Actions of ZD0947, a novel ATP-sensitive K+ channel opener, on membrane currents in human detrusor myocytes.

BACKGROUND AND PURPOSE: ATP-sensitive K+ channels (K(ATP) channels) play important roles in regulating the resting membrane potential of detrusor smooth muscle. Actions of ZD0947, a novel KATP channel opener, on both carbachol (CCh)-induced detrusor contractions and membrane currents in human urinary bladder myocytes were investigated. EXPERIMENTAL APPROACH: Tension measurements and patch-clamp techniques were utilized to study the effects of ZD0947 in segments of human urinary bladder. Immunohistochemistry was also performed to detect the expression of the sulphonylurea receptor 1 (SUR1) and the SUR2B antigens in human detrusor muscle. KEY RESULTS: ZD0947 (> or = 0.1 microM) caused a concentration-dependent relaxation of the CCh-induced contraction of human detrusor, which was reversed by glibenclamide. The rank order of the potency to relax the CCh-induced contraction was pinacidil > ZD0947 > diazoxide. In conventional whole-cell configuration, ZD0947 (> or = 1 microM) caused a concentration-dependent inward K+ current which was suppressed by glibenclamide at -60 mV. When 1 mM ATP was included in the pipette solution, application of pinacidil or ZD0947 caused no inward K+ current at -60 mV. Gliclazide (< or =1 microM), a selective SUR1 blocker, inhibited the ZD0947-induced currents (Ki = 4.0 microM) and the diazoxide-induced currents (high-affinity site, Ki1 = 42.4 nM; low-affinity site, Ki2 = 84.5 microM) at -60 mV. Immunohistochemical studies indicated the presence of SUR1 and SUR2B proteins, which are constituents of KATP channels, in the bundles of human detrusor smooth muscle. CONCLUSIONS AND IMPLICATIONS: These results suggest that ZD0947 caused a glibenclamide-sensitive detrusor relaxation through activation of glibenclamide-sensitive KATP channels in human urinary bladder.

Imidazoline receptors, novel agents and therapeutic potential.

The initial realization that agents containing an imidazoline structure may interact with a distinct class of receptors, has led to a major class of cardiovascular agents, which now has the potential to enter a third generation. There is now general acceptance that there are three main imidazoline receptor classes, the I(1) imidazoline receptor which mediates the sympatho-inhibitory actions to lower blood pressure, the I(2) receptor which is an important allosteric binding site of monoamine oxidase and the I(3) receptor which regulates insulin secretion from pancreatic beta cells. Thus all three represent important targets for cardiovascular research. Interestingly, an I(1)- receptor candidate has been cloned (IRAS, imidazoline receptor antisera selected) which is a homologue of the mouse cell adhesion integrin binding protein Nischarin. There has been range of new agonists and antagonists with very high selectivity for I(1), I(2) and I(3) receptors developed. Three different endogenous ligands have been characterized including agmatine (decarboxylated arginine), a range of beta-carbolines including harman and harmane, and more recently imidazoleacetic acid-ribotide. The imidazoline field has recently seen an enormous diversification with discoveries that I(1) and I(2) receptors also play a role in cell proliferation, regulation of body fat, neuroprotection, inflammation and some psychiatric disorders such as depression. This diversification has continued with the addition of effective agents with imidazoline affinity in the fields of cancer, pain and opioid addiction, stress, cell adhesion, epilepsy and appetite. The imidazoline field has maturated considerably with a range of highly selective leader molecules, candidate receptors and endogenous ligands. We are therefore only at the threshold of an exciting new era as we begin to understand the diverse and complex nature of their function.

Identification of the central imidazoline receptor subtype involved in modulation of halothane-epinephrine arrhythmias in rats.

