M1 Receptor Antagonist Research Articles

Differential effects of m1 and m2 receptor antagonists in perirhinal cortex on visual recognition memory in monkeys

Microinfusions of the nonselective muscarinic antagonist scopolamine into perirhinal cortex impairs performance on visual recognition tasks, indicating that muscarinic receptors in this region play a pivotal role in recognition memory. To assess the mnemonic effects of selective blockade in perirhinal cortex of muscarinic receptor subtypes, we locally infused either the m1-selective antagonist pirenzepine or the m2-selective antagonist methoctramine in animals performing one-trial visual recognition, and compared these scores with those following infusions of equivalent volumes of saline. Compared to these control infusions, injections of pirenzepine, but not of methoctramine, significantly impaired recognition accuracy. Further, similar doses of scopolamine and pirenzepine yielded similar deficits, suggesting that the deficits obtained earlier with scopolamine were due mainly, if not exclusively, to blockade of m1 receptors. The present findings indicate that m1 and m2 receptors have functionally dissociable roles, and that the formation of new visual memories is critically dependent on the cholinergic activation of m1 receptors located on perirhinal cells.

Antagonism of 5‐HT2A receptors attenuates the effects of amphetamine and 3,4‐methylenedioxymethamphetamine on wakefulness and dopamine overflow

The neuropharmacology of the sleep and arousal systems involves a complex interplay between multiple neurotransmitter systems. Previous work has downplayed the role of dopamine in sleep and wakefulness, at least in part, because dopamine neurons do not appear to change their firing rates across arousal states. In the present study, we examined the wake promoting effects of amphetamine and 3,4‐methylenedioxymethamphetamine (MDMA) in rhesus monkeys using noninvasive telemetric monitoring. To evaluate the importance of dopamine, we used in vivo microdialysis targeting the caudate nucleus and pretreatment experiments with the selective 5‐HT2A receptor antagonist M100907. We targeted 5‐HT2A receptors because previous studies found that antagonizing this receptor attenuates some of the behavioral and neurochemical effects of amphetamine and MDMA. We found that amphetamine and MDMA significantly and dose‐dependently increased wakefulness. The effects of amphetamine were more pronounced than those of MDMA, but this may have been the result of different durations of action. Antagonism of 5‐HT2A receptors attenuated the effects of amphetamine and MDMA on both wakefulness and dopamine overflow. These findings suggest that increased dopamine neurotransmission may be necessary for the wake promoting effects of amphetamines. These studies were supported by USPHS grants (DA 00517 and RR 00165).

Opioid μ‐receptors in the rostral medullary raphe modulate hypoxia‐induced hyperpnea in unanesthetized rats

It has been suggested that the medullary raphe (MR) plays a key role in the physiological responses to hypoxia. As the rostral MR (rMR) expresses an extensive population of opioid μ‐receptors, we studied the putative role of opioid μ‐receptors in the rMR region on ventilation in normal and 7% hypoxic conditions. We measured pulmonary ventilation and the body temperatures of male Wistar rats before and after the selective opioid μ‐receptor antagonist CTAP (D‐Phe‐Cys‐Tyr‐D‐Trp‐Arg‐Thr‐Pen‐Thr‐NH2, cyclic, 0.1 μg/0.1 μL) was microinjected into the rMR during normoxia or after 60 minutes of hypoxia. The animals treated with intra‐rMR CTAP exhibited an attenuation of the ventilatory response to hypoxia (430 ± 86 mL kg−1 min−1) compared to the control group (790 ± 82 mL kg−1 min−1) (P<0.05) due to a reduced tidal volume (3.0 ± 0.41 versus 6.1 ± 0.6 ml kg−1; P<0.05). During hypoxic recovery, the ventilation remained reduced in the CTAP intra‐rRM group. No differences in the Tb were observed among groups during hypoxia. These data suggest that opioids acting on μ‐receptors in the rMR exert an excitatory modulation of hyperventilation induced by hypoxia.Financial support: FAPESP, CNPq, PRONEX.

