Contribution of the M3 muscarinic receptors to the vasodilator response to acetylcholine in the human forearm vascular bed

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

• Available evidence suggests that the M₃ receptor on endothelial cells is responsible for acetylcholine (Ach)-dependent vasodilatation.
• Data from human studies only provide indirect evidence for this, and results are more difficult to interpret.

WHAT THIS STUDY ADDS

• This study used the new M₃ receptor antagonist darifenacin as a pharmacological ‘tool’ to investigate the role of M₃ receptor in the human forearm circulation.
• It provides evidence for a major role for the M₃ receptors in ACh-dependent vasodilatation in the forearm vascular bed.

AIMS

Acetylcholine (ACh) is a muscarinic agonist that causes receptor-mediated, endothelium-dependent vasodilatation in the forearm vasculature. Previous indirect evidence suggests this effect may be mediated by muscarinic M₃ receptors. Darifenacin is a recently developed antimuscarinic drug with greater M₃ selectivity, and our main objective was to investigate whether darifenacin affects dose-dependent vasodilatation to ACh in the forearm circulation.

METHODS

Healthy subjects were enrolled in two studies designed to assess the effects of atropine and darifenacin on the forearm blood flow (FBF) response to ACh.

RESULTS

In both studies ACh caused similar dose-dependent vasoditation in the forearm vasculature. In study I (5 subjects), the FBF response to ACh was largely attenuated by pretreatment with the nonselective muscarinic antagonist atropine. In study II (10 subjects), oral administration of darifenacin 15 mg for 1 week significantly reduced the FBF dose-dependent response to ACh 20 µg min⁻¹ {mean difference from placebo 5.8 [95% confidence interval (CI) 3.1, 8.7] ml min⁻¹ per 100 ml of forearm volume, P < 0.001} and to ACh 60 µg min⁻¹[mean difference from placebo 5.9 (95% CI 3.1, 8.7) ml min⁻¹ per 100 ml of forearm volume, P < 0.001]. After darifenacin, the AUC of change in FBF from baseline was reduced by almost 50% compared with placebo.

CONCLUSIONS

These results suggest that, in the forearm vasculature, muscarinic M₃ receptors play a major role in ACh-induced endothelium-mediated vasodilatation.     

Involvement of decreased muscarinic receptor function in prepulse inhibition deficits in mice reared in social isolation

BACKGROUND AND PURPOSE

We have previously reported that galantamine, a weak acetylcholinesterase inhibitor, improves prepulse inhibition (PPI) deficits in mice reared in social isolation. ACh receptors are involved in the underlying mechanism of PPI, but whether rearing in social isolation causes dysfunction of the cholinergic system is unknown. In this study, we examined the involvement of muscarinic receptors in the improvement of PPI deficits induced by galantamine, and whether the cholinergic system is altered in mice reared in isolation.

EXPERIMENTAL APPROACH

Three-week-old male ddY mice were housed in isolated cages for 6 weeks before the initiation of experiments to create PPI deficits. Cholinergic functions were determined by measuring the behavioural and neurochemical responses to nicotinic and muscarinic receptor agonists.

KEY RESULTS

The improvement by galantamine of social isolation-induced PPI deficits was blocked by scopolamine, a non-selective muscarinic antagonist, and telenzepine, a preferential M₁ receptor antagonist. Activation of M₁ receptors improved social isolation-induced PPI deficits. Social isolation did not affect choline acetyltransferase and acetylcholinesterase activities in the prefrontal cortex and hippocampus, but it reduced the locomotor-suppressive response to muscarinic agonist oxotremorine, but not to nicotine. The isolation also attenuated the M₁ receptor agonist N-desmethylclozapine-induced increase in prefrontal dopamine release.

CONCLUSIONS AND IMPLICATIONS

Galantamine improves PPI deficits of mice reared in social isolation via activation of M₁ receptors. Social isolation reduces the muscarinic, especially M₁, receptor function and this is involved in PPI deficits.     

Etanercept prevents airway hyperresponsiveness by protecting neuronal M2 muscarinic receptors in antigen-challenged guinea pigs

Background and purpose

Increased tumour necrosis factor-α (TNF-α) is associated with airway hyperreactivity in antigen-challenged animals. In human asthmatics, TNF-α is increased and blocking it prevents airway hyperreactivity in some asthmatic patients. However, the mechanisms by which TNF-α mediates hyperreactivity are unknown. Airway hyperreactivity can be caused by dysfunction of neuronal M₂ muscarinic receptors that normally limit acetylcholine release from parasympathetic nerves. Here we test whether blocking TNF-α receptors with etanercept prevents M₂ receptor dysfunction and airway hyperreactivity in antigen-challenged guinea pigs.

