μM Atropine Research Articles

Opposite Contractile Effects of Amphetamine-Related Hallucinogenic Drugs in the Isolated Human Atrium.

The present study examined three hallucinogenic amphetamine derivatives, namely, 2,5-dimethoxy-4-iodoamphetamine (DOI) as well as 2,5-dimethoxy-4-methylamphetamine (DOM) and 4-methylmethcathinone (mephedrone). The objective of this study was to test the hypothesis that DOI, DOM, and mephedrone would increase the contractile force in isolated human atrial preparations in a manner similar to amphetamine. To this end, we measured contractile force under isometric conditions in electrically stimulated (1 Hz) human atrial preparations obtained during open surgery. DOI and DOM alone or in the presence of isoprenaline reduced the contractile force concentration-dependently in human atrial preparations. These negative inotropic effects of DOM and DOI were not attenuated by 10 µM atropine. However, mephedrone increased the contractile force in human atrial preparations in a concentration- and time-dependent manner. Furthermore, these effects were attenuated by the subsequent addition of 10 µM propranolol or pretreatment with 10 µM cocaine in the organ bath. Therefore, it can be concluded that amphetamine derivatives may exert opposing effects on cardiac contractile force. The precise mechanism by which DOI and DOM exert their negative inotropic effects remains unknown at present. The cardiac effects of mephedrone are probably due to the release of cardiac noradrenaline.

Muscarinic Receptors as Targets for Metronomic Therapy in Ovarian Cancer

Aim: In this study, the effects of muscarinic acetylcholine receptor (mAChR) agonist carbachol on the proliferation of cisplatin-resistant (A2780cis) and cisplatin-free (SKOV-3) ovarian cancer cell line were for the first time investigated to further evaluate the potential therapeutic effect of metronomic chemotherapy.
 Material and Methods: The inhibitory effect of carbachol on cell proliferation was detected using the xCELLigence Real-Time Cell Analyzer (RTCA) dual plate (DP) system. A preliminary study was conducted to determine the dose of carbachol 100 µM, cisplatin 1 µM, and two combination studies were carried out with 100 µM carbachol + cisplatin 1 µM and 100 µM carbachol + 10 µM atropine, over cancer cells without drugs was used as the control group. The cell proliferation curve was monitored for 96 hours. The cell index value of inhibition in cell proliferation was automatically measured every hour for each well using RTCA 1.2.1 software.
 Results: Co-administration of carbachol with cisplatin caused a decrease in cell number in both A2780cis and SKOV-3 cell lines in a time-dependent manner (p

P014 Impact of experimental ileitis and Toll-Like Receptor 4 signaling on enteric inhibitory neurotransmission

Abstract Background Alterations in Toll-like receptor 4 (TLR4) expression and changes in enteric neurotransmissions influence the onset and severity of inflammatory bowel disease (IBD). We aimed to assess the interaction between the enteric inhibitory pathways and TLR4 signaling in a mouse model of dinitrobenzene sulfonic acid (DNBS)-induced small intestine inflammation. Methods Male TLR4-/- mice (9±2 weeks old) were intrarectally treated with 2.5% DNBS. Pro-inflammatory cytokines (IL-6 and TNF-α) were measured in ileal samples. Changes in ileal muscle tension were recorded following exposure to: i) 10-Hz electric field stimulation in non-adrenergic-non-cholinergic (NANC) conditions (1 μM guanethidine + 1 μM atropine) with or without 10 µM 1400W (a inducible nitric oxide synthase (iNOS) inhibitor), or 0.1 µM Nω-nitro-L-arginine methyl ester (L-NAME; a pan-NOS inhibitor); ii) 30 μM dopamine or 30 μM SKF38393 (a dopamine 1 receptor (D1R) agonist) or bromocriptine (a D2R agonist). Immunoreactivity of neuronal (HuC/D) and glial (S100β) markers as well as nitrergic chemical coding (i.e. nNOS and iNOS) were determined in longitudinal muscle-myenteric plexus whole-mount preparations (LMMPs) by confocal microscopy. Results No changes in IL-6 and TNF-α ileal mRNA transcripts were found after DNBS administration. The development of ileitis did not affect the NANC-inhibitory response, that was partially reduced in presence of 1400W or L-NAME, suggesting that TLR4 signaling influences NO-mediated inhibitory responses. DNBS-treated mice showed a significant increase in dopamine-induced relaxation (+31%) and in D1R- and D2R-mediated inhibitory responses (by +57% and +75%, respectively). A significant reduction of the total number of HuC/D+ neurons (-25%), with a proportional increase in nNOS+ neurons (+11%) was found in LMMPs of DNBS mice, associated with a significant reduction of S100β immunofluorescence (-12%) and no changes in iNOS immunoreactivity, highlighting a potential involvement of TLR4 signaling in the development of neuroplasticity and reactive gliosis. Conclusion The present data suggest that the severity of small intestine ileitis and neuronal dysfunction depend on the interplay occurring between TLR4 signaling and enteric inhibitory pathways. This evidence further strengthens the importance of characterizing neuroimmune interactions to identify potential molecular mechanism(s) representing putative pharmacological targets for the development of efficacious IBD therapies.