We previously reported that imidazoline receptors in the central nervous system are involved in modulation of halothane-epinephrine arrhythmias. These receptors have been subclassified as I1 and I2 subtypes, but it is not known which receptor subtype is involved in halothane-epinephrine-induced arrhythmias. We designed the present study to clarify the involvement of central imidazoline receptor subtype in the modulation of halothane-epinephrine-induced arrhythmias. Rats were anesthetized with halothane and monitored continuously for systemic arterial blood pressure and premature ventricular contractions. The arrhythmogenic dose of epinephrine was defined as the smallest dose that produces three or more premature ventricular contractions within a 15-s period. Intracisternal moxonidine dose-dependently inhibited the epinephrine-induced arrhythmias during halothane anesthesia. Intracisternal efaroxan, a selective I1 antagonist with little affinity for I2 subtype, but not rauwolscine, an alpha2 antagonist without affinity for imidazoline receptors, blocked the antiarrhythmic effect of moxonidine. Intracisternal BU 224 and 2-BFI, selective I2 ligands, also inhibited the epinephrine-induced arrhythmias dose-dependently; however, these effects were abolished by efaroxan. We conclude that central I1, but not I2, receptors play an important role in inhibition of halothane-epinephrine arrhythmia.

Current status of drug receptor nomenclature: receptor closure? The role of NC-IUPHAR.

Pharmacology is at a crucial point, because we now have access to sequences, by homology, for almost all of the receptors in the human genome. The International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR) has set up > 50 subcommittees to define the receptors, and their recommendations, when approved, are posted on a website freely available to all scientists. A major new effort is to functionally define relevant receptor polymorphisms. This initiative is open to all, and works only because of the freely given voluntary effort of scientists.

Therapeutic potential of cannabinoids in CNS disease.

The major psychoactive constituent of Cannabis sativa, delta(9)-tetrahydrocannabinol (delta(9)-THC), and endogenous cannabinoid ligands, such as anandamide, signal through G-protein-coupled cannabinoid receptors localised to regions of the brain associated with important neurological processes. Signalling is mostly inhibitory and suggests a role for cannabinoids as therapeutic agents in CNS disease where inhibition of neurotransmitter release would be beneficial. Anecdotal evidence suggests that patients with disorders such as multiple sclerosis smoke cannabis to relieve disease-related symptoms. Cannabinoids can alleviate tremor and spasticity in animal models of multiple sclerosis, and clinical trials of the use of these compounds for these symptoms are in progress. The cannabinoid nabilone is currently licensed for use as an antiemetic agent in chemotherapy-induced emesis. Evidence suggests that cannabinoids may prove useful in Parkinson's disease by inhibiting the excitotoxic neurotransmitter glutamate and counteracting oxidative damage to dopaminergic neurons. The inhibitory effect of cannabinoids on reactive oxygen species, glutamate and tumour necrosis factor suggests that they may be potent neuroprotective agents. Dexanabinol (HU-211), a synthetic cannabinoid, is currently being assessed in clinical trials for traumatic brain injury and stroke. Animal models of mechanical, thermal and noxious pain suggest that cannabinoids may be effective analgesics. Indeed, in clinical trials of postoperative and cancer pain and pain associated with spinal cord injury, cannabinoids have proven more effective than placebo but may be less effective than existing therapies. Dronabinol, a commercially available form of delta(9)-THC, has been used successfully for increasing appetite in patients with HIV wasting disease, and cannabinoid receptor antagonists may reduce obesity. Acute adverse effects following cannabis usage include sedation and anxiety. These effects are usually transient and may be less severe than those that occur with existing therapeutic agents. The use of nonpsychoactive cannabinoids such as cannabidiol and dexanabinol may allow the dissociation of unwanted psychoactive effects from potential therapeutic benefits. The existence of other cannabinoid receptors may provide novel therapeutic targets that are independent of CB(1) receptors (at which most currently available cannabinoids act) and the development of compounds that are not associated with CB(1) receptor-mediated adverse effects. Further understanding of the most appropriate route of delivery and the pharmacokinetics of agents that act via the endocannabinoid system may also reduce adverse effects and increase the efficacy of cannabinoid treatment. This review highlights recent advances in understanding of the endocannabinoid system and indicates CNS disorders that may benefit from the therapeutic effects of cannabinoid treatment. Where applicable, reference is made to ongoing clinical trials of cannabinoids to alleviate symptoms of these disorders.

Nonpsychotropic cannabinoid receptors regulate microglial cell migration.