Involvement of NMDA receptors in the beneficial effects of pioglitazone on scopolamine-induced memory impairment in mice

IntroductionPioglitazone, a peroxisome proliferator activated receptor γ (PPARγ) agonist, is widely used in clinical medicine as a treatment for type 2 diabetes and is recently proved to have beneficial effects on improving cognition in early stages of Alzheimer's disease (AD). Moreover, it has been shown that pioglitazone reduces N-methyl-d-aspartate (NMDA, a glutamate agonist) mediated calcium currents and transients. Since enhanced calcium transients are present in AD models, we tested the hypothesis whether pioglitazone manifests its acquisition memory enhancement role through glutamatergic pathway. Material and methodsMemory performance was evaluated in a two-trial recognition Y-maze test and passive avoidance in mice. Pioglitazone (20 or 40mg/kg, p.o.) was administered 2h before each trial, NMDA (75mg/kg i.p.), 15min before pioglitazone, and scopolamine, an M1 (muscarinic) receptor antagonist (0.3 or 1.0mg/kg i.p.) and MK-801 (dizocilpine) (0.01, 0.03 or 0.1mg/kg, i.p.), the highly selective, non-competitive NMDA antagonist – 30min beforehand. Results(1) We induced the memory impairment by scopolamine or MK-801 before trials. (2) Pioglitazone did not improve the memory impairment induced by MK-801. (3) Pioglitazone significantly improved the memory impairment induced by scopolamine. (4) Subeffective dose of MK-801 nullified the beneficial effects of pioglitazone in scopolamine induced memory impaired mice. (5) NMDA promoted the effects of subeffective dose of pioglitazone on memory impaired by scopolamine. DiscussionIn conclusion, the present study suggests that glutamatergic pathway is involved in the pioglitazone induced memory performance.

Effects of the 5-HT2C receptor agonist Ro60-0175 and the 5-HT2A receptor antagonist M100907 on nicotine self-administration and reinstatement

The reinforcing effects of nicotine are mediated in part by brain dopamine systems. Serotonin, acting via 5-HT2A and 5-HT2C receptors, modulates dopamine function. In these experiments we examined the effects of the 5-HT2C receptor agonist Ro60-0175 and the 5-HT2A receptor antagonist (M100907, volinanserin) on nicotine self-administration and reinstatement of nicotine-seeking. Male Long-Evans rats self-administered nicotine (0.03 mg/kg/infusion, IV) on either a FR5 or a progressive ratio schedule of reinforcement. Ro60-0175 reduced responding for nicotine on both schedules. While Ro60-0175 also reduced responding for food reinforcement, response rates under drug treatment were several-fold higher than in animals responding for nicotine. M100907 did not alter responding for nicotine, or food, on either schedule. In tests of reinstatement of nicotine-seeking, rats were first trained to lever press for IV infusions of nicotine; each infusion was also accompanied by a compound cue consisting of a light and tone. This response was then extinguished over multiple sessions. Injecting rats with a nicotine prime (0.15 mg/kg) reinstated responding; reinstatement was also observed when responses were accompanied by the nicotine associated cue. Ro60-0175 attenuated reinstatement of responding induced by nicotine and by the cue. The effects of Ro60-0175 on both forms of reinstatement were blocked by the 5-HT2C receptor antagonist SB242084. M100907 also reduced reinstatement induced by either the nicotine prime or by the nicotine associated cue. The results indicate that 5-HT2C and 5-HT2A receptors may be potential targets for therapies to treat some aspects of nicotine dependence.