The Effect of Low-Dose Atropine on Alpha Ganglion Cell Signaling in the Mouse Retina.

Low-dose atropine helps to control myopia progression with few side effects. However, the impact of atropine, a non-selective muscarinic Acetylcholine (ACh) receptor antagonist, on retinal ganglion cells (RGCs) remains unclear. After immersing the cornea and adjacent conjunctiva of enucleated eyes in 0.05% (approximately 800 μM) atropine solution for 30 min, the atropine concentration reached in the retina was below 2 μM. After direct superfusion of the retina with 1 μM atropine (considering that the clinical application of 0.05% atropine eye drops will be diluted over time due to tear flow for 30 min), no noticeable changes in the morphology of ON and OFF alpha RGCs (αRGCs) were observed. Atropine affected the light-evoked responses of ON and OFF αRGCs in a dose- and time-dependent fashion. Direct application of less than 100 μM atropine on the retina did not affect light-evoked responses. The time latency of light-induced responses of ON or OFF αRGCs did not change after the application of 0.05–100 μM atropine for 5 min. However, 50 μM atropine extended the threshold of joint inter-spike interval (ISI) distribution of the RGCs. These results indicated that low-dose atropine (<0.5 μM; equal to 1% atropine topical application) did not interfere with spike frequency, the pattern of synchronized firing between OFF αRGCs, or the threshold of joint ISI distribution of αRGCs. The application of atropine unmasked inhibition to induce ON responses from certain OFF RGCs, possibly via the GABAergic pathway, potentially affecting visual information processing.

Acetylcholine-induced Ciliary Beat of the Human Nasal Mucosa Is Regulated by the Pannexin-1 Channel and Purinergic P2X Receptor.

These results indicate that the regulatory mechanism of the Ach-induced ciliary beat is dependent on extracellular Ca2+ and involves the muscarinic Ach receptor, IP3 receptor, pannexin-1 channel, purinergic P2X receptor, and connexin channel. We proposed a tentative intracellular signaling pathway of the Ach-induced ciliary beat, in which the pannexin-1-P2X unit may play a central role in ciliary beat regulation.

Inducible Loss Of Integrin β1 From Bladder Smooth Muscle Causes Increased Voiding Frequency And Impaired Muscarinic Contractility

