Exogenous ACh Research Articles

Two Biological Active Fractions Isolated from Buthotus schach (BS)Scorpion Venom Examined on Striated Muscle Preparation, In-vitro.

Buthotus schach is one of the most dangerous scorpions in tropical part of Iran. The effects of its crude venom at 1, 3, 10 μg/mL and its obtained fractions by gel filtrations were investigated on neuromuscular transmission. CBC and MHD indirectly and directly stimulated preparations techniques were used to study their possible pre or post junctional activities. At 3 and 10 μg/mL (not at 1 μg/mL), BS venom caused initiall increase in twitch height followed by blockage due to large contraction that responded gradually at the same time. Contracture responses to exogenous Ach (1-2 mM, 30 sec) and Carb (30-40 μM, 60 sec) in the presence of the venom were not increased which does show no anticholinstrease effects. Furthermore Contracture response to KCl (20-40 mM, 30 sec) does changed exposure to venom in CBC preparations. On the other hand the effects of the venom in response to directly stimulated preparations was shallower than in indirect stimulated preparations. So in agreement with KCL response BS venom affects mostly prejunctionally to facilitate the neurotransmitter release rather than postjunctionally effects. To access bioactive components, seven fractions were collected by gel filtrations techniques. Among the fractions F6, LD50=21 μg < F4, LD50= 35.5 μg < Venom LD50= 84 μg per mice were more toxic respectively. Both fractions show the same effects but stronger than venom on twitch height responses in indirectly stimulated CBC preparations. Finally, according to our results venom as well as fractions F4 and F6 act mostly prejunctionally on Ach release. More attempt is carrying out to study their effects on ion channel activities.

Neuromuscular activity of Bothrops alcatraz snake venom in chick biventer cervicis preparations

Venom (10–100 μg/ml) from Bothrops alcatraz, a pitviper from the Alcatrazes Archipelago off the coast of southeastern Brazil, caused progressive, irreversible neuromuscular blockade in chick isolated biventer cervicis preparations. The venom also inhibited contractures to exogenous ACh (110 μM) and KCl (20 mM), caused myofiber damage and increased creatine kinase release. Commercial bothropic antivenom raised against mainland Bothrops species neutralized the neuromuscular activity, depending on the venom concentration.

Neuromuscular activity of the venoms of the Colombian coral snakes Micrurus dissoleucus and Micrurus mipartitus: An evolutionary perspective

The venoms of coral snakes (genus Micrurus) are known to induce a broad spectrum of pharmacological activities. While some studies have investigated their potential human effects, little is known about their mechanism of action in terms of the ecological diversity and evolutionary relationships among the group. In the current study we investigated the neuromuscular blockade of the venom of two sister species Micrurus mipartitus and Micrurus dissoleucus, which exhibit divergent ecological characteristics in Colombia, by using the chick biventer cervicis nerve-muscle preparation. We also undertook a phylogenetic analysis of these species and their congeners, in order to provide an evolutionary framework for the American coral snakes. The venom of M. mipartitus caused a concentration-dependant inhibition (3–10 μg/ml) of nerve-mediated twitches and significantly inhibited contractile responses to exogenous ACh (1 mM), but not KCl (40 mM), indicating a postsynaptic mechanism of action. The inhibition of indirect twitches at the lower venom dose (3 μg/ml) showed to be triphasic and the effect was further attenuated when PLA2 was inhibited. M. dissoleucus venom (10–50 μg/ml) failed to produce a complete blockade of nerve-mediated twitches within a 3 h time period and significantly inhibited contractile responses to exogenous ACh (1 mM) and KCl (40 mM), indicating both postsynaptic and myotoxic mechanisms of action. Myotoxic activity was confirmed by morphological studies of the envenomed tissues. Our results demonstrate a hitherto unsuspected diversity of pharmacological actions in closely related species which exhibit divergent ecological characteristics; these results have important implications for both the clinical management of Coral snake envenomings and the design of Micrurus antivenom.