The effects of galanin on neuropathic pain in streptozotocin-induced diabetic rats

Diabetic neuropathy is a common complication associated with diabetes and is frequently painful. However, mechanisms responsible for diabetic neuropathic pain are still unclear. Experimental evidence has shown that the galanin and its receptor are involved in pain sensitization. The objective of the present study was to investigate the role of galanin and its receptor antagonist or agonist on neuropathic pain in streptozotocin-induced diabetic rats. The expression of galanin, galanin receptors 1 and 2 in dorsal root ganglion (DRG) and spinal dorsal horn (SDH) in diabetic rats were detected by Western blot assay. The effects of galanin, galanin receptor antagonist M35, galanin receptor 1 agonist M617, and galanin receptor 2 agonist AR-M1896 on neuropathic pain were evaluated by mechanical stimuli. The results showed that (1) the diabetic rats showed a significant mechanical hyperalgesia between 4 and 12weeks; (2) galanin receptor 1 expression decreased in SDH in diabetic rats; (3) galanin receptor 2 expression decreased in DRG and SDH in diabetic rats; (4) intrathecal administration of exogenous galanin attenuated diabetic neuropathic pain, this effect could be blocked by pre-treatment with galanin receptor antagonist M35; and (5) intrathecal administration of galanin receptor 1 agonist M617, but not galanin receptor 2 agonist AR-M1896, attenuated diabetic neuropathic pain. These results imply that galanin acts through receptor 1, but not galanin receptor 2, to exert analgesic effect in diabetic neuropathic pain and is one of the potential therapeutic targets on diabetic neuropathic pain sensitization.

Opiate-induced constipation related to activation of small intestine opioid μ2-receptors

To investigate the role of opioid μ-receptor subtype in opiate-induced constipation (OIC). The effect of loperamide on intestinal transit was investigated in mice. Ileum strips were isolated from 12-wk-old male BALB/c mice for identification of isometric tension. The ileum strips were precontracted with 1 μmol/L acetylcholine (ACh). Then, decrease in muscle tone (relaxation) was characterized after cumulative administration of 0.1-10 μmol/L loperamide into the organ bath, for a concentration-dependent study. Specific blockers or antagonists were used for pretreatment to compare the changes in loperamide-induced relaxation. In addition to the delay in intestinal transit, loperamide produced a marked relaxation in isolated ileum precontracted with ACh, in a dose-dependent manner. This relaxation was abolished by cyprodime, a selective opioid μ-receptor antagonist, but not modified by naloxonazine at a dose sufficient to block opioid μ-1 receptors. Also, treatment with opioid μ-1 receptor agonist failed to modify the muscle tone. Moreover, the relaxation by loperamide was attenuated by glibenclamide at a dose sufficient to block ATP-sensitive K+ (K(ATP)) channels, and by protein kinase A (PKA) inhibitor, but was enhanced by an inhibitor of phosphodiesterase for cyclic adenosine monophosphate (cAMP). Loperamide induces intestinal relaxation by activation of opioid μ-2 receptors via the cAMP-PKA pathway to open K(ATP) channels, relates to OIC.

The neuropharmacology of prolactin secretion elicited by 3,4-methylenedioxymethamphetamine (“ecstasy”): A concurrent microdialysis and plasma analysis study

3,4-methylenedioxymethamphetamine (MDMA) is a substituted phenethylamine that is widely abused as the street drug “ecstasy”. Racemic MDMA (S,R(+/−)-MDMA) and its stereoisomers elicit complex spectrums of psychobiological, neurochemical, and hormonal effects. In this regard, recent findings demonstrated that S,R(+/−)-MDMA and its stereoisomer R(−)-MDMA elicit increases in striatal extracellular serotonin levels and plasma levels of the hormone prolactin in rhesus monkeys. In the present mechanistic study, we evaluated the role of the serotonin transporter and the 5-HT2A receptor in S,R(+/−)-MDMA- and R(−)-MDMA-elicited prolactin secretion in rhesus monkeys through concurrent microdialysis and plasma analysis determinations and drug interaction experiments. Concurrent neurochemical and hormone determinations showed a strong positive temporal correlation between serotonin release and prolactin secretion. Consistent with their distinct mechanisms of action and previous studies showing that the serotonin transporter inhibitor fluoxetine attenuates the behavioral and neurochemical effects of S,R(+/−)-MDMA, pretreatment with fluoxetine attenuated serotonin release elicited by either S,R(+/−)-MDMA or R(−)-MDMA. As hypothesized, at a dose that had no significant effects on circulating prolactin levels when administered alone, fluoxetine also attenuated prolactin secretion elicited by S,R(+/−)-MDMA. In contrast, combined pretreatment with both fluoxetine and the selective 5-HT2A receptor antagonist M100907 was required to attenuate prolactin secretion elicited by R(−)-MDMA, suggesting that this stereoisomer of S,R(+/−)-MDMA elicits prolactin secretion through both serotonin release and direct agonism of 5-HT2A receptors. Accordingly, these findings inform our understanding of the neuropharmacology of both S,R(+/−)-MDMA and R(−)-MDMA and the regulation of prolactin secretion.