Integrins are integral membrane proteins which, in dynamic ways, physically link, the actin cytoskeleton to the extracellular matrix. They exhibit broad functional diversity and are involved in cellular differentiation, adhesion, proliferation and mechanotransduction. Diabetic bladder dysfunction (DBD) is a very common progressive complication of diabetes mellitus resulting in overactivity during the early compensatory phase and ultimately in an atonic bladder which is unable to empty properly. Both genomic and proteomic approaches focused on bladder smooth muscle (BSM) have demonstrated altered expression of cell adhesion proteins and extracellular matrix as well as compromised contractility, as molecular markers of DBD. To begin to understand the role of cell adhesion and mechanosensitive signaling in BSM we created a mouse with tamoxifen (TMX) inducible, smooth muscle‐conditional deletion of β1‐integrin using Cre‐lox (Txβ1‐cKO). Loss of β1‐integrin mRNA and protein following TMX induction by i.p. injection (5 × 2 mg on consecutive days) were confirmed by PCR and Western blotting. Void‐spotting assays on filter paper revealed a progressive increase in number of voids from 2.5/4 h to 8.3/4 h over a period of 60 days following Cre‐induction (n=5 mice/group, P&lt;0.05). Littermate controls injected with vehicle (no TMX) showed no change. The mean volume/void decreased from 264 μl to 158 μl over 60 days in the integrin knockouts. An increase in the number of voids with reduced volume/void is often taken to indicate some form of overactivity. Urodynamic measurements using cystometry under anesthesia, revealed significantly reduced peak bladder pressures during voiding (20 cmH2O vs. 26 cmH2O, P&lt;0.05), reduced intercontraction interval (44 s vs. 249 s, P&lt;0.01), and reduced compliance (17 s/cmH2O vs. 68 s/cmH2O, N.S.) in TMX injected vs. control mice. Thus, in vivo voiding frequency was dramatically increased at the same time as the bladders showed increased resistance to filling and lower expulsive pressures. Urothelium dissected muscle‐strip force measurements by myography revealed a significant loss of contractile force in integrin‐deficient bladders in response to electrical field stimulation (45% and 62% mean reductions at 25 and 60 days post Cre‐induction respectively, for stimulation frequencies of 1 – 50 Hz). Selective inhibition of the muscarinic force component by incubation of strips with 0.5 μM atropine, indicated that muscarinic contractility was severely inhibited in integrin‐deficient BSM (17.2% of force generation was due to muscarinic receptors compared to 50% in controls) while purinergic contractility was largely intact. Masson trichrome staining of paraffin embedded bladder sections revealed a substantial expansion of collagenous connective tissue in BSM and disruption to close packing of smooth muscle bundles. We conclude that β1‐integrin is necessary for BSM structural integrity and for muscarinic signaling during force generation. These findings begin to offer insights into the physiological role of integrin mechanical signaling in BSM and may be relevant to certain bladder pathologies in which integrin expression levels have been shown to be dysregulated.Support or Funding InformationSupport for this work was provided by NIH grants DK‐083299 and DK‐095922.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

AB314. SPR-41 Localization of neuromuscular nicotinic receptors in the functionally reinnervated canine bladder after prolonged decentralization

ObjectiveWe previously found that intravenous succinylcholine, a depolarizing neuromuscular nicotinic receptors blocker, prevents bladder contractions induced by new neuronal pathways established by nerve transfer in decentralized dogs. We studied the detrusor pressure response in vivo and contractile response of bladder smooth muscle strips in vitro from sham, decentralized and reinnervated animals to localize the neuromuscular nicotinic receptors involved.MethodsThree groups of female mongrel hound dogs were used: sham (N=4), 12-month decentralized (N=3) and 6-month reinnervated (N=3). Decentralization was created by bilateral transection of all spinal roots caudal to L7, including the dorsal roots of L7 and the hypogastric nerves. Reinnervation was created by bilateral transfer of the obturator nerve to anterior vesical branches of the pelvic nerve. Two-way ANOVAs and Sidak post-hoc tests were used to determine group differences.ResultsIn reinnervated dogs, blockade of neuromuscular nicotinic receptor with intravenous injections of the competitive antagonist atracurium besylate significantly reduced the increase in detrusor pressure induced by electrical stimulation of the transferred obturator nerve (L1 or L2). Atracurium did not block the increase in pressure induced by stimulation of sacral nerve roots in sham-operated controls. In vitro, neither the competitive neuromuscular nicotinic receptor antagonist d-tubocurarine nor the ganglionic antagonist hexamethonium inhibited electric field stimulation (EFS)-induced contractions of reinnervated or sham-operated control bladder strips. No contractile response was elicited in the presence of 1 µM tetrodotoxin (TTX) across groups. Similarly, EFS-evoked contractions were strongly reduced by 10 µM alpha, beta-methylene ATP (α,β-mATP) and 1 µM atropine in all groups relative to the vehicle (water).ConclusionsIn vivo blockade of nerve-evoked bladder pressure by atracurium in the reinnervated, but not sham operated controls, suggests that neuromuscular nicotinic receptors become involved in bladder contractions induced by the new neuronal pathway. Because d-tubocurarine did not block in vitro contractions induced by EFS in the reinnervated bladders, the neuromuscular nicotinic receptors involved in the new neuronal pathway must not be located in the bladder muscle or intramural ganglia and therefore, are likely in preganglionic neurons. TTX blockade validates that EFS-induced contractions at all frequencies were nerve-evoked. Both muscarinic and purinergic components contributed similarly to neurotransmission based on response to blockade of nerve evoked muscle strip contractions with a combination of atropine and α,β-mATP.Funding Source(s)NIH-NINDS NS070267

AB318. SPR-45 Decentralization reduces nicotinic receptor-mediated canine bladder contractions in vitro