During neuroinflammation, activated microglial cells migrate toward dying neurons, where they exacerbate local cell damage. The signaling molecules that trigger microglial cell migration are poorly understood. In this paper, we show that pathological overstimulation of neurons by glutamate plus carbachol dramatically increases the production of the endocannabinoid 2-arachidonylglycerol (2-AG) but only slightly increases the production of anandamide and does not affect the production of two putative endocannabinoids, homo-gamma-linolenylethanolamide and docosatetraenylethanolamide. We further show that pathological stimulation of microglial cells with ATP also increases the production of 2-AG without affecting the amount of other endocannabinoids. Using a Boyden chamber assay, we provide evidence that 2-AG triggers microglial cell migration. This effect of 2-AG occurs through CB2 and abnormal-cannabidiol-sensitive receptors, with subsequent activation of the extracellular signal-regulated kinase 1/2 signal transduction pathway. It is important to note that cannabinol and cannabidiol, two nonpsychotropic ingredients present in the marijuana plant, prevent the 2-AG-induced cell migration by antagonizing the CB2 and abnormal-cannabidiol-sensitive receptors, respectively. Finally, we show that microglial cells express CB2 receptors at the leading edge of lamellipodia, which is consistent with the involvement of microglial cells in cell migration. Our study identifies a cannabinoid signaling system regulating microglial cell migration. Because this signaling system is likely to be involved in recruiting microglial cells toward dying neurons, we propose that cannabinol and cannabidiol are promising nonpsychotropic therapeutics to prevent the recruitment of these cells at neuroinflammatory lesion sites.

Cannabinoid pharmacology: implications for additional cannabinoid receptor subtypes.

Delta(9)-Tetrahydrocannabinol (delta(9)-THC), the primary psychoactive constituent of marijuana (Cannabis sativa), is known to bind to two cannabinoid receptors: CB(1) receptors, located primarily in the brain, and CB(2) receptors, located primarily in the periphery. Recent research has suggested that other cannabinoids, including anandamide and WIN 55212-2, may also act at novel non-CB(1), non-CB(2) cannabinoid receptor(s). Anandamide produces a number of in vivo pharmacological effects in CB(1) knockout mice that are not produced by delta(9)-THC and cannot be explained by anandamide's rapid metabolism. In addition, in vitro anandamide and WIN 55212-2 stimulate [35S]GTPgammaS binding in both CB(1) knockout and wildtype mice while delta(9)-THC stimulates this binding only in wildtype mice. Although anandamide and vanilloid agonists share pharmacological effects, anandamide's actions in CB(1) knockout mice do not appear to be mediated by vanilloid VR(1) receptors. While not yet conclusive, these results suggest the possibility of additional cannabinoid receptors in the brain and periphery.

Spinal and peripheral mechanisms of cannabinoid antinociception: behavioral, neurophysiological and neuroanatomical perspectives.

A large body of literature indicates that cannabinoids suppress behavioral responses to acute and persistent noxious stimulation. This review examines behavioral, neurophysiological and neuroanatomical evidence supporting a role for cannabinoids in suppressing nociceptive transmission at spinal and peripheral levels. The development of subtype-selective competitive antagonists and high-affinity agonists provides the pharmacological tools required to study cannabinoid antinociceptive mechanisms. These studies provide insight into the functional roles of cannabinoid receptor subtypes, CB1 and CB2, in cannabinoid antinociceptive mechanisms as revealed in animal models of acute and persistent (somatic inflammatory, visceral inflammatory, neuropathic) pain. Localization studies employing receptor binding and quantitative autoradiography, immunocytochemistry and in situ hybridization are reviewed to examine the distribution of cannabinoid receptors at these levels and provide a neuroanatomical framework with which to understand the roles of endogenous cannabinoids in sensory processing.

Receptor classification: post genome.

Most of the G-protein-coupled receptors (GPCRs) in the human genome have been described. Investigation will now shift from discovery to analysis. Like many other genes, those encoding GPCRs are frequently found adjacent to each other in clusters. Duplicated genes often share ligands, signalling pathways and amino acid sequence. But, GPCRs do not have to be adjacent to be similar to each other. Phylogenetic analysis divides Family A GPCRs into many clusters that, more often than not, share similar types of ligands. Communication of these types of data for hundreds of GPCRs requires a robust and accepted nomenclature, Locus Link symbols are suggested.