Modulation of Peripheral Inflammatory Pain Thresholds by M1 and Nicotinic Receptor Antagonists

The study used the paw withdrawal test to investigate the role of the cholinergic system on the modulation of inflammatory pain induced by carrageenan (Cg) at the peripheral level, through activation of muscarinic and nicotinic receptors. Intraplantar administration of the specific M₁ receptor antagonist telenzepine (TEL; 6, 12 and 24 µg/paw) caused a dose-dependent reduction in the nociceptive threshold induced by Cg (125 µg/paw). This effect was not observed with increasing doses (4, 10 and 40 µg) of other specific receptor antagonists: M₂ (dimethindene), M₃ (4-DAMP) and M₄ (tropicamide). The nicotinic antagonist mecamylamine (MEC; 25, 50 and 100 µg/paw) also caused a dose-dependent reduction in the nociceptive threshold induced by Cg (125 µg). To exclude a non-local effect, Cg (125 µg) was injected into both hind paws, while TEL (12 µg) and MEC (50 µg) were administered only in the right paw. At these doses, the muscarinic antagonists increased inflammatory pain only in the treated right paw, suggesting a peripheral effect. In the presence of prostaglandin E₂ (1 µg/paw), TEL (12 µg) and MEC (50 µg) did not reduce the nociceptive threshold, suggesting that this hyperalgesic agent does not induce the release of endogenous acetylcholine. These data suggest that muscarinic M₁ and nicotinic receptors participate in the modulation of endogenous cholinergic inflammatory pain at the peripheral level.

Alpha-cobratoxin inhibits T-type calcium currents through muscarinic M4 receptor and G o-protein βγ subunits-dependent protein kinase A pathway in dorsal root ganglion neurons

The long-chain neurotoxic protein, alpha-cobratoxin (α-CTx), has been shown to have analgesic effects. However, the underlying mechanisms still remain unclear. In this study, we examined the effects of α-CTx on T-type calcium channel currents (T-currents) and elucidated the relevant mechanisms in mouse dorsal root ganglion (DRG) neurons. Our results showed that α-CTx reversibly inhibited T-currents in a dose-dependent manner. This inhibitory effect was blocked by the selective muscarinic M4 receptor antagonist tropicamide, while methyllycaconitine, a specific antagonist for the α7 subtype of nicotinic receptor had no effect. siRNA targeting the M4 receptor in small DRG neurons abolished α-CTx-induced T-current inhibition. Intracellular application of GDP-β-S or a selective antibody against the G oα-protein, as well as pretreatment of the cells with pertussis toxin, abolished the inhibitory effects of α-CTx. The M4 receptor-mediated response was blocked by dialyzing cells with QEHA peptide or anti-G β antibody. Pretreatment of the cells with protein kinase A (PKA) inhibitor H89 or intracellular application of PKI 6-22 abolished α-CTx-induced T-current inhibition in small DRG neurons, whereas inhibition of phosphatidylinositol 3-kinase or PKC elicited no such effects. In addition, α-CTx significantly increased PKA activity in DRG neurons, whereas pretreatment of the cells with tropicamide abolished this effect. In summary, our results suggest that activation of muscarinic M4 receptor by α-CTx inhibits T-currents via the G βγ of G o-protein and PKA-dependent pathway. This article is part of a Special Issue entitled ‘Post-Traumatic Stress Disorder’.