Pharmacological characterization of cannabinoid receptor activity in the rat‐isolated ileum myenteric plexus‐longitudinal muscle preparation

Cannabinoid effects on intestinal transit are commonly evaluated in rats. We characterized the cannabinoid receptors mediating the inhibitory effect of 5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)-cyclohexyl]-phenol (CP 55,940), (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN 55,212-2), arachidonylethanolamide (AEA) and Delta(9)-tetrahydrocannabinol (Delta(9)-THC) on contractions of the rat ileum myenteric plexus-longitudinal muscle (MPLM) preparation. The interaction of each agonist was examined with the CB(1) and CB(2) receptor antagonist rimonabant and SR 144,528 respectively, on contractions elicited by electrical field stimulation (EFS) or exogenous ACh. The interaction of AEA with capsazepine, a TRPV(1) receptor antagonist, was also investigated. EFS with single and trains of pulses evoked neurogenic ACh-mediated twitch and rebound contractions respectively. The rank order of potency for inhibition was CP 55,940 = WIN 55,212-2 > AEA > Delta(9)-THC and AEA > WIN 55,212-2 =Delta(9)-THC = CP 55,940 respectively. The stereoisomer WIN 55,212-3 was without effect. Rimonabant antagonized the inhibition of the twitches with pK(B) values of around 8.60, but only antagonized rebound contractions induced by WIN 55,212-2, AEA and Delta(9)-THC, with pA(2) values of around 6.80. Rimonabant increased the twitches but inhibited the rebound contractions. Contractions to exogenous ACh were not altered. These observations extended to the guinea pig ileum MPLM. The rat MPLM contains CB(1) receptors and at least two non-CB(1)-non-CB(2)-non-TRPV(1) receptors attenuating EFS-evoked ACh-mediated contractions in an EFS frequency-dependent pre-synaptic and stereo-specific manner. Augmentation of the twitches by rimonabant may be through antagonism of an endocannabinoid tone or inverse agonism, whereas inhibition of the rebound contractions involved partial agonism.

Peripheral 5-HT1Aand 5-HT7Serotonergic Receptors Modulate Parasympathetic Neurotransmission in Long-Term Diabetic Rats

We analyzed the modulation of serotonin on the bradycardia induced in vivo by vagal electrical stimulation in alloxan-induced long-term diabetic rats. Bolus intravenous administration of serotonin had a dual effect on the bradycardia induced either by vagal stimulation or exogenous Ach, increasing it at low doses and decreasing it at high doses of 5-hydroxytryptamine (5-HT), effect reproduced by 5-carboxamidotryptamine maleate (5-CT), a 5-HT1/7 agonist. The enhancement of the bradycardia at low doses of 5-CT was reproduced by 5-HT1A agonist 8-hydroxy-2-dipropylaminotetralin hydrobromide (8-OH-DPAT) and abolished by WAY-100,635, 5-HT1A antagonist. Pretreatment with 5-HT1 antagonist methiothepin blocked the stimulatory and inhibitory effect of 5-CT, whereas pimozide, 5-HT7 antagonist, only abolished 5-CT inhibitory action. In conclusion, long-term diabetes elicits changes in the subtype of the 5-HT receptor involved in modulation of vagally induced bradycardia. Activation of the 5-HT1A receptors induces enhancement, whereas attenuation is due to 5-HT7 receptor activation. This 5-HT dual effect occurs at pre- and postjunctional levels.

Cholinesterases in development and disease

Cholinesterases (ChEs) including acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are abundant in the nervous system and other tissues. Here we describe two different aspects of ChEs and the cholinergic system. The first aspect concerns the role of cholinergic transmission in central pattern generation in the neonatal rat spinal cord and the second one describes the involvement of ChEs in the pathologies of dystrophin-deficient mutant ( mdx) mice, the animal model of Duchenne muscular dystrophy. Thus, this study is divided into two distinct parts. In the first part we show that AChE is abundant in ventral horn neurons, central canal-adjacent and partition neurons in all the observed segments (L2, L5, S1, and S2). AChE was also found in the intermediolateral and sacral parasympathetic nuclei of L2 and S1, respectively. Blocking the AChE by edrophonium produced non-stationary bursting in spinal cord preparations of developing rats. Cross-wavelet/coherence analyses of the data revealed epochs of locomotor-like activity (left–right and flexor–extensor alternation) followed by other rhythmic or non-rhythmic bursting patterns. Addition of exogenous ACh stabilized the rhythm and increased the incidence of locomotor-like pattern in the preparations. Thus, the cholinergic system in the spinal cord is capable of producing and modulating functional rhythmic bursts. Moreover, bath-applied edrophonium and exogenous ACh were found as potent means of modulation of the locomotor rhythm produced by stimulation of sacrocaudal afferents (SCAs). We show that a subclass of sacral neurons with contralateral funicular projections to the thoracolumbar cord is associated with the cholinergic system. This group of neurons may play a major role in the observed enhancement of the SCA-induced motor rhythm. In the second part we show that adult mdx-muscles are malformed with distorted neuromuscular junctions (nmjs) and impaired regulation of acetylcholine receptors. Examination of circulating ChE levels revealed that in mdx-sera, while AChE activity was elevated, BuChE activity was markedly lower than in wild-type ( wt) sera. Thus, BuChE to AChE ratio in mouse sera decreased from 6:1 in wt control to 3:1 in mdx. Because serum ChE levels may be modulated by gonadal steroids, it is possible that lack of dystrophin in mdx-mice may affect this regulation. Further studies are in progress to determine the potential endocrine regulation of ChEs in circulation and at the nmjs of mdx- and wt-mice. These studies will help clarify whether the hormonal regulation is impaired in the mdx mutant, and whether changes in circulating ChE reflect or influence the functional deficits observed in excitable tissues of diseased states.