Experimental approach

Ovalbumin-sensitized guinea pigs were challenged by inhalation of antigen. Some animals received etanercept (3 mg kg⁻¹ i.p.) 3 h before challenge. 24 h after challenge, airway hyperreactivity and M₂ receptor function were tested. Inflammatory cells in bronchoalveolar lavage, blood and lung were counted. TNF-α and its receptors were detected by real-time RT-PCR and immunocytochemistry in parasympathetic nerves from humans and guinea pigs and in human neuroblastoma cells.

Key results

Antigen-challenged animals were hyperreactive to vagal stimulation and neuronal M₂ receptors were dysfunctional. Both M₂ receptor dysfunction and airway hyperreactivity were prevented by etanercept. Etanercept reduced eosinophils around airway nerves, and in blood, bronchoalveolar lavage and airway smooth muscle. Also, TNF-α decreased M₂ receptor mRNA in human and guinea pig parasympathetic neurons.

Conclusions and implications

Tumour necrosis factor-α may contribute to M₂ receptor dysfunction and airway hyperreactivity directly by decreasing receptor expression and indirectly by promoting recruitment of eosinophils, containing major basic protein, an M₂ antagonist. This suggests that etanercept may be beneficial in treatment of allergic asthma.     

Effects of excitatory and inhibitory neurotransmission on motor patterns of human sigmoid colon in vitro

Background and purpose:

To characterize the in vitro motor patterns and the neurotransmitters released by enteric motor neurons (EMNs) in the human sigmoid colon.

Experimental approach:

Sigmoid circular strips were studied in organ baths. EMNs were stimulated by electrical field stimulation (EFS) and through nicotinic ACh receptors.

Key results:

Strips developed weak spontaneous rhythmic contractions (3.67±0.49 g, 2.54±0.15 min) unaffected by the neurotoxin tetrodotoxin (TTX; 1 μM). EFS induced strong contractions during (on, 56%) or after electrical stimulus (off, 44%), both abolished by TTX. Nicotine (1–100 μM) inhibited spontaneous contractions. Latency of off-contractions and nicotine responses were reduced by N^G-nitro-L-arginine (1 mM) and blocked after further addition of apamin (1 μM) or the P2Y₁ receptor antagonist MRS 2179 (10 μM) and were unaffected by the P2X antagonist NF279 (10 μM) or α-chymotrypsin (10 U mL⁻¹). Amplitude of on- and off-contractions was reduced by atropine (1 μM) and the selective NK₂ receptor antagonist Bz-Ala-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH₂ (1 μM). MRS 2179 reduced the amplitude of EFS on- and off-contractions without altering direct muscular contractions induced by ACh (1 nM–1 mM) or substance P (1 nM–10 μM).

Conclusions and implications:

Latency of EFS-induced off-contractions and inhibition of spontaneous motility by nicotine are caused by stimulation of inhibitory EMNs coreleasing NO and a purine acting at muscular P2Y₁ receptors through apamin-sensitive K⁺ channels. EFS-induced on- and off-contractions are caused by stimulation of excitatory EMNs coreleasing ACh and tachykinins acting on muscular muscarinic and NK₂ receptors. Prejunctional P2Y₁ receptors might modulate the activity of excitatory EMNs. P2Y₁ and NK₂ receptors might be therapeutic targets for colonic motor disorders.     

Identification of muscarinic receptor subtypes involved in catecholamine secretion in adrenal medullary chromaffin cells by genetic deletion

Background and Purpose

Activation of muscarinic receptors results in catecholamine secretion in adrenal chromaffin cells in many mammals, and muscarinic receptors partly mediate synaptic transmission from the splanchnic nerve, at least in guinea pigs. To elucidate the physiological functions of muscarinic receptors in chromaffin cells, it is necessary to identify the muscarinic receptor subtypes involved in excitation.

Experimental Approach

To identify muscarinic receptors, pharmacological tools and strains of mice where one or several muscarinic receptor subtypes were genetically deleted were used. Cellular responses to muscarinic stimulation in isolated chromaffin cells were studied with the patch clamp technique and amperometry.