Effects of oral loperamide on efficacy of naltrexone, baclofen and AM-251 in blocking ethanol self-administration in rats

Naltrexone is a μ-opioid receptor antagonist that has been extensively studied for its ability to block the rewarding effects of ethanol. Opioid receptors are widely distributed within the gastrointestinal tract (GIT). Typically, naltrexone is administered by parenteral routes in nonclinical studies. We initially tested if opioid receptors within the GIT would influence the ability of oral naltrexone to inhibit ethanol oral self-administration in rats using the co-administration of oral loperamide, a peripherally restricted opioid agonist. As expected, oral naltrexone only had modest effects on ethanol intake, and the response was not dose-dependent. However in rats, treatment with loperamide prior to the administration of naltrexone resulted in a suppression of ethanol intake which approached that observed with naltrexone given by the subcutaneous (SC) route. Importantly, administration of loperamide prior to administration of naltrexone did not alter blood concentrations of naltrexone. We then evaluated if oral loperamide would enhance effects of baclofen (a GABAB receptor agonist) and AM-251 (a CB-1 receptor antagonist) and found that pre-treatment with loperamide did potentiate the action of both drugs to reduce ethanol self-administration. Finally, the specific opioid receptor type involved was investigated using selective μ- and κ-receptor antagonists to determine if these would affect the ability of the AM-251 and loperamide combination to block ethanol drinking behavior. The effect of loperamide was blocked by ALKS 37, a peripherally restricted μ-receptor antagonist. These data suggest an important role for opioid receptors within the GIT in modulating central reward pathways and may provide new insights into strategies for treating reward disorders, including drug dependency.

Opioid μ‐receptors in the rostral medullary raphe modulate hypoxia‐induced hyperpnea in unanesthetized rats

It has been suggested that the medullary raphe (MR) plays a key role in the physiological responses to hypoxia. As opioid μ-receptors have been found in the MR, we studied the putative role of opioid μ-receptors in the rostral MR (rMR) region on ventilation in normal and 7% hypoxic conditions. We measured pulmonary ventilation (VE) and the body temperatures (Tb) of male Wistar rats before and after the selective opioid μ-receptor antagonist CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2, cyclic, 0.1 μg per 0.1 μL) was microinjected into the rMR during normoxia or after 60 min of hypoxia. The animals treated with intra-rMR CTAP exhibited an attenuation of the ventilatory response to hypoxia (430 ± 86 mL kg(-1) min(-1)) compared with the control group (790 ± 82 mL kg(-1) min(-1) ) (P < 0.05). No differences in the Tb were observed between groups during hypoxia. These data suggest that opioids acting on μ-receptors in the rMR exert an excitatory modulation of hyperventilation induced by hypoxia.

Role of TNF‐α in virus‐induced airway hyperresponsiveness and neuronal M2 muscarinic receptor dysfunction

Infections with respiratory viruses induce exacerbations of asthma, increase acetylcholine release and potentiate vagally mediated bronchoconstriction by blocking inhibitory M₂ muscarinic receptors on parasympathetic neurons. Here we test whether virus-induced M₂ receptor dysfunction and airway hyperresponsiveness are tumour necrosis factor-alpha (TNF-α) dependent. Guinea pigs were pretreated with etanercept or phosphate-buffered saline 24 h before intranasal infection with parainfluenza. Four days later, pulmonary inflation pressure, heart rate and blood pressure were measured. M₂ receptor function was assessed by the potentiation by gallamine (an M₂ receptor antagonist) of bronchoconstriction caused by electrical stimulation of the vagus nerves and measured as increased pulmonary inflation pressure. Human airway epithelial cells were infected with influenza and TNF-α concentration in supernatant was measured before supernatant was applied to human neuroblastoma cells. M₂ receptor expression in these neuroblastoma cells was measured by qRT-PCR. Influenza-infected animals were hyperresponsive to vagal stimulation but not to intravenous ACh. Gallamine did not potentiate vagally induced bronchoconstriction in virus-infected animals, indicating M₂ receptor dysfunction. Etanercept prevented virus-induced airway hyperresponsiveness and M₂ receptor dysfunction, without changing lung viral titres. Etanercept caused a non-significant decrease in total cells, macrophages and neutrophils in bronchoalveolar lavage. Influenza infection significantly increased TNF-α release from isolated epithelial cells, sufficient to decrease M₂ receptors in neuroblastoma cells. This ability of supernatants from infected epithelial cells to inhibit M₂ receptor expression was blocked by etanercept. TNF-α is a key mediator of virus-induced M₂ muscarinic receptor dysfunction and airway hyperresponsiveness.