Skin Grafting Impairs Postsynaptic Cutaneous Vasodilator and Sweating Responses

This study tested the hypothesis that postsynaptic cutaneous vascular responses to endothelial-dependent and -independent vasodilators, as well as sweat gland function, are impaired in split-thickness grafted skin 5 to 9 months after surgery. Intradermal microdialysis membranes were placed in grafted and adjacent control skin, thereby allowing local delivery of the endothelial-dependent vasodilator, acetylcholine (ACh; 1 x 10(-7) to 1 x 10(-1) M at 10-fold increments) and the endothelial-independent nitric oxide donor, sodium nitroprusside (SNP; 5 x 10(-8) to 5 x 10(-2) M at 10-fold increments). Skin blood flow and sweat rate were simultaneously assessed over the semipermeable portion of the membrane. Cutaneous vascular conductance (CVC) was calculated from the ratio of laser Doppler-derived skin blood flow to mean arterial blood pressure. deltaCVC responses from baseline to these drugs were modeled via nonlinear regression curve fitting to identify the dose of ACh and SNP causing 50% of the maximal vasodilator response (EC50). A rightward shift in the CVC dose response curve for ACh was observed in grafted (EC50 = -2.61 +/- 0.44 log M) compared to adjacent control skin (EC50 = -3.34 +/- 0.46 log M; P = .003), whereas the mean EC50 for SNP was similar between grafted (EC50 = -4.21 +/- 0.94 log M) and adjacent control skin (EC50 = -3.87 +/- 0.65 log M; P = 0.332). Only minimal sweating to exogenous ACh was observed in grafted skin whereas normal sweating was observed in control skin. Increased EC50 and decreased maximal CVC responses to the exogenous administration of ACh suggest impairment of endothelial-dependent cutaneous vasodilator responses in grafted skin 5 to 9 months after surgery. Greatly attenuated sweating responses to ACh suggests either abnormal or an absence of functional sweat glands in the grafted skin.

Excitatory muscarinic M1 receptors mediate in the regulation of guinea-pig terminal ileum motility

In vitro experiments were performed on smooth muscle strips cut out longitudinally or circularly from the terminal ileum about 5 cm from the ileo-coecal sphincter. The aim of this study was to determine the functional role of M1 muscarinic receptor subtype in the regulation of cholinergic contractions of the guinea-pig terminal ileum. Electrical field stimulation (EFS; 16 Hz) produced in vitro contractile responses of the guinea-pig terminal ileum muscle strips (longitudinal and circular muscle layer). Those contractions were inhibited by 1 μM tetrodotoxin (2±2% of the control response) and 1 μM atropine (1±1% of the control response), thus indicating the activation of intrinsic cholinergic nerves. Exogenous ACh (1 μM) induced contractions that were inhibited by 1 μM atropine, but not by 1 μM tetrodotoxin, indicating a direct effect on the smooth muscle. MCN-343 specific agonist of M1 receptor subtype (5 μM) induced contractions that were inhibited by 1 μM atropine and by 1 μM tetrodotoxin. M1 receptor antagonist pirenzepine (10 nM) had no effect on ACh - induced contractions but reduced EFS - induced contractions by 11±3% and MCN-A-343 induced contractions by 17±4%. In conclusion, specific M1 receptors modulate terminal ileum contractions by regulating the release of ACh from cholinergic nerves. M1 receptors, present on cholinergic nerves, function as prejunctional facilitatory autoreceptors.