ObjectiveBladder function depends upon several complex signaling pathways that induce either contraction or relaxation. We performed nerve re-routing surgery on bladder of decentralized dogs (reinnervated) as a model with the goal of restoring bladder function in spinal cord injured patients. The neuromuscular nicotinic receptor blocker, succinylcholine blocks spinal root-stimulated bladder contraction in vivo in reinnervated dogs, but not in sham-operated dogs. Our lab explored the function and location of nicotinic receptors involved in bladder contraction, with emphasis on their possible role in the release of other neurotransmitters such as acetylcholine or ATP in sham-operated, 12-month-decentralized and immediate-reinnervated bladders.MethodsSmooth muscle strips (mucosa denuded) were isolated from the region just rostral to the trigone and suspended in muscle baths. Strips were ranked based on their contractile response to a 3-min exposure to 120 mM KCl and sorted so that the average response in each group was equal. Strips were incubated with either 1 µM atropine (ATR), 1 µM tetrodotoxin (TTX), 10 µM alpha, beta-methylene ATP (α,β-ATP) or vehicle (water) and then induced to contract with the nicotinic receptor agonists 1,1 dimethyl-4-phenyl-piperazinium iodide (DMPP, 100 µM), TC2559 (100 µM) or epibatidine (10 µM) or 1 mM nicotine itself.ResultsThe DMPP-induced contraction was not different between sham, reinnervated or decentralized bladders (11%, 3.3%, and 3.6% of KCl contraction respectively). While the epibatidine-induced contraction in shams was not different relative to that in the reinnervated (41% vs. 27% KCl respectively), it was significantly greater than that in decentralized bladders (13% of KCl). TC2559 did not induce bladder contractions. Nicotine-induced contractions in sham-operated controls were 16% of KCl. ATR completely blocked nicotine-induced contraction while α,β-ATP had no statistically significant effect in shams. TTX had no significant inhibitory effect on DMPP, epibatidine or nicotine-induced contraction in any group.ConclusionsNicotinic receptors mediate contraction in sham, reinnervated and decentralized bladders. This nicotinic receptor-mediated contraction is decreased after decentralization. TTX does not block nicotinic receptor-mediated contractions, indicating that action potentials are not required to induce contraction. In sham-operated dog bladders, the nicotine-induced contraction is blocked by ATR, suggesting that these nicotinic receptors are located on cholinergic nerve terminals and induce the release of acetylcholine, which activates muscarinic receptors on the smooth muscle.Funding Source(s)NIH-NINDS NS070267

Inhibitory Effects of Botulinum Toxin Type A on Pyloric Cholinergic Muscle Contractility of Rat.

Botulinum toxin type A (BTX-A) selectively cleaves synaptosomal-associated protein of 25 kDa (SNAP-25) and results in inhibition of the fusion of synaptic vesicles containing neurotransmitters with the presynaptic membrane to undergo exocytosis and release. The aim of this study was to investigate whether BTX-A inhibited the pyloric smooth muscle contractility induced by acetylcholine (ACh) after BTX-A-mediated cleavage of SNAP-25 antagonized by toosendanin (TSN). Three groups of rat pyloric muscle strips were studied in vitro. All strips were allowed to equilibrate for 52 min under a basal loading tension of 1 g in Krebs solution and spontaneous contractile waves were recorded as their own controls before adding each drug. According to experimental protocols, 100 μM ACh, 1 μM atropine, 29.6 μM TSN and 10 U/ml BTX-A was added, respectively. BTX-A directly inhibited pyloric spontaneous contraction and ACh-induced contractile response. Addition of 10 U/ml BTX-A still inhibited pyloric smooth muscle contractility following incubation of TSN, while subsequent administration of 100 μM ACh had no effect. BTX-A inhibits pyloric smooth muscle contractility in our study suggesting BTX-A inhibits not only ACh release from cholinergic nerves but also muscarinic cholinergic muscular transmission.