Activation of Opioid μ-Receptors in the Commissural Subdivision of the Nucleus Tractus Solitarius Abolishes the Ventilatory Response to Hypoxia in Anesthetized Rats

: The commissural subnucleus of the nucleus tractus solitarius (comNTS) is a key region in the brainstem responsible for the hypoxic ventilatory response (HVR) because it contains the input terminals of the carotid chemoreceptor. Because opioids inhibit the HVR via activating central μ-receptors that are expressed abundantly in the comNTS, the authors of the current study asked whether activating local μ-receptors attenuated the carotid body-mediated HVR. : To primarily stimulate the carotid body, brief hypoxia (100% N2) and hypercapnia (15% CO2) for 10 s and/or intracarotid injection of NaCN (10 μg/100 μl) were performed in anesthetized and spontaneously breathing rats. These stimulations were repeated after: (1) microinjecting three doses of μ-receptor agonist [d-Ala2, N-Me-Phe4, Gly-ol]-Enkephalin (DAMGO) (approximately 3.5 nl) into the comNTS; (2) carotid body denervation; and (3) systemic administration of DAMGO (300 μg/kg) without and with previous intracomNTS injection of d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2, a μ-receptor antagonist. : Study results showed that DAMGO at 0.25 and 2.5, but not 0.025 mM, caused a similar decrease in baseline ventilation (approximately 12%). DAMGO at 0.25 mM largely reduced (64%) the HVR, whereas DAMGO at 2.5 mM abolished the HVR (and the VE response to NaCN) and moderately attenuated (31%) the hypercapnic ventilatory response. Interestingly, similar HVR abolition and depression of the hypercapnic ventilatory response were observed after carotid body denervation. Blocking comNTS μ-receptors by d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2 significantly attenuated the HVR depression by systemic DAMGO with little change in the DAMGO modulatory effects on baseline ventilation and the hypercapnic ventilatory response. : The data suggest that opioids within the comNTS, via acting on μ-receptors, are able to abolish the HVR by affecting the afferent pathway of the carotid chemoreceptor.

Activation of Peripheral Opioid µ-Receptors in Blood Vessel May Lower Blood Pressure in Spontaneously Hypertensive Rats

Background/Aims: The role of opioid receptors in the regulation of vascular function remains unclear. In the current study, we evaluated the ability of loperamide, a peripheral opioid receptor agonist, to regulate blood pressure in spontaneously hypertensive rats (SHRs) and examined the mechanism(s) by which loperamide exerts its effects. Methods: In male SHRs, mean arterial pressure (MAP) was measured and hemodynamic analysis was recorded. Additionally, the isometric tension of aortic rings isolated from SHRs was determined. Results: Loperamide dose-dependently decreased MAP in SHRs but not in the normal group of Wistar-Kyoto rats. This reduction of MAP in conscious SHRs was abolished by the selective opioid µ-receptor antagonist cyprodime, but not by naloxonazine, the µ₁-opioid receptor antagonist. However, cardiac output was not altered by loperamide in anesthetized SHRs. Moreover, loperamide-induced relaxation in isolated aortic rings precontracted with phenylephrine or vasopressin. This relaxation was abolished by cyprodime, but not by naloxonazine. Loperamide-induced relaxation was also attenuated by glibenclamide, an ATP-sensitive potassium (K_ATP) channel blocker. Additionally, vasodilatation by loperamide was reduced by an inhibitor of protein kinase A (PKA) and enhanced by an inhibitor of phosphodiesterases. Conclusion: We suggest that loperamide can lower MAP in SHRs via µ₂-opioid receptor-dependent cAMP-PKA pathway that induces vascular relaxation by opening K_ATP channels.