Key Results

Muscarinic M₁, M₄ and M₅ receptors were immunologically detected in mouse chromaffin cells, and these receptors disappeared after the appropriate gene deletion. Mouse cells secreted catecholamines in response to muscarinic agonists, angiotensin II and a decrease in external pH. Genetic deletion of M₁, but not M₃, M₄ or M₅, receptors in mice abolished secretion in response to muscarine, but not to other stimuli. The muscarine-induced secretion was suppressed by MT7, a snake peptide toxin specific for M₁ receptors. Similarly, muscarine failed to induce an inward current in the presence of MT7 in mouse and rat chromaffin cells. The binding affinity of VU0255035 for the inhibition of muscarine-induced currents agreed with that for the M₁ receptor.

Conclusions and Implications

Based upon the effects of genetic deletion of muscarinic receptors and MT7, it is concluded that the M₁ receptor alone is responsible for muscarine-induced catecholamine secretion.     

Investigating the interaction of McN-A-343 with the M1 muscarinic receptor using its nitrogen mustard derivative and ACh mustard

Background and purpose:

We investigated how McN-A-343 inhibited the alkylation of the M₁ muscarinic receptor by its nitrogen mustard derivative and that of ACh to identify whether it interacts allosterically or orthosterically.

Experimental approach:

We incubated the M₁ muscarinic receptor expressed in Chinese hamster ovary cells with ACh mustard for various periods of time in the presence of McN-A-343 or known allosteric and orthosteric ligands. After stopping the reaction and removing unreacted ligands, unalkylated receptors were measured using [³H]N-methylscopolamine. Analogous experiments were done using a nitrogen mustard analog of McN-A-343. Affinity constants, cooperativity values for allosteric interactions and rate constants for receptor alkylation were estimated using a mathematical model.

Key results:

The kinetics of receptor alkylation by the nitrogen mustard derivatives of ACh and McN-A-343 were consistent with a two-step model in which the aziridinium ion rapidly forms a reversible receptor complex, which converts to a covalent complex at a slower rate. The inhibition of receptor alkylation by acetycholine, N-methylscopolamine and McN-A-343 was consistent with competitive inhibition, whereas that caused by gallamine was consistent with allosterism. Affinity constants estimated from alkylation kinetics agreed with those measured by displacement of [³H]N-methylscopolamine binding.

Conclusions and implications:

Our results suggest that McN-A-343 and its nitrogen mustard derivative interact competitively with ACh and N-methylscopolamine at the orthosteric site on the M₁ muscarinic receptor. Measuring how drugs modulate the kinetics of receptor alkylation by an irreversible ligand is a powerful approach for distinguishing between negative allosteric modulators and competitive inhibitors.     

Central muscarinic receptor subtypes involved in pilocarpine-induced salivation, hypertension and water intake

Background and purpose:

Recent evidence has suggested that pilocarpine (ACh receptor agonist) injected peripherally may act centrally producing salivation and hypertension. In this study, we investigated the effects of specific M₁ (pirenzepine), M₂/M₄ (methoctramine), M₁/M₃ (4-DAMP) and M₄ (tropicamide) muscarinic receptor subtype antagonists injected into the lateral cerebral ventricle (LV) on salivation, water intake and pressor responses to peripheral pilocarpine.

Experimental approach:

Male Holtzman rats with stainless steel cannulae implanted in the LV were used. Salivation was measured in rats anaesthetized with ketamine (100 mg per kg body weight) and arterial pressure was recorded in unanaesthetized rats.

Key results:

Salivation induced by i.p. pilocarpine (4 μmol per kg body weight) was reduced only by 4-DAMP (25–250 nmol) injected into the LV, not by pirenzepine, methoctramine or tropicamide at the dose of 500 nmol. Pirenzepine (0.1 and 1 nmol) and 4-DAMP (5 and 10 nmol) injected into the LV reduced i.p. pilocarpine-induced water intake, whereas metoctramine (50 nmol) produced nonspecific effects on ingestive behaviours. Injection of pirenzepine (100 nmol) or 4-DAMP (25 and 50 nmol) into the LV reduced i.v. pilocarpine-induced pressor responses. Tropicamide (500 nmol) injected into the LV had no effect on pilocarpine-induced salivation, pressor responses or water intake.

Conclusions and implications:

The results suggest that central M₃ receptors are involved in peripheral pilocarpine-induced salivation and M₁ receptors in water intake and pressor responses. The involvement of M₃ receptors in water intake and pressor responses is not clear because 4-DAMP blocks both M₁ and M₃ receptors.     