Effects of Nerve Stimulation in Mouse Lungs

Mouse pulmonary airways and blood vessels are innervated by autonomic and sensory nerves. Stimulation of mouse pulmonary nerves can activate a variety of neurotransmitter systems to control airway and blood vessel diameter. The objective of this study was to identify the neurotransmitter systems causing constriction and/or dilatation of mouse airways and pulmonary arteries. Precision cut lung slices from CF‐1 mice were exposed to electric field stimulation (EFS) using parameters known to activate neurotransmitter release (1.0 ms, 50 V, 1–30 Hz). Changes in airway or pulmonary artery luminal area were captured as video images and quantified using ImageJ. EFS caused constriction in both airways and pulmonary arteries in a frequency‐dependent manner. Maximum constriction to 30 Hz EFS was 52.8 ± 3.5% in airways and 42.4 ± 3.7% in pulmonary arteries. Interestingly, the ability of the muscarinic receptor antagonist atropine to block EFS induced constriction in airways was frequency dependent. In airways, 1 μM atropine blocked 70.6 ± 19.2% of EFS induced constriction at 3 Hz, but only blocked 22.1 ± 12.6% of EFS induced constriction at 30 Hz. Treatment of pulmonary arteries with 1 μM prazosin, an α1‐adrenoceptor antagonist, reduced constriction by 62.6 ± 12.3% at 30 Hz EFS. These data are consistent with parasympathetic constriction of airways by acetylcholine and sympathetic constriction of pulmonary arteries by norepinephrine. In addition, an atropine‐resistant EFS constriction in airways and a prazosin‐resistant EFS constriction in pulmonary arteries were identified. Understanding the neurotransmitter systems causing nerve mediated constriction and/or dilatation of airways and pulmonary arteries may identify novel therapeutic targets for treatment of asthma, pulmonary hypertension, and other lung diseases.Support or Funding InformationSupported by NIH R01HL116849 and Nebraska State LB595 to Y. Tu and P.W. Abel

An Accessory Agonist Binding Site Promotes Activation of α4β2* Nicotinic Acetylcholine Receptors

Neuronal nicotinic acetylcholine receptors containing α4, β2, and sometimes other subunits (α4β2* nAChRs) regulate addictive and other behavioral effects of nicotine. These nAChRs exist in several stoichiometries, typically with two high affinity acetylcholine (ACh) binding sites at the interface of α4 and β2 subunits and a fifth accessory subunit. A third low affinity ACh binding site is formed when this accessory subunit is α4 but not if it is β2. Agonists selective for the accessory ACh site, such as 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283), cannot alone activate a nAChR but can facilitate more efficient activation in combination with agonists at the canonical α4β2 sites. We therefore suggest categorizing agonists according to their site selectivity. NS9283 binds to the accessory ACh binding site; thus it is termed an accessory site-selective agonist. We expressed (α4β2)2 concatamers in Xenopus oocytes with free accessory subunits to obtain defined nAChR stoichiometries and α4/accessory subunit interfaces. We show that α2, α3, α4, and α6 accessory subunits can form binding sites for ACh and NS9283 at interfaces with α4 subunits, but β2 and β4 accessory subunits cannot. To permit selective blockage of the accessory site, α4 threonine 126 located on the minus side of α4 that contributes to the accessory site, but not the α4β2 sites, was mutated to cysteine. Alkylation of this cysteine with a thioreactive reagent blocked activity of ACh and NS9283 at the accessory site. Accessory agonist binding sites are promising drug targets.

Allosteric Modulation of M1 Muscarinic Acetylcholine Receptor Internalization and Subcellular Trafficking

Allosteric modulators are an attractive approach to achieve receptor subtype-selective targeting of G protein-coupled receptors. Benzyl quinolone carboxylic acid (BQCA) is an unprecedented example of a highly selective positive allosteric modulator of the M1 muscarinic acetylcholine receptor (mAChR). However, despite favorable pharmacological characteristics of BQCA in vitro and in vivo, there is limited evidence of the impact of allosteric modulation on receptor regulatory mechanisms such as β-arrestin recruitment or receptor internalization and endocytic trafficking. In the present study we investigated the impact of BQCA on M1 mAChR regulation. We show that BQCA potentiates agonist-induced β-arrestin recruitment to M1 mAChRs. Using a bioluminescence resonance energy transfer approach to monitor intracellular trafficking of M1 mAChRs, we show that once internalized, M1 mAChRs traffic to early endosomes, recycling endosomes and late endosomes. We also show that BQCA potentiates agonist-induced subcellular trafficking. M1 mAChR internalization is both β-arrestin and G protein-dependent, with the third intracellular loop playing an important role in the dynamics of β-arrestin recruitment. As the global effect of receptor activation ultimately depends on the levels of receptor expression at the cell surface, these results illustrate the need to extend the characterization of novel allosteric modulators of G protein-coupled receptors to encapsulate the consequences of chronic exposure to this family of ligands.