BK channel β1 subunits regulate airway contraction secondary to M2 muscarinic acetylcholine receptor mediated depolarization

The large conductance calcium- and voltage-activated potassium channel (BK channel) and its smooth muscle-specific β1 subunit regulate excitation–contraction coupling in many types of smooth muscle cells. However, the relative contribution of BK channels to control of M2- or M3-muscarinic acetylcholine receptor mediated airway smooth muscle contraction is poorly understood. Previously, we showed that knockout of the BK channel β1 subunit enhances cholinergic-evoked trachea contractions. Here, we demonstrate that the enhanced contraction of the BK β1 knockout can be ascribed to a defect in BK channel opposition of M2 receptor-mediated contractions. Indeed, the enhanced contraction of β1 knockout is eliminated by specific M2 receptor antagonism. The role of BK β1 to oppose M2 signalling is evidenced by a greater than fourfold increase in the contribution of L-type voltage-dependent calcium channels to contraction that otherwise does not occur with M2 antagonist or with β1 containing BK channels. The mechanism through which BK channels oppose M2-mediated recruitment of calcium channels is through a negative shift in resting voltage that offsets, rather than directly opposes, M2-mediated depolarization. The negative shift in resting voltage is reduced to similar extents by BK β1 knockout or by paxilline block of BK channels. Normalization of β1 knockout baseline voltage with low external potassium eliminated the enhanced M2-receptor mediated contraction. In summary, these findings indicate that an important function of BK/β1 channels is to oppose cholinergic M2 receptor-mediated depolarization and activation of calcium channels by restricting excitation–contraction coupling to more negative voltage ranges.

Impulsive action induced by amphetamine, cocaine and MK801 is reduced by 5-HT2C receptor stimulation and 5-HT2A receptor blockade

Previous work has shown that 5-HT2C receptor agonists and 5-HT2A receptor antagonists reduce impulsive action, as well as the locomotor stimulant effect of psychomotor stimulants. Since psychomotor stimulants also increase impulsive action we examined the effects of the 5-HT2C receptor agonist Ro60-0175, and the 5-HT2A receptor antagonist M100907 on impulsive action induced by amphetamine, cocaine and the NMDA receptor antagonist MK801 (dizocilpine). Impulsive action was measured in adult male Long-Evans rats as premature responding in the 5-choice serial reaction time (5-CSRT) test. Initially, we determined that amphetamine (0.3 mg/kg), cocaine (15 mg/kg) and MK801 (0.03 mg/kg) induced comparable premature response rates of approximately 50–70 per session, compared to 10–15 responses under baseline conditions. Each drug and its vehicle were then tested in combination with Ro60-0175 (0.1 and 0.6 mg/kg) or its vehicle, or M100907 (0.5 mg/kg) or its vehicle. At 0.1 mg/kg Ro60-0175 did not modify the effects of amphetamine, cocaine or MK801. In contrast, the 0.6 mg/kg dose reduced premature responses induced by amphetamine, cocaine and MK801. M100907 also reduced premature responding induced by all three of these drugs. In general, treatment with Ro60-0175 or M100907 by itself did not consistently alter any of the other aspects of task performance in the 5-CSRT test including number of trials completed, and accuracy of responding. These data show that activation of 5-HT2C receptors and blockade of 5-HT2A receptors have seemingly similar functional effects on a measure of impulsive action.