International Union of Pharmacology. XXVII. Classification of cannabinoid receptors.

Two types of cannabinoid receptor have been discovered so far, CB(1) (2.1: CBD:1:CB1:), cloned in 1990, and CB(2) (2.1:CBD:2:CB2:), cloned in 1993. Distinction between these receptors is based on differences in their predicted amino acid sequence, signaling mechanisms, tissue distribution, and sensitivity to certain potent agonists and antagonists that show marked selectivity for one or the other receptor type. Cannabinoid receptors CB(1) and CB(2) exhibit 48% amino acid sequence identity. Both receptor types are coupled through G proteins to adenylyl cyclase and mitogen-activated protein kinase. CB(1) receptors are also coupled through G proteins to several types of calcium and potassium channels. These receptors exist primarily on central and peripheral neurons, one of their functions being to inhibit neurotransmitter release. Indeed, endogenous CB(1) agonists probably serve as retrograde synaptic messengers. CB(2) receptors are present mainly on immune cells. Such cells also express CB(1) receptors, albeit to a lesser extent, with both receptor types exerting a broad spectrum of immune effects that includes modulation of cytokine release. Of several endogenous agonists for cannabinoid receptors identified thus far, the most notable are arachidonoylethanolamide, 2-arachidonoylglycerol, and 2-arachidonylglyceryl ether. It is unclear whether these eicosanoid molecules are the only, or primary, endogenous agonists. Hence, we consider it premature to rename cannabinoid receptors after an endogenous agonist as is recommended by the International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification. Although pharmacological evidence for the existence of additional types of cannabinoid receptor is emerging, other kinds of supporting evidence are still lacking.

The diversity in the vanilloid (TRPV) receptor family of ion channels.

Following cloning of the vanilloid receptor 1 (VR1) at least four other related proteins have been identified. Together, these form a distinct subgroup of the transient receptor potential (TRP) family of ion channels. Members of the vanilloid receptor family (TRPV) are activated by a diverse range of stimuli, including heat, protons, lipids, phorbols, phosphorylation, changes in extracellular osmolarity and/or pressure, and depletion of intracellular Ca2+ stores. However, VR1 remains the only channel activated by vanilloids such as capsaicin. These channels are excellent molecular candidates to fulfil a range of sensory and/or cellular roles that are well characterized physiologically. Furthermore, as novel pharmacological targets, the vanilloid receptors have potential for the development of many future disease treatments.

Different binding properties and affinities for ATP and ADP among sulfonylurea receptor subtypes, SUR1, SUR2A, and SUR2B.

ATP-sensitive potassium (K(ATP)) channels, composed of sulfonylurea receptor (SURx) and Kir6.x, play important roles by linking cellular metabolic state to membrane potential in various tissues. Pancreatic, cardiac, and vascular smooth muscle K(ATP) channels, which consist of different subtypes of SURx, differ in their responses to cellular metabolic state. To explore the possibility that different interactions of SURx with nucleotides cause differential regulation of K(ATP) channels, we analyzed the properties of nucleotide-binding folds (NBFs) of SUR1, SUR2A, and SUR2B. SURx in crude membrane fractions was incubated with 8-azido-[alpha-(32)P]ATP or 8-azido-[gamma-(32)P]ATP under various conditions and was photoaffinity-labeled. Then, SURx was digested mildly with trypsin, and partial tryptic fragments were immunoprecipitated with antibodies against NBF1 and NBF2. Some nucleotide-binding properties were different among SUR subtypes as follows. 1) Mg(2+) dependence of nucleotide binding of NBF2 of SUR1 was high, whereas those of SUR2A and SUR2B were low. 2) The affinities of NBF1 of SUR1 for ATP and ADP, especially for ATP, were significantly higher than those of SUR2A and SUR2B. 3) The affinities of NBF2 of SUR2B for ATP and ADP were significantly higher than those of SUR2A. This is the first biochemical study to analyze and compare the nucleotide-binding properties of NBFs of three SUR subtypes, and our results suggest that their different properties may explain, in part, the differential regulation of K(ATP) channel subtypes. The high nucleotide-binding affinities of SUR1 may explain the high ability of SUR1 to stimulate pancreatic K(ATP) channels. It is also suggested that the C-terminal 42 amino acids affect the physiological roles of SUR2A and SUR2B by changing the nucleotide-binding properties of their NBFs.