Novel Structural and Functional Insights into M3 Muscarinic Receptor Dimer/Oligomer Formation

Class A G protein-coupled receptors (GPCRs) are able to form homodimers and/or oligomeric arrays. We recently proposed, based on bioluminescence resonance energy transfer studies with the M3 muscarinic receptor (M3R), a prototypic class A GPCR, that the M3R is able to form multiple, structurally distinct dimers that are probably transient in nature (McMillin, S. M., Heusel, M., Liu, T., Costanzi, S., and Wess, J. (2011) J. Biol. Chem. 286, 28584-28598). To provide more direct experimental support for this concept, we employed a disulfide cross-linking strategy to trap various M3R dimeric species present in a native lipid environment (transfected COS-7 cells). Disulfide cross-linking studies were carried out with many mutant M3Rs containing single cysteine (Cys) substitutions within two distinct cytoplasmic M3R regions, the C-terminal portion of the second intracellular loop (i2) and helix H8 (H8). The pattern of cross-links that we obtained, in combination with molecular modeling studies, was consistent with the existence of two structurally distinct M3R dimer interfaces, one involving i2/i2 contacts (TM4-TM5-i2 interface) and the other one characterized by H8-H8 interactions (TM1-TM2-H8 interface). Specific H8-H8 disulfide cross-links led to significant impairments in M3R-mediated G protein activation, suggesting that changes in the structural orientation or mobility of H8 are critical for efficient receptor-G protein coupling. Our findings provide novel structural and functional insights into the mechanisms involved in M3R dimerization (oligomerization). Because the M3R shows a high degree of sequence similarity with many other class A GPCRs, our findings should be of considerable general interest.

Reciprocal regulation of inhibitory synaptic transmission by nicotinic and muscarinic receptors in rat nucleus accumbens shell

Medium spiny neurones (MSNs) in the nucleus accumbens (NAc) are the principal neurones whose activities are regulated by GABAergic inputs from MSNs and fast-spiking interneurones (FSNs). Cholinergic interneurones play important roles in the regulation of activity in MSNs; however, how acetylcholine modulates inhibitory synaptic transmission from MSNs/FSNs to MSNs remains unknown. We performed paired whole-cell patch-clamp recordings from MSNs and FSNs in rat NAc shell slice preparations and examined cholinergic effects on unitary inhibitory postsynaptic currents (uIPSCs). Carbachol (1 μM) suppressed uIPSC amplitude by 58.3 ± 8.0% in MSN→MSN connections, accompanied by increases in paired-pulse ratio and failure rate, suggesting that acetylcholine reduces the probability of GABA release from the synaptic terminals of MSNs. Carbachol-induced uIPSC suppression was antagonised by 100 μM atropine, and was mimicked by pilocarpine (1 μM) and acetylcholine (1 μM) but not nicotine (1 μM). Application of AM251 slightly reduced carbachol-induced uIPSC suppression (30.8 ± 8.9%), suggesting an involvement of endocannabinoid signalling in muscarinic suppression of uIPSCs. In contrast, FSN→MSN connections showed that pilocarpine had little effect on the uIPSC amplitude, whereas both nicotine and acetylcholine facilitated uIPSC amplitude, with decreases in failure rate and paired-pulse ratio, suggesting that nicotine-induced uIPSC facilitation is mediated by presynaptic mechanisms. Miniature IPSC recordings support these hypotheses of presynaptic cholinergic mechanisms. These results suggest a differential role for muscarinic and nicotinic receptors in GABA release, which depends on presynaptic neuronal subtypes in the NAc shell.

Neurotensin and cholecystokinin contract gallbladder circular muscle in chickens

The contractile effects of neurotensin (NT) and cholecystokinin octapeptide (CCK-8) on isolated circular smooth muscle strips of chicken gallbladder were investigated. The NT (0.25-300 nM) produced concentration-dependent contractions on smooth muscle with an EC50 of 8.5 nM (95% confidence limits = 5.3-13.6 nM). In comparison, CCK-8 produced concentration-dependent contractions with an EC50 of 13 nM (95% confidence limits of 9-20 nM). There were no statistical differences in contractile responses when comparing NT and CCK-8 at equimolar concentrations. The NT appears to act directly on smooth muscle tissue in the chicken; the contractile responses were not blocked by 10 µM atropine or tetrodotoxin. A portion of the activity is mediated by extracellular calcium as 100 nM nifedipine inhibited 30% of peptide-induced muscle tension. The NT receptor (NTR) type 1 antagonist SR 48692 (0.1 µM) did not significantly reduce NT potency. The contractile effects of CCK-8 remained unaltered in tissues pretreated with atropine, TTX, or nifedipine. The CCK-A antagonist lorglumide, at a concentration of 1 µM, reduced the contractile potency of CCK-8 by one-half. Avian receptors for NT and CCK may differ pharmacologically from their mammalian counterparts, but their contractile actions on the gallbladder resulting in increased biliary output by flow are further evidence of their role in the postprandial regulation of lipid digestion in chickens.