Galanin receptor/Neuropeptide Y receptor interactions in the dorsal raphe nucleus of the rat

The aim of this study was to evaluate by quantitative receptor autoradiography the interactions between Neuropeptide Y Y1 (NPY Y1) and Galanin (GAL) receptors in the dorsal raphe nucleus (DRN) where both GAL receptors and NPY Y1 receptors exist. The ability of the GAL receptor antagonist M35 to block the GAL action was also evaluated. Double immunocytochemical staining of 5-hydroxytryptmine and c-Fos and stereology techniques were used to study the specific cell activation in the DRN after the intracerebroventricular coinjections of GAL and the NPY Y1/Y5 agonist [ 125I] Leu 31,Pro 34PYY. GAL (0.3 nM) decreases [ 125I] Leu 31,Pro 34PYY binding in the DRN by 48% ( p < 0.01) as shown by quantitative receptor autoradiography. This effect was reversed with the GAL receptor antagonist M35. Intracerebroventricular coinjections of NPY Y1/Y5 agonist and GAL reduced the c-Fos expression in the serotoninergic cells induced by the NPY Y1/Y5 agonist in DRN. These results indicate the existence of antagonistic interactions between GAL receptors and NPY Y1 receptors in the DRN that may be of relevance in mood disorders.

Galanin Receptor Antagonist M35 but Not M40 or C7 Ameliorates Cerulein-Induced Acute Pancreatitis in Mice

Background/Aims: We compared the galanin antagonists C7, M35, M40 and galantide, for their ability to ameliorate acute pancreatitis (AP). Methods: Galanin antagonists were co-administered with 7 hourly cerulein injections used to induce AP. Plasma amylase and lipase activities were measured as indices of AP, and pancreata were harvested at 12 h for histological examination and estimation of myeloperoxidase (MPO) activity. Results: Treatment with galantide, M35 and C7 ameliorated the AP-induced plasma hyperenzymemia by 40–75%. Administration of M40 did not significantly alter plasma hyperenzymemia. Galantide, M35 and M40 significantly reduced the pancreatic MPO activity by 65–80%, whereas C7 increased MPO activity. Galantide and M35 but not C7 or M40 treatment significantly reduced the AP-induced necrosis score by 30–50% compared to the AP alone group. C7 alone increased plasma lipase activity and the pancreatic necrosis score compared with saline treatment alone, whereas the other antagonists were without effect. Conclusion: Galantide and M35 ameliorated the severity of AP, but M40 and C7 had mixed effects. Complex galanin pathways may be involved in cerulein-induced AP. M35 and galantide are potential therapeutic peptides for the treatment of AP and further evaluation should be considered.

β-Endorphin inhibits phagocytic activity of lizard splenic phagocytes through μ receptor-coupled adenylate cyclase-protein kinase A signaling pathway

The receptor-coupled intracellular signaling mechanism of endogenous opioid peptide β-endorphin (β-end) is explored for the first time in ectothermic vertebrates using wall lizard as a model. β-End inhibited the percentage phagocytosis and phagocytic index of lizard splenic phagocytes in a dose-dependent manner. The inhibitory effect of β-end on phagocytosis was completely antagonized by non-selective opioid receptor antagonist naltrexone and also by selective μ-receptor antagonist CTAP. However, selective antagonists for other opioid receptors like NTI for δ-receptor and NorBNI for κ-receptor did not alter the effect of β-end on phagocytosis. This suggests that β-end mediated its inhibitory effect on phagocytic activity of splenic phagocytes exclusively through μ opioid receptors. The μ opioid receptor-coupled downstream signaling cascade was subsequently explored using inhibitors of adenylate cyclase (SQ 22536) and protein kinase A (H-89). Both SQ 22536 and H-89 abolished the inhibitory effect of β-end on phagocytosis in a concentration-related manner. Implication of cAMP as second messenger was corroborated by cAMP assay where an increase in intracellular cAMP level was observed in response to β-end treatment. It can be concluded that β-end downregulated the phagocytic activity of lizard splenic phagocytes through μ opioid receptor-coupled adenylate cyclase-cAMP-protein kinase A pathway.