Evidence that 2-arachidonoylglycerol but not N-palmitoylethanolamine or anandamide is the physiological ligand for the cannabinoid CB2 receptor. Comparison of the agonistic activities of various cannabinoid receptor ligands in HL-60 cells.

We examined the effect of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, on the intracellular free Ca(2+) concentrations in HL-60 cells that express the cannabinoid CB2 receptor. We found that 2-arachidonoylglycerol induces a rapid transient increase in intracellular free Ca(2+) concentrations in HL-60 cells. The response was affected by neither cyclooxygenase inhibitors nor lipoxygenase inhibitors, suggesting that arachidonic acid metabolites are not involved. Consistent with this notion, free arachidonic acid was devoid of any agonistic activity. Importantly, the Ca(2+) transient induced by 2-arachidonoylglycerol was blocked by pretreatment of the cells with SR144528, a CB2 receptor-specific antagonist, but not with SR141716A, a CB1 receptor-specific antagonist, indicating the involvement of the CB2 receptor but not the CB1 receptor in this cellular response. G(i) or G(o) is also assumed to be involved, because pertussis toxin treatment of the cells abolished the response. We further examined the structure-activity relationship. We found that 2-arachidonoylglycerol is the most potent compound among a number of naturally occurring cannabimimetic molecules. Interestingly, anandamide and N-palmitoylethanolamine, other putative endogenous ligands, were found to be a weak partial agonist and an inactive ligand, respectively. These results strongly suggest that the CB2 receptor is originally a 2-arachidonoylglycerol receptor, and 2-arachidonoylglycerol is the intrinsic natural ligand for the CB2 receptor that is abundant in the immune system.

MCC-134, a novel vascular relaxing agent, is an inverse agonist for the pancreatic-type ATP-sensitive K(+) channel.

The effects of a novel vasorelaxant agent, MCC-134 (1-[4-(1H-imidazol-1-yl)benzoyl]-N-methyl-cyclobutanecarbothioamide++ +), were examined on reconstituted ATP-sensitive K(+) (K(ATP)) channels, which are composed of an inwardly rectifying K(+) channel, Kir6.2, and three types of sulfonylurea receptors (SUR): SUR1, SUR2A, and SUR2B. Each type of K(ATP) channel was heterologously expressed in human embryonic kidney 293T cells. The expressed K(ATP) channel currents were measured with the whole-cell configuration of the patch-clamp method. MCC-134 activated the SUR2B/Kir6.2 channel, was a weak activator of the SUR2A/Kir6.2 channel, but did not activate the SUR1/Kir6.2 channel. MCC-134 suppressed SUR1/Kir6.2 channel currents that had been fully activated by either diazoxide or NaCN, whereas it did not affect the fully activated SUR2A/Kir6.2 or SUR2B/Kir6.2 channel currents. Thus, MCC-134, which is a relatively effective opener of the vascular smooth muscle type (SUR2B) of K(ATP) channel, is an antagonist of the pancreatic type (SUR1) of K(ATP) channel. Therefore, depending on the subtype of SUR, a pharmacological agent can cause either activation or inhibition of K(ATP) channel activity.

Cannabis and cannabinoids: pharmacology and rationale for clinical use.