Relaxing Effect of Acetylcholine on Phenylephrine-Induced Contraction of Isolated Rabbit Prostate Strips Is Mediated by Neuronal Nitric Oxide Synthase

PurposeThe location of acetylcholinesterase-containing nerve fibers suggests a role for acetylcholine in both contractility and secretion in the prostate gland. The colocalization of nitrergic nerves with cholinergic nerves, and the cotransmission of nitric oxide with acetylcholine in cholinergic nerves, has been demonstrated in the prostate glands of various species. Thus, we investigated the effects of acetylcholine on phenylephrine-induced contraction and the correlation between cholinergic transmission and nitric oxide synthase by using isolated prostate strips of rabbits.Materials and MethodsIsolated prostate strips were contracted with phenylephrine and then treated with cumulative concentrations of acetylcholine. Changes in acetylcholine-induced relaxation after preincubation with NG-nitroarginine methyl ester, 7-nitroindazole, and aminoguanidine were measured. The effects of selective muscarinic receptor antagonists were also evaluated.ResultsIn the longitudinal phenylephrine-contracted strip, the cumulative application of acetylcholine (10-9 to 10-4 M) elicited a concentration-dependent relaxation effect. Acetylcholine-induced relaxation was inhibited not only by nitric oxide synthase inhibitors (10 µM L-NAME or 10 µM 7-nitroindazole) but also by 10 µM atropine and some selective muscarinic receptor antagonists (10-6 M 11-([2-[(diethylamino)methyl]-1-piperdinyl]acetyl)-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one and 10-6 M 4-diphenylacetoxy-N-methyl-piperidine). In contrast, relaxation was significantly increased by pretreatment of the strips with 10 mM L-arginine.ConclusionsAcetylcholine relaxed phenylephrine-induced contractions of isolated rabbit prostate strips. This relaxation may be mediated via both cholinergic and constitutive nitric oxide synthase with both the M2 and M3 receptors possibly playing key roles.

Modulation of Vagus Nerve Stimulation via Ganglionic Plexus activation and Ventricular Ischaemia

Discreet regions in the heart, called ganglionic plexuses (GPs) are a network of integration centres that process afferent and efferent nerve activity to modulate the heart. We aimed to investigate the role of the right atrial GP (RAGP) in controlling heart rate (HR).MethodsThe decentralised innervated Langendorff perfused rabbit heart model was used (n=6). 1) RAGP was stimulated using 0.1–0.4mg nicotine (0.1ml) in 2) the presence of 0.1μm Atropine (At) or 1.8μm Metoprolol (Met), 3) The right vagus nerve was stimulated (VNS, 7H10V) before and after RAGP stimulation and 4) VNS before and following coronary artery occlusion. Data are mean±SEM, repeated measures ANOVA or Students t‐test.Resultsi) RAGP stimulation elicited an initial bradycardia (145±9 to 109±3bpm, P&lt;0.05) and secondary tachycardia (226±12bpm, P&lt;0.001, n=5) ii) At abolished the bradycardia (139±8 to 138±10bpm, P=NS), but not the tachycardia (247±9bpm, P&lt;0.001). Met did not affect the bradycardia (135±7 to 106±4, P&lt;0.05) but abolished the tachycardia (128±6bpm, P=NS, N=5. iii) VNS‐bradycardia was augmented after RAGP stimulation (−81±8 vs 142±9bpm, P&lt;0.01, n=3). iv) Coronary artery occlusion increased VNS‐bradycardia (−45±6 vs −68±8bpm, P&lt;0.05, n‐4).ConclusionThis is the first data representing GP modification in the rabbit showing that RAGP activation elicits a mixed autonomic HR response, via vagal and sympathetic activation that enhanced VNS‐bradycardia. In addition, novel data shows that acute ventricular ischemia augments the HR effect of cervical VNS confirming that the RAGP is important in controlling HR in this species.Research support from the University of Leicester

Endogenously Expressed Muscarinic Receptors in HEK293 Cells Augment Up-regulation of Stably Expressed α4β2 Nicotinic Receptors