Galantamine improves apomorphine-induced deficits in prepulse inhibition via muscarinic ACh receptors in mice

Background and purpose

Galantamine, a weak acetylcholine esterase (AChE) inhibitor and allosteric potentiator of nicotinic ACh receptors (nAChRs), improves apomorphine-induced deficits in prepulse inhibition (PPI), sensory information-processing deficits, via a nAChR-independent mechanism. The present study examined the role of muscarinic ACh receptors (mAChRs) in the effect of galantamine, and studied the mechanism of galantamine-induced increases in prefrontal ACh levels in mice.

Experimental approach

Apomorphine (1 mg kg⁻¹) was administered to male ddY mice (9–10 weeks old) to create a PPI deficit model. Extracellular ACh concentrations in the prefrontal cortex were measured by in vivo microdialysis.

Key results

Galantamine- and donepezil-mediated improvements in apomorphine-induced PPI deficits were blocked by the preferential M₁ mAChR antagonist telenzepine. The mAChR agonist oxotremorine also improved apomorphine-induced PPI deficits. Galantamine, like donepezil, increased extracellular ACh concentrations in the prefrontal cortex. Galantamine-induced increases in prefrontal ACh levels were partially blocked by the dopamine D₁ receptor antagonist {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390, but not by antagonists of mAChRs (telenzepine) and nAChRs (mecamylamine). Galantamine increased dopamine, but not 5-HT, release in the prefrontal cortex.

Conclusions and implications

Galantamine improves apomorphine-induced PPI deficits by stimulating mAChRs through increasing brain ACh levels via a dopamine D₁ receptor-dependent mechanism and AChE inhibition.     

Effect of age and COX-2-derived prostanoids on the progression of adult vascular dysfunction in the offspring of diabetic rats

BACKGROUND AND PURPOSE

The present study was designed to determine how diabetes in pregnancy affects vascular function in their offspring, the influence of age and whether COX activation is involved in this effect.

EXPERIMENTAL APPROACH

Relaxation responses to ACh were analysed in mesenteric resistance arteries from the offspring of control rats (O-CR) and those of diabetic rats (O-DR) at 3, 6 and 12 months of age. TxB₂, PGE₂ and PGF_2α release were determined by enzyme immunoassay. COX-1 and COX-2 expression were measured by Western blot analysis.

KEY RESULTS

O-DR developed hypertension from 6 months of age compared with O-CR. In O-DR, relaxation responses to ACh were impaired in all ages studied and were restored by COX-2 inhibition. TP receptor blockade (SQ29548) restored ACh relaxation in arteries from 3-month-old O-DR while TP and EP receptor blockade (SQ29548 + AH6809) was required to restore it in 6-month-old O-DR. In 12-month-old O-DR, ACh relaxation was restored when TP, EP and FP receptors were blocked (SQ29548 + AH6809 + AL8810). ACh-stimulated TxB₂ was higher in all O-DR. ACh-stimulated PGE₂ release was increased in arteries from 6- and 12-month-old O-DR, whereas PGF_2α was increased only in 12-month-old O-DR. COX-2, but not COX-1, expression was higher in O-DR than O-CR.

CONCLUSIONS AND IMPLICATIONS

The results indicate an age-dependent up-regulation of COX-2 coupled to an enhanced formation of vasoconstrictor prostanoids in resistance arteries from O-DR. This effect plays a key role in the pathogenesis of endothelial dysfunction, which in turn could contribute to the progression of vascular dysfunction in these rats.     

Impact of species variability and 'probe-dependence' on the detection and in vivo validation of allosteric modulation at the M4 muscarinic acetylcholine receptor

BACKGROUND AND PURPOSE

We recently characterized LY2033298 as a novel allosteric modulator and agonist at M₄ muscarinic acetylcholine receptors (mAChRs). Evidence also suggested a difference in the potency of LY2033298 at rodent relative to human M₄ mAChRs. The current study investigated the basis for the species difference of this modulator and used this knowledge to rationalize its in vivo actions.

EXPERIMENTAL APPROACH

LY2033298 was investigated in vitro in CHO cells stably expressing human or mouse M₄ mAChRs, using assays of agonist-induced ERK1/2 or GSK-3α phosphorylation, [³⁵S]-GTPγS binding, or effects on equilibrium binding of [³H]-NMS and ACh. The in vivo actions of LY2033298 were investigated in a mouse model of amphetamine-induced locomotor activity. The function of LY2033298 was examined in combination with ACh, oxotremorine or xanomeline.