It is now known that there are at least two types of cannabinoid receptors. These are CB1 receptors, present mainly on central and peripheral neurones, and CB2 receptors, present mainly on immune cells. Endogenous cannabinoid receptor agonists ('endocannabinoids') have also been identified. The discovery of this 'endogenous cannabinoid system' has led to the development of selective CB1 and CB2 receptor ligands and fueled renewed interest in the clinical potential of cannabinoids. Two cannabinoid CB1 receptor agonists are already used clinically, as antiemetics or as appetite stimulants. These are D 9 - tetrahydrocannabinol (THC) and nabilone. Other possible uses for CB1 receptor agonists include the suppression of muscle spasm/spasticity associated with multiple sclerosis or spinal cord injury, the relief of chronic pain and the management of glaucoma and bronchial asthma. CB1 receptor antagonists may also have clinical applications, e. g. as appetite suppressants and in the management of schizophrenia or disorders of cognition and memory. So too may CB2 receptor ligands and drugs that activate cannabinoid receptors indirectly by augmenting endocannabinoid levels at cannabinoid receptors. When taken orally, THC seems to undergo variable absorption and to have a narrow 'therapeutic window' (dose range in which it is effective without producing significant unwanted effects). This makes it difficult to predict an oral dose that will be both effective and tolerable to a patient and indicates a need for better cannabinoid formulations and modes of administration. For the therapeutic potential of cannabis or CB1 receptor agonists to be fully exploited, it will be important to establish objectively and conclusively (a) whether these agents have efficacy against selected symptoms that is of clinical significance and, if so, whether the benefits outweigh the risks, (b) whether cannabis has therapeutic advantages over individual cannabinoids, (c) whether there is a need for additional drug treatments to manage any of the disorders against which cannabinoids are effective, and (d) whether it will be possible to develop drugs that have reduced psychotropic activity and yet retain the ability to act through CB1 receptors to produce their sought-after effects.

Cannabinoid receptors can activate and inhibit G protein-coupled inwardly rectifying potassium channels in a xenopus oocyte expression system.

In this study, we focused on the pharmacological characterization of cannabinoid receptor coupling to G protein-gated inwardly rectifying potassium (GIRK) channels. Cannabinoids were tested on Xenopus laevis oocytes coexpressing the CB(1) receptor and GIRK1 and GIRK4 channels (CB(1)/GIRK1/4) or the CB(2) receptor and GIRK1/4 channels (CB(2)/GIRK1/4). WIN 55,212-2 enhanced currents carried by GIRK channels in the CB(1)/GIRK1/4 and CB(2)/GIRK1/4 system; however, the CB(2) receptor did not couple efficiently to GIRK1/4 channels. In the CB(1)/GIRK1/4 system, WIN 55,212-2 was the most efficacious compound tested. CP 55,940 and anandamide acted as partial agonists. The rank order of potency was CP 55,940 > WIN 55,212-2 = anandamide. The CB(1)-selective antagonist SR141716A alone acted as a inverse agonist by inhibiting GIRK currents in oocytes expressing CB(1)/GIRK1/4, suggesting the CB(1) receptor is constitutively activated. A conserved aspartate residue, which was previously shown to be critical for G protein coupling in cannabinoid receptors, was mutated (to asparagine, D163N) and analyzed. Oocytes coexpressing CB(1)/GIRK1/4 or D163N/GIRK1/4 were compared. The potency of WIN 55, 212-2 at the mutant receptor was similar to wild type, but its efficacy was substantially reduced. CP 55,940 did not elicit currents in oocytes expressing D163N/GIRK1/4. In summary, it appears the CB(1) and CB(2) receptors couple differently to GIRK1/4 channels. In the CB(1)/GIRK1/4 system, cannabinoids evaluated demonstrated the ability to enhance or inhibit GIRK currents. Furthermore, a conserved aspartate residue in the CB(1) receptor is required for normal communication with GIRK channels in oocytes demonstrating the interaction between receptor and channels is G protein dependent.

Peripheral and central actions of capsaicin and VR1 receptor.

Vanilloid receptor subtype 1 (VR1), a capsaicin receptor, is expressed in primary sensory neurons and vagal nerves. Heat and protons as well as capsaicin activate VR1 to induce the influx of cations, particularly Ca2+ and Na+ ions. Characteristic effects of capsaicin are the induction of a burning sensation after acute administration and the desensitization of sensory neurons after large doses and prolonged administration. The latter feature made capsaicin cream applicable for the treatment of chronic pain and pruritus. Capsaicin alters several visceral functions, which may be mediated by action on vagal nerves and central neurons. Capsaicin affects thermoregulation after intra-hypothalamic injection and releases glutamate from the hypothalamus and cerebral cortex slices, while VR1-like immunoreactivity is not apparent in these regions. These findings taken together suggest the existence of other subtypes of vanilloid receptors in the brain.