Nicotine-induced up-regulation of neuronal nicotinic receptors (nAChRs) has been known and studied for more than 25 years. Other nAChR ligands can also up-regulate nAChRs, but it is not known if these ligands induce up-regulation by mechanisms similar to that of nicotine. In this study, we compared up-regulation by three different nicotinic agonists and a competitive antagonist of several different nAChR subtypes expressed in HEK293 cells. Nicotine markedly increased α4β2 nAChR binding site density and β2 subunit protein. Carbachol, a known nAChR and muscarinic receptor agonist, up-regulated both α4β2 nAChR binding sites and subunit protein 2-fold more than did nicotine. This increased up-regulation was shown pharmacologically to involve endogenously expressed muscarinic receptors, and stimulation of these muscarinic receptors also correlated with a 2-fold increase in α4 and β2 mRNA. Muscarinic receptor activation in these cells appears to affect CMV promoter activity only minimally (∼1.2 fold), suggesting that the increase in α4 and β2 nAChR mRNA may not be dependent on enhanced transcription. Instead, other mechanisms may contribute to the increase in mRNA and a consequent increase in receptor subunits and binding site density. These studies demonstrate the possibility of augmenting nAChR expression in a cell model through mechanisms and targets other than the nAChR receptor itself.

Activation of beta3 adrenoreceptors decreases the amplitude of the electrical field stimulation induced contractions in rat detrusor smooth muscle isolated strips

It is believed that during bladder filling phase, β-adrenoceptors (β-AR) facilitate relaxation of the detrusor smooth muscle (DSM). Our previous studies showed that BRL37344, a specific β3-AR agonist, caused a concentration-dependant decrease in the myogenic contractions of rat isolated DSM strips. Here, using tissue baths equipped with electrodes for electrical field stimulation (EFS), we investigated the role of β3-AR in rat DSM neurogenic contractions. Neurogenic contractions were generated by EFS pulses (20 V amplitude, 0.75 ms width, and 3 s duration) with increasing frequencies (0.5–50 Hz). BRL37344 (100 μM) significantly decreased the amplitude of the EFS-induced contractions of rat DSM (n=12, N=6). We further used specific inhibitors to separate the cholinergic and purinergic components of the EFS-induced contractions of rat DSM. The BRL37344 inhibitory effect was also observed in the presence of 1 μM atropine (n=15, N=8) as well as in the presence of 10 μM suramin and 10 μM α,β-methylene-ATP (n=13, N=5). BRL37344 (100 μM) inhibitory effect was blocked by SR59230A (10 μM), a specific β3-AR antagonist (n=8, N=3). SR59230A per se did not have any effect on the neurogenic contractions (n=8, N=3). In conclusion, activation of β3-AR decreases DSM neurogenic contractions, thus making β3-AR a new pharmacological target for the treatment of overactive bladder.

Effects of stimulation of muscarinic receptors on bladder afferent nerves in the in vitro bladder–pelvic afferent nerve preparation of the rat

Effects of a muscarinic receptor agonist oxotremorine-M (oxo-M) on bladder afferent nerve (BAN) activity were studied in an in vitro bladder–pelvic nerve preparation. Distension of the bladder induced rhythmic bladder contractions that were accompanied by multiunit afferent firing. Intravesical administration of 25 and 50 μM oxo-M significantly increased afferent firing from 41 ± 2 spikes/s to 51 ± 4 spikes/s and 60.5 ± 5 spikes/s, respectively, but did not change the maximum amplitude of spontaneous bladder contractions. The afferent nerve firing induced by isotonic distension of the bladder (10–40 cmH 2O) was increased 22–100% by intravesical administration of 50 μM oxo-M. Electrical stimulation on the surface of the bladder elicited action potentials (AP) in BAN. Oxo-M significantly decreased the voltage threshold by 40% (p < 0.05) and increased by 157% (p < 0.05) the area of the AP evoked at a submaximal stimulus intensity. These effects were blocked by intravesical injection of 5 μM atropine methyl nitrate (AMN). Intravesical administration of 5 μM AMN alone did not alter BAN firing or the amplitude of bladder contractions. The facilitatory effects induced by oxo-M on BAN activity were also suppressed (p < 0.05) by intravesical administration of 2′,3′-0-trinitrophenyl-ATP (TNP-ATP) (30 μM). In preparations pretreated with capsaicin (125 mg/kg, s.c.) the facilitatory effects of 50 μM oxo-M on BAN activity were absent. These results suggest that activation of muscarinic receptors facilitates mechano-sensitive, capsaicin-sensitive BAN activity in part by mechanisms involving purinergic receptors located near the luminal surface of the bladder and ATP release which presumably occurs in the urothelium.