Synaptically Released and Exogenous ACh Activates Different Nicotinic Receptors to Enhance Evoked Glutamatergic Transmission in the Lateral Geniculate Nucleus

The effects of activation of nicotinic acetylcholine receptors (nAChRs) on glutamatergic transmission in the ventral lateral geniculate nucleus (LGNv) were examined in chick brain slices. Whole cell recordings showed that monosynaptic postsynaptic currents (PSCs) evoked in LGNv neurons by optic tract stimulation were blocked by glutamate receptor antagonists. Exogenously applied nicotine (0.5 microM), choline (1 mM), or acetylcholine (ACh, 100 microM) markedly increased (>3-fold) these evoked PSCs. Potentiation by ACh was dose-dependent and did not desensitize during a 5-min application. In a second set of experiments, the effect of releasing endogenous ACh by stimulating the lateral portion of the LGNv through a separate conditioning electrode before optic tract stimulation was examined. Conditioning stimulation trains increased PSCs by an average of 5.2-fold, an effect dependent on both the intensity and number of conditioning pulses. This increase in PSC amplitude was most likely caused by released ACh activating alpha6- and/or alpha3-containing nAChRs because it was blocked by 100 nM alpha-conotoxin MII, 100 nM dihydro-beta-erythroidine (DHbetaE), and 0.1-1.0 microM methyllycaconitine (MLA). In contrast, exogenously applied ACh increased PSC amplitude by activating a pharmacologically different population of nAChRs because this effect was inhibited by 100 nM alpha-bungarotoxin, 50 nM MLA, and a high concentration (30 microM) of DHbetaE, indicating that alpha7- and/or alpha8-containing receptors were involved. The results are consistent with a model whereby alpha6- and/or alpha3-containing nAChRs on retinal ganglion cell nerve terminals are located preferentially at cholinergic synapses, whereas alpha7- and/or alpha8-containing receptors are primarily extrasynaptic.

Loss of enteric motor neurotransmission in the gastric fundus of Sl/Sl(d) mice.

Studies of W/W(V) mice, which lack intramuscular interstitial cells of Cajal (IC-IM), have suggested that IC-IM act as mediators of enteric motor neurotransmission in the gastrointestinal tract. We have studied Sl/Sl(d) mice, which lack the ability to make membrane-bound stem cell factor, to determine the consequences of inappropriate stem cell factor expression on IC-IM populations and on enteric motor neurotransmission. IC-IM were found within the circular and longitudinal muscles of the gastric fundus of wild-type mice. IC-IM were intimately associated with motor nerve terminals and nerve varicosities formed synaptic structures with these cells. IC-IM were also connected with neighbouring smooth muscle cells via gap junctions. Immunohistochemistry and electron microscopy showed that IC-IM were absent from fundus muscles of Sl/Sl(d) mice, but the density of excitatory and inhibitory nerves was not significantly different than in wild-type muscles. Loss of IC-IM was associated with decreased membrane noise (unitary potentials) and significant reductions in post-junctional excitatory and inhibitory enteric nerve responses. Reductions in neural responses were not due to defects in smooth muscle cells as responses to exogenous ACh and K(+)-induced depolarization were normal in Sl/Sl(d) mice. Responses to neurally released ACh were revealed in Sl/Sl(d) mice by inhibiting ACh breakdown with the acetylcholinesterase inhibitor neostigmine. Inhibitory nerve stimulation elicited inhibitory junction potentials (IJPs) and relaxations in wild-type mice. IJPs were reduced in amplitude and relaxation responses were absent in Sl/Sl(d) mice. These observations suggest that membrane-bound stem cell factor is essential for development of IC-IM and that the close, synaptic-like relationship between nerve terminals and IC-IM may be the primary site of innervation by enteric motor neurons in gastric muscles.

Mechanical efficiency of isolated in situ perfused hearts of the eel Anguillaaustralis

Administration of exogenous Ach to isolated in situ saline-perfused hearts of Anguilla australis via the perfusate, resulted in reduction in cardiac frequency (F). This reduction in F became significant at Ach concentrations of 10 −11 M or greater. A weak inotropic effect of Ach at the lowest concentration tested (10 −13 M) was also observed. Maximum power output of preparations was 2.94±0.26 mW gVM −1. The mechanical efficiency of A. australis hearts working at 25 and 50% of maximum power under different conditions of stroke volume (SV) and F was investigated. Cardiac frequency was manipulated using a combination of temperature and Ach administration. Stroke volume was manipulated by regulating input pressure of the perfusate supplying the preparations (pre-load). Values of MEF from preparations generating flow under conditions of low F/ high SV (treated with Ach 10 −7 M) were significantly greater than under conditions of high F/ low SV (untreated). The MEF of preparations appears to be related to inotropic state. The negative inotropy and increased mechanical efficiency produced by Ach appears to be the opposite of the so-called ‘oxygen-wasting’ effect produced by the positive inotropic agents, the catecholamines. This effect of Ach may be related to the dependence of teleost myocardium on extracellular Ca 2+ for excitation-contraction coupling during the cardiac cycle.

