ and no inhibitory effect on 2-arachidonoylglycerol and anandamide hydrolysis and cellular uptake.

Conclusions and implications:

The inhibitory effect of salvinorin A on motility reveals a functional interaction between cannabinoid CB₁ receptors and KORs in the inflamed—but not in the normal—gut in vivo.     

Use of the A(2A) adenosine receptor as a physiological immunosuppressor and to engineer inflammation in vivo

Inflammation must be inhibited in order to treat, e.g., sepsis or autoimmune diseases or must be selectively enhanced to improve, for example, immunotherapies of tumors or the development of vaccines. Predictable enhancement of inflammation depends upon the knowledge of the "natural" pathways by which it is down-regulated in vivo. Extracellular adenosine and A(2A) adenosine (purinergic) receptors were identified recently as anti-inflammatory signals and as sensors of excessive inflammatory tissue damage, respectively (Ohta A and Sitkovsky M, Nature 2001;414:916-20). These molecules may function as an important part of a physiological "metabolic switch" mechanism, whereby the inflammatory stimuli-produced local tissue damage and hypoxia cause adenosine accumulation and signaling through cyclic AMP-elevating A(2A) adenosine receptors in a delayed negative feedback manner. Patterns of A(2A) receptor expression are activation- and differentiation-dependent, thereby allowing for the "acquisition" of an immunosuppressive "OFF button" and creation of a time-window for immunomodulation. Identification of A(2A) adenosine receptors as "natural" brakes of inflammation provided a useful framework for understanding how tissues regulate inflammation and how to enhance or decrease (engineer) inflammation by targeting this endogenous anti-inflammatory pathway. These findings point to the need of more detailed testing of anti-inflammatory agonists of A(2A) receptors and create a previously unrecognized strategy to enhance inflammation and targeted tissue damage by using antagonists of A(2A) receptors. It is important to further identify the contributions of different types of immune cells at different stages of the inflammatory processes in different tissues to enable the "tailored" treatments with drugs that modulate the signaling through A(2A) purinergic receptors.     

Modification of the structure of 4, 6-disubstituted 2-(4-alkyl-1-piperazinyl)pyridines: synthesis and their 5-HT2A receptor activity

Structure-activity relationship studies of a series of novel 4, 6-disubstituted 2-(1-piperazinyl)pyridines were conducted to revise our model of serotonin 5-HT(2A) receptor antagonist. Target compounds were synthesized using the benzotriazole-assisted Katritzky method. The majority of those compounds were found to be selective 5-HT(2A)/5-HT(1A) receptor ligands, though less potent than their previously described pyrimidine counterparts. In particular, the three compounds 6-8 showed the highest 5-HT(2A) receptor affinity (K(i) = 34-78 nM) and were classified as 5-HT(2A) antagonists in in vivo experiments. The influence of the structural modifications on the in vitro results was discussed; however, the elucidation of the role of the central core system requires further studies.     

Effects of clozapine on rat striatal muscarinic receptors coupled to inhibition of adenylyl cyclase activity and on the human cloned m4 receptor

1. Clozapine has recently been claimed to behave as a selective and full agonist at the cloned m4 muscarinic receptor artificially expressed in Chinese hamster ovary (CHO) cells. In the present study we have investigated whether clozapine could activate the rat striatal muscarinic receptors coupled to the inhibition of adenylyl cyclase activity, considered as pharmacologically equivalent to the m4 gene product. In addition, we have examined the effect of the drug on various functional responses following the activation of the cloned m4 receptor expressed in CHO cells.
2. In rat striatum, clozapine (1 nM–10 μM) caused a slight inhibition of forskolin-stimulated adenylyl cyclase activity, which was not counteracted by 10 μM atropine. On the other hand, clozapine antagonized the inhibitory effect of acetylcholine with a pA₂ value of 7.51. Moreover, clozapine (1 μM) failed to inhibit dopamine D₁ receptor stimulation of adenylyl cyclase activity, but counteracted the inhibitory effect of carbachol (CCh). Clozapine displaced [³H]-N-methylscopolamine ([³H]-NMS) bound to striatal M₄ receptors with a monophasic inhibitory curve and a pK_i value of 7.69. The clozapine inhibition was not affected by the addition of guanosine-5′-O-(thio)triphosphate (GTPγS).
3. In intact CHO cells, clozapine inhibited forskolin-stimulated cyclic AMP accumulation with an EC₅₀ of 31 nM. This effect was antagonized by atropine. CCh produced a biphasic effect on cyclic AMP levels, inhibiting at concentrations up to 1 μM (EC₅₀=50 nM) and stimulating at higher concentrations (EC₅₀=7 μM). Clozapine (0.3–5 μM) antagonized the CCh stimulation of cyclic AMP with a pK_i value of 7.47. Similar results were obtained when the adenylyl cyclase activity was assayed in CHO cell membranes.
4. In CHO cells pretreated with the receptor alkylating agent 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (10 μM), the maximal inhibitory effect of clozapine on cyclic AMP formation was markedly reduced, whereas the CCh inhibitory curve was shifted to the right with no change in the maximum.
5. As in rat striatum, in CHO cell membranes the displacement of [³H]-NMS binding by clozapine yielded a monophasic curve which was not affected by GTPγS.
6. Clozapine (10 nM–10 μM) had a small stimulant effect (∼20%) on the binding of [³⁵S]-GTPγS to CHO cell membranes, whereas CCh caused a 250% increase of radioligand binding. Moreover, clozapine (50 nM–5 μM) antagonized the CCh-stimulated [³⁵S]-GTPγS binding with a pA₂ value of 7.48.
7. These results show that at the striatal M₄ receptors clozapine is a potent and competitive antagonist, whereas at the cloned m4 receptor it elicits both agonist and antagonist effects. Thus, clozapine behaves as a partial agonist, rather than as a full agonist, at the m4 receptor subtype, with intrinsic activity changing as a function of the coupling efficiency of the receptor to effector molecules.