KEY RESULTS

LY2033298 had similar affinities for the human and mouse M₄ mAChRs. However, LY2033298 had a lower positive co-operativity with ACh at the mouse relative to the human M₄ mAChR. At the mouse M₄ mAChR, LY2033298 showed higher co-operativity with oxotremorine than with ACh or xanomeline. The different degrees of co-operativity between LY2033298 and each agonist at the mouse relative to the human M₄ mAChR necessitated the co-administration of LY2033298 with oxotremorine in order to show in vivo efficacy of LY2033298.

CONCLUSIONS AND IMPLICATIONS

These results provide evidence for species variability when comparing the allosteric interaction between LY2033298 and ACh at the M₄ mAChR, and also highlight how the interaction between LY2033298 and different orthosteric ligands is subject to ‘probe dependence’. This has implications for the validation of allosteric modulator actions in vivo.     

AE9C90CB: a novel, bladder-selective muscarinic receptor antagonist for the treatment of overactive bladder

Background and purpose:

AE9C90CB (N- [(1R, 5S, 6R)-3-azabicyclo [3.1.0] hex-6-ylmethyl]-2-hydroxy-N-methyl-2, 2-diphenylacetamide), a novel muscarinic receptor antagonist, was synthesized for the treatment of overactive bladder. Here we describe the in vitro and in vivo profiles of AE9C90CB for action in bladder over salivary gland and compare it with four agents already in clinical use (tolterodine, oxybutynin, solifenacin and darifenacin).

Experimental approach:

Radioligand binding assay and isolated tissue-based functional assay were used to evaluate affinity, potency, and receptor subtype selectivity of compounds. Inhibition of carbachol-induced increase in intravesicular pressure and salivary secretion were measured in anaesthetized rabbits to assess the functional selectivity.

Key results:

In vitro radioligand binding study using human recombinant muscarinic receptors showed that AE9C90CB had greater affinity for M₃ muscarinic receptors with pKi of 9.90 ± 0.11 and was 20-fold more selective for M₃ than for M₂ muscarinic receptors. AE9C90CB exhibited an unsurmountable antagonism on rat bladder strips (pK_B, 9.13 ± 0.12). In anaesthetized rabbits after intravenous administration, AE9C90CB dose dependently inhibited carbachol-induced increase in intravesicular pressure and salivary secretion, and exhibited functional selectivity for urinary bladder over salivary gland which was ninefold better than that of oxybutynin.

Conclusions and implications:

We have identified AE9C90CB, a compound exhibiting moderate selectivity for M₃ over M₂ receptors but greater selectivity for urinary bladder over salivary gland than oxybutynin, tolterodine, solifenacin and darifenacin. Therefore, AE9C90CB may be a promising compound for the treatment of overactive bladder with reduced potential to cause dry mouth than currently available antimuscarinic drugs.     

Divergence of allosteric effects of rapacuronium on binding and function of muscarinic receptors

Background

Many neuromuscular blockers act as negative allosteric modulators of muscarinic acetylcholine receptors by decreasing affinity and potency of acetylcholine. The neuromuscular blocker rapacuronium has been shown to have facilitatory effects at muscarinic receptors leading to bronchospasm. We examined the influence of rapacuronium on acetylcholine (ACh) binding to and activation of individual subtypes of muscarinic receptors expressed in Chinese hamster ovary cells to determine its receptor selectivity.

Results

At equilibrium rapacuronium bound to all subtypes of muscarinic receptors with micromolar affinity (2.7-17 μM) and displayed negative cooperativity with both high- and low-affinity ACh binding states. Rapacuronium accelerated [³H]ACh association with and dissociation from odd-numbered receptor subtypes. With respect to [³⁵S]GTPγS binding rapacuronium alone behaved as an inverse agonist at all subtypes. Rapacuronium concentration-dependently decreased the potency of ACh-induced [³⁵S]GTPγS binding at M₂ and M₄ receptors. In contrast, 0.1 μM rapacuronium significantly increased ACh potency at M₁, M₃, and M₅ receptors. Kinetic measurements at M₃ receptors showed acceleration of the rate of ACh-induced [³⁵S]GTPγS binding by rapacuronium.

Conclusions

Our data demonstrate a novel dichotomy in rapacuronium effects at odd-numbered muscarinic receptors. Rapacuronium accelerates the rate of ACh binding but decreases its affinity under equilibrium conditions. This results in potentiation of receptor activation at low concentrations of rapacuronium (1 μM) but not at high concentrations (10 μM). These observations highlight the relevance and necessity of performing physiological tests under non-equilibrium conditions in evaluating the functional effects of allosteric modulators at muscarinic receptors. They also provide molecular basis for potentiating M₃ receptor-mediated bronchoconstriction.     