A pharmacological examination of venoms from three species of death adder ( Acanthophis antarcticus, Acanthophis praelongus and Acanthophis pyrrhus)

The common ( A. antarcticus), northern ( A. praelongus) and desert ( A. pyrrhus) death adders are species belonging to the Acanthophis genus. The present study compared some pharmacological aspects of the venoms of these species and examined the in vitro efficacy of death adder antivenom. Neurotoxicity was determined by the time to produce 90% inhibition ( t 90) of indirect (0.1 Hz, 0.2 ms, supramaximal voltage) twitches in the chick biventer cervicis nerve-muscle (3–10 μg/ml) and mouse phrenic nerve-diaphragm (10 μg/ml) preparations. A. praelongus venom was significantly less neurotoxic than A. antarcticus venom but was not significantly different from A. pyrrhus venom. In the biventer muscle, all three venoms (3–10 μg/ml) abolished responses to exogenous ACh (1 mM) and carbachol (20 μM), but not KCl (40 mM), indicating activity at post-synaptic nicotinic receptors. All venoms (30 μg/ml) failed to produce significant inhibition of direct twitches (0.1 Hz, 2.0 ms, supramaximal voltage) in the chick biventer cervicis nerve-muscle preparation. However, A. praelongus (30 μg/ml) venom initiated a significant direct contracture of muscle, indicative of some myotoxic activity. The prior (10 min) administration of death adder antivenom (1 unit/ml), which is raised against A. antarcticus venom, markedly attenuated the twitch blockade produced by all venoms (10 μg/ml). Administration of antivenom (1.5 units/ml) at t 90 markedly reversed, over a period of 4 h, the inhibition of twitches produced by A. praelongus (3 μg/ml, 72±6% recovery) and A. pyrrhus (3 μg/ml, 51±9% recovery) but was less effective against A. antarcticus venom (3 μg/ml, 22±7% recovery). These results suggest that all three venoms contain postsynaptic neurotoxins. Death adder antivenom displayed differing efficacy against the in vitro neurotoxicity of the three venoms.

Neurotoxic activity of venom from the Australian eastern mouse spider (Missulena bradleyi) involves modulation of sodium channel gating.

Mouse spiders represent a potential cause of serious envenomation in humans. This study examined the activity of Missulena bradleyi venom in several in vitro preparations. Whilst female M. bradleyi venom at doses up to 0.05 microl ml(-1) failed to alter twitch or resting tension in all preparations used, male venom (0.02 and 0.05 microl ml(-1)) produced potent effects on transmitter release in both smooth and skeletal neuromuscular preparations. In the mouse phrenic nerve diaphragm preparation, male M. bradleyi venom (0.02 microl ml(-1)) caused rapid fasciculations and an increase in indirectly evoked twitches. Male venom (0.02 and 0.05 microl ml(-1)) also caused a large contracture and rapid decrease in indirectly evoked twitches in the chick biventer cervicis muscle, however had no effect on responses to exogenous ACh (1 mM) or potassium chloride (40 mM). In the chick preparation, contractile responses to male M. bradleyi venom (0.05 microl ml(-1)) were attenuated by (+)-tubocurarine (100 microM) and by tetrodotoxin (TTX, 1 microM). Both actions of male M. bradleyi venom were blocked by Atrax robustus antivenom (2 units ml(-1)). In the unstimulated rat vas deferens, male venom (0.05 microl ml(-1)) caused contractions which were inhibited by a combination of prazosin (0.3 microM) and P(2X)-receptor desensitization (with alpha,beta-methylene ATP 10 microM). In the rat stimulated vas deferens, male venom (0.05 microl ml(-1)) augmented indirectly evoked twitches. Male venom (0.1 microl ml(-1)) causes a slowing of inactivation of TTX-sensitive sodium currents in acutely dissociated rat dorsal root ganglion neurons. These results suggest that venom from male M. bradleyi contains a potent neurotoxin which facilitates neurotransmitter release by modifying TTX-sensitive sodium channel gating. This action is similar to that of the delta-atracotoxins from Australian funnel-web spiders.