Presynaptic cannabinoid and imidazoline receptors in the human heart and their potential relationship.

Segments of human right atrial appendages preincubated with [3H]noradrenaline and superfused with physiological salt solution containing desipramine and corticosterone were used to examine whether the cardiac sympathetic nerves are endowed with cannabinoid receptors and to further study pharmacological properties of presynaptic imidazoline receptors. The cannabinoid CB1 receptor agonists CP55,940, HU210 and anandamide inhibited evoked [3H]noradrenaline release. The inhibition by CP55,940 and anandamide was abolished by the CB1 receptor antagonists SR141716A (1 microM) and LY320135 (1 microM). Rauwolscine at the imidazoline receptor-blocking concentration of 30 microM abolished the inhibitory effect of CP55,940 and anandamide. After blockade of alpha2-adrenoceptors with 1 microM rauwolscine, the imidazoline binding site ligand S23230, which is the (-)-enantiomer of the racemic oxazoline derivative S22687, exhibited low potency in inhibiting electrically evoked [3H]noradrenaline release (pIC30%=4.96), whereas the (+)-enantiomer S23229 and the racemate S22687 were ineffective. In the presence of 30 microM rauwolscine, S23230 did not significantly inhibit evoked release. The imidazoline receptor-mediated inhibitory effect of BDF 6143 and aganodine on evoked [3H]noradrenaline release was abolished by 1 microM SR141716A and by 1 microM LY320135. The inhibitory effect of moxonidine on evoked [3H]noradrenaline release, which is exclusively mediated via activation of alpha2-autoreceptors, was not antagonized by 1 microM SR141716A. In conclusion, inhibitory cannabinoid CB1 receptors are present on the sympathetic axon terminals of human atrial appendages. Presynaptic imidazoline receptors share the property of other receptors in that they can be stereoselectively activated. The cross-antagonism of imidazoline receptor agonists/antagonists with CB1 receptor antagonists/agonists suggests that these receptors may have certain binding domains in common or that they interact with each other in an unknown manner.

Pharmacologic and molecular discrimination of I2-imidazoline receptor subtypes.

I2-imidazoline receptors (I2-IR) are characterized by their high affinity for imidazolines and guanidines and medium affinity for imidazolidines. The differential recognition of I2-IR by amiloride led to subtype these sites as amiloride-sensitive (I2A-IR) and amiloride-insensitive (I2B-IR). I2-IR labeled with [3H]idazoxan or [3H]2-BFI in the rabbit cerebral cortex (I2A-IR) displayed higher affinities for amiloride and amiloride analogs than in the rat cerebral cortex (I2B-IR). Other drugs tested displayed biphasic curves in competition experiments, indicating the existence of high and low affinity sites for both I2-IR subtypes. The drugs (+)- and (-)-medetomidine, bromoxidine, moxonidine, and clorgyline were more potent on the high and/or low affinity sites of I2B-IR than on I2A-IR. Preincubation (30 min at 25 degrees C) with 10(-6) M isothiocyanatobenzyl imidazoline (IBI) or with 10(-6) M clorgyline reduced by 40% and 26%, respectively, the binding of [3H]2-BFI to I2B-IR, but it did not alter the binding of the radioligand to I2A-IR. These results indicated that the I2-IR subtypes differ in their pharmacologic profiles and in the nature of the imidazoline binding site involved in clorgyline and IBI alkylation. In rat cortical membranes, western blot detection of immunoreactive imidazoline receptor proteins revealed a double band of approximately 29/30 kD and three less intense bands of approximately 45, approximately 66, and approximately 85 kD. In rabbit cortical membranes the antibody detected proteins of approximately 30, approximately 57, approximately 66, and approximately 85 kD. It is suggested that I2-IR may be related to more than one receptor protein and that I2-IR subtypes differ in the nature of the proteins implicated.