Effects of tachykinin NK1 receptor antagonists on vagal hyperreactivity and neuronal M2 muscarinic receptor function in antigen challenged guinea-pigs

1. The role of tachykinin NK₁ receptors in the recruitment of eosinophils to airway nerves, loss of inhibitory neuronal M₂ muscarinic receptor function and the development of vagal hyperreactivity was tested in antigen-challenged guinea-pigs.
2. In anaesthetized guinea-pigs, the muscarinic agonist, pilocarpine (1–100 μg kg⁻¹, i.v), inhibited vagally induced bronchoconstriction, in control, but not in antigen-challenged guinea-pigs 24 h after antigen challenge. This indicates normal function of neuronal M₂ muscarinic receptors in controls and loss of neuronal M₂ receptor function in challenged guinea-pigs. Pretreatment of sensitized guinea-pigs with the NK₁ receptor antagonists CP99994 (4 mg kg⁻¹, i.p.), SR140333 (1 mg kg⁻¹, s.c.) or CP96345 (15 mg kg⁻¹, i.p.) before antigen challenge, prevented M₂ receptor dysfunction.
3. Neither administration of the NK₁ antagonists after antigen challenge, nor pretreatment with an NK₂ receptor antagonist, MEN10376 (5 μmol kg⁻¹, i.p.), before antigen challenge, prevented M₂ receptor dysfunction.
4. Electrical stimulation of the vagus nerves caused a frequency-dependent (2–15 Hz, 10 V, 0.2 ms for 5 s) bronchoconstriction that was significantly increased following antigen challenge. Pretreatment with the NK₁ receptor antagonists CP99994 or SR140333 before challenge prevented this increase.
5. Histamine (1–20 nmol kg⁻¹, i.v.) caused a dose-dependent bronchoconstriction, which was vagally mediated, and was significantly increased in antigen challenged guinea-pigs compared to controls. Pretreatment of sensitized animals with CP99994 before challenge prevented the increase in histamine-induced reactivity.
6. Bronchoalveolar lavage and histological studies showed that after antigen challenge significant numbers of eosinophils accumulated in the airways and around airway nerves. This eosinophilia was not altered by pretreatment with the NK₁ receptor antagonist CP99994.
7. These data indicate that pretreatment of antigen-sensitized guinea-pigs with NK₁, but not with NK₂ receptor antagonists before antigen challenge prevented the development of hyperreactivity by protecting neuronal M₂ receptor function. NK₁ receptor antagonists do not inhibit eosinophil accumulation around airway nerves.

     

Magnesium sulfate enhances non-depolarizing muscle relaxant vecuronium action at adult muscle-type nicotinic acetylcholine receptor in vitro

Aim:

To investigate the effect of magnesium sulfate and its interaction with the non-depolarizing muscle relaxant vecuronium at adult muscle-type acetylcholine receptors in vitro.

Methods:

Adult muscle-type acetylcholine receptors were expressed in HEK293 cells. Drug-containing solution was applied via a gravity-driven perfusion system. The inward currents were activated by brief application of acetylcholine (ACh), and recorded using whole-cell voltage-clamp technique.

Results:

Magnesium sulfate (1–100 mmol/L) inhibited the inward currents induced ACh (10 μmol/L) in a concentration-dependent manner (IC₅₀=29.2 mmol/L). The inhibition of magnesium sulfate was non-competitive. In contrast, vecuronium produced a potent inhibition on the adult muscle-type acetylcholine receptor (IC₅₀=8.7 nmol/L) by competitive antagonism. Magnesium sulfate at the concentrations of 1, 3, and 6 mmol/L markedly enhanced the inhibition of vecuronium (10 nmol/L) on adult muscle-type acetylcholine receptors.

Conclusion:

Clinical enhancement of vecuronium-induced muscle relaxation by magnesium sulfate can be attributed partly to synergism between magnesium sulfate and non-depolarizing muscle relaxants at adult muscle-type acetylcholine receptors.     

Activation of ��-opioid receptors and block of KIR3 potassium channels and NMDA receptor conductance by l- and d-methadone in rat locus coeruleus

BACKGROUND AND PURPOSE

Methadone activates opioid receptors to increase a potassium conductance mediated by G-protein-coupled, inwardly rectifying, potassium (K_IR3) channels. Methadone also blocks K_IR3 channels and N-methyl-D-aspartic acid (NMDA) receptors. However, the concentration dependence and stereospecificity of receptor activation and channel blockade by methadone on single neurons has not been characterized.

EXPERIMENTAL APPROACH

Intracellular and whole-cell recording were made from locus coeruleus neurons in brain slices and the activation of µ-opioid receptors and blockade of K_IR3 and NMDA channels with l- and d-methadone was examined.

KEY RESULTS

The potency of l-methadone, measured by the amplitude of hyperpolarization was 16.5-fold higher than with d-methadone. A maximum hyperpolarization was caused by both enantiomers (∼30 mV); however, the maximum outward current measured with whole-cell voltage-clamp recording was smaller than the current induced by [Met]⁵enkephalin. The K_IR3 conductance induced by activation of α₂-adrenoceptors was decreased with high concentrations of l- and d-methadone (10–30 µM). In addition, methadone blocked the resting inward rectifying conductance (K_IR). Both l- and d-methadone blocked the NMDA receptor-dependent current. The block of NMDA receptor-dependent current was voltage-dependent suggesting that methadone acted as a channel blocker.

CONCLUSIONS AND IMPLICATIONS

Methadone activated µ-opioid receptors at low concentrations in a stereospecific manner. K_IR3 and NMDA receptor channel block was not stereospecific and required substantially higher concentrations. The separation in the concentration range suggests that the activation of µ-opioid receptors rather than the channel blocking properties mediate both the therapeutic and toxic actions of methadone.     

Functional, morphological and molecular characterization of bladder dysfunction in streptozotocin-induced diabetic mice: evidence of a role for L-type voltage-operated Ca2+ channels

BACKGROUND AND PURPOSE

Diabetic cystopathy is one of the most common and incapacitating complications of diabetes mellitus. This study aimed to evaluate the functional, structural and molecular alterations of detrusor smooth muscle (DSM) in streptozotocin-induced diabetic mice, focusing on the contribution of Ca²⁺ influx through L-type voltage-operated Ca²⁺ channels (L-VOCC).

EXPERIMENTAL APPROACH

Male C57BL/6 mice were injected with streptozotocin (125 mg·kg⁻¹). Four weeks later, contractile responses to carbachol, α,β-methylene ATP, KCl, extracellular Ca²⁺ and electrical-field stimulation were measured in urothelium-intact DSM strips. Cystometry and histomorphometry were performed, and mRNA expression for muscarinic M₂/M₃ receptors, purine P2X1 receptors and L-VOCC in the bladder was determined.

KEY RESULTS

Diabetic mice exhibited higher bladder capacity, frequency, non-void contractions and post-void pressure. Increased bladder weight, wall thickness, bladder volume and neural tissue were observed in diabetic bladders. Carbachol, α,β-methylene ATP, KCl, extracellular Ca²⁺ and electrical-field stimulation all produced greater DSM contractions in diabetic mice. The L-VOCC blocker nifedipine almost completely reversed the enhanced DSM contractions in bladders from diabetic animals. The Rho-kinase inhibitor Y27632 had no effect on the enhanced carbachol contractions in the diabetic group. Expression of mRNA for muscarinic M₃ receptors and L-VOCC were greater in the bladders of diabetic mice, whereas levels of M₂ and P2X1 receptors remained unchanged.

CONCLUSIONS AND IMPLICATIONS

Diabetic mice exhibit features of urinary bladder dysfunction, as characterized by overactive DSM and decreased voiding efficiency. Functional and molecular data suggest that overactive DSM in diabetes is the result of enhanced extracellular Ca²⁺ influx through L-VOCC.     

Negative cooperativity in binding of muscarinic receptor agonists and GDP as a measure of agonist efficacy

BACKGROUND AND PURPOSE

Conventional determination of agonist efficacy at G-protein coupled receptors is measured by stimulation of guanosine-5′-γ−thiotriphosphate (GTPγS) binding. We analysed the role of guanosine diphosphate (GDP) in the process of activation of the M₂ muscarinic acetylcholine receptor and provide evidence that negative cooperativity between agonist and GDP binding is an alternative measure of agonist efficacy.

EXPERIMENTAL APPROACH

Filtration and scintillation proximity assays measured equilibrium binding as well as binding kinetics of [³⁵S]GTPγS and [³H]GDP to a mixture of G-proteins as well as individual classes of G-proteins upon binding of structurally different agonists to the M₂ muscarinic acetylcholine receptor.

KEY RESULTS

Agonists displayed biphasic competition curves with the antagonist [³H]-N-methylscopolamine. GTPγS (1 µM) changed the competition curves to monophasic with low affinity and 50 µM GDP produced a similar effect. Depletion of membrane-bound GDP increased the proportion of agonist high-affinity sites. Carbachol accelerated the dissociation of [³H]GDP from membranes. The inverse agonist N-methylscopolamine slowed GDP dissociation and GTPγS binding without changing affinity for GDP. Carbachol affected both GDP association with and dissociation from G_i/o G-proteins but only its dissociation from G_s/olf G-proteins.

CONCLUSIONS AND IMPLICATIONS

These findings suggest the existence of a low-affinity agonist-receptor conformation complexed with GDP-liganded G-protein. Also the negative cooperativity between GDP and agonist binding at the receptor/G-protein complex determines agonist efficacy. GDP binding reveals differences in action of agonists versus inverse agonists as well as differences in activation of G_i/o versus G_s/olf G-proteins that are not identified by conventional GTPγS binding.     

Biomechanical properties and innervation of the female caveolin-1-deficient detrusor

BACKGROUND AND PURPOSE

Caveolin-1-deficiency is associated with substantial urogenital alterations. Here, a mechanical, histological and biochemical characterization of female detrusors from wild-type and caveolin-1-deficient (KO) mice was made to increase the understanding of detrusor changes caused by lack of caveolae.

EXPERIMENTAL APPROACH

Length–tension relationships were generated, and we recorded responses to electrical field stimulation, the muscarinic receptor agonist carbachol and the purinoceptor agonist ATP. Tyrosine nitration and the contents of caveolin-1, cavin-1, muscarinic M₃ receptors, phospholipase C_β1, muscle-specific kinase (MuSK) and L-type Ca²⁺ channels were determined by immunoblotting. Innervation was assessed by immunohistochemistry.

KEY RESULTS

Bladder to body weight ratio was not changed, nor was there any change in the optimum circumference for force development. Depolarization- and ATP-induced stress was reduced, as was carbachol-induced stress between 0.1 and 3 µM, but the supramaximal relative (% K⁺) response to carbachol was increased, as was M₃ expression. The scopolamine-sensitive component of the electrical field stimulation response was impaired, and yet bladder nerves contained little caveolin-1. The density of cholinergic nerves was unchanged, whereas CART- and CGRP-positive nerves were reduced. Immunoblotting revealed loss of MuSK.

CONCLUSIONS AND IMPLICATIONS

Ablation of caveolae in the female detrusor leads to generalized impairment of contractility, ruling out prostate hypertrophy as a contributing factor. Cholinergic neuroeffector transmission is impaired without conspicuous changes in the density of cholinergic nerves or morphology of their terminals, but correlating with reduced expression of MuSK.     

Conditional involvement of muscarinic M1 receptors in vagally mediated contraction of guinea-pig bronchi

The involvement of ganglionic muscarinic M₁ receptors in vagally induced bronchoconstriction in guinea-pig airways is controversial. Therefore, we studied the effects of the M₁-selective muscarinic receptor antagonist pirenzepine on vagus nerve (VNS, preganglionic) and electrical field stimulation (EFS, postganglionic)-induced contractions of the guinea-pig main bronchus under various experimental conditions.Using identical stimulation parameters for VNS and EFS (8V, 30 Hz, 0.5 ms, 5s every min), the amplitude of the VNS-induced twitch contractions was 30.4% of the EFS-induced responses, and pirenzepine showed 2.3-fold selectivity (pIC₅₀-values 6.45 and 6.09, respectively) to inhibit vagally induced contractions. With the stimulation frequency for EFS lowered to match contraction levels obtained using VNS, pirenzepine was equipotent to inhibit both types of response at M₃ receptor-selective concentrations, suggesting that M₁ receptors are not involved. By contrast, when the stimulation episode was prolonged until plateau contraction (10–20 s), in the presence of the nicotinic antagonist hexamethomum (5 M), the M₂ receptor antagonist AQ-RA 741 (0.1 M) and the -adrenoceptor antagonist timolol (1 M), and again using matched VNS- and EFS-induced contraction levels, pirenzepine inhibited nerve stimulation-evoked responses in a biphasic manner, yielding (pIC₅₀-values of 8.12 indicative of M₁ receptor blockade) and 6.43 (indicative of M₃ receptor blockade) for the first and second phase, respectively, while postganglionic stimulation showed a purely monophasic inhibition (pIC₅₀ = 6.32).These results show that facilitatory muscarinic M₁ receptors are involved in vagally mediated contraction of guinea-pig bronchi, under conditions of elevated neurotransmission and partial nicotinic receptor blockade.