Pre- and postjunctional effects of inflammatory mediators in horse airways.

In addition to their direct contractile effects, histamine (Hist), serotonin [5-hydroxytryptamine (5-HT)], and leukotriene (LT) D(4), in low concentrations, dramatically augment electrical field stimulation (EFS)-induced smooth muscle contractions in equine airways. To determine the mechanism of their action, we studied, in trachealis strips, the effect of these mediators on both cholinergically induced tension and the release of ACh from cholinergic nerves. All three mediators synergistically augmented the contraction of the trachealis that was due to release of endogenous ACh, i.e., EFS-induced contraction. These same mediators caused only a small but parallel shift of the ACh concentration-response curve. Comparison of the mediator effects on the responses to endogenous and exogenous ACh suggested a prejunctional effect. However, release of ACh was augmented only by Hist and 5-HT but not by LTD(4). Hist-induced contraction of trachealis was abolished by pyrilamine (H(1)-receptor antagonist) but not by ranitidine (H(2)-receptor antagonist), whereas thioperamide (H(3)-receptor antagonist) shifted the Hist response curve to the left. The augmenting effect of Hist on EFS-induced contraction was abolished by pyrilamine and unaffected by ranitidine or thioperamide. We conclude that inflammatory mediators can increase endogenous cholinergic responses of equine airways via both prejunctional and postjunctional mechanisms. LTD(4) acts solely on smooth muscle, whereas 5-HT and Hist additionally act on neuronal receptors to facilitate release of ACh. Excitatory effects of Hist, i.e., direct contractile effect, and augmentation of endogenous cholinergic response are both mediated via H(1) receptors, whereas the inhibitory H(3) receptors partially oppose the direct contractile effect of this mediator.

Subtypes of muscarinic receptors regulating gallbladder cholinergic contractions.

The aim of this study was to determine the functional role of muscarinic receptor subtypes regulating gallbladder cholinergic contractions. Electrical field stimulation (EFS; 16 Hz) produced contractile responses of guinea pig gallbladder muscle strips in vitro that were inhibited by 1 microM tetrodotoxin (2 +/- 2% of control) and 1 microM atropine (1 +/- 1% of control), indicating activation of intrinsic cholinergic nerves. Exogenous ACh (5 microM)-induced contractions were inhibited by atropine (1 +/- 1% of control) but not tetrodotoxin (102 +/- 1% of control), indicating a direct effect on smooth muscle. The M1 receptor antagonist pirenzepine (10 nM) had no effect on ACh-induced contractions but inhibited EFS-induced contractions by 11 +/- 3%. The M2 antagonist methoctramine (10 nM) had no effect on ACh-induced contractions but augmented EFS-induced contractions by 5 +/- 2%. The M3 antagonist 4-DAMP (10 nM) inhibited ACh-induced contractions by 14 +/- 4% and EFS-induced contractions by 22 +/- 5%. In conclusion, specific M1, M2, and M3 receptors modulate gallbladder muscle contractions by regulating ACh release from cholinergic nerves and mediating the contraction. Cholinergic contractions are mediated by M3 receptors directly on the smooth muscle. M2 receptors are on cholinergic nerves and function as prejunctional inhibitory autoreceptors. M1 receptors are on cholinergic nerves and function as prejunctional facilitatory autoreceptors.

Mediators of anaphylaxis but not activated neutrophils augment cholinergic responses of equine small airways.

Neutrophilic inflammation in small airways (SA) and bronchospasm mediated via muscarinic receptors are features of chronic obstructive pulmonary disease in horses (COPD). Histamine, serotonin, and leukotrienes (LTs) are reported to be involved in the exacerbation of COPD, and currently, histamine has been shown to increase tension response to electrical field simulation (EFS) in equine SA. We tested the effects of these mediators and the effects of activated neutrophils on the cholinergic responses in SA. Histamine, serotonin, and LTD4 had a synergistic effect on EFS responses and only an additive effect on the tension response to exogenous ACh or methacholine. Atropine and TTX entirely eliminated the EFS-induced tension response in the presence of all three inflammatory mediators, indicating that augmentation of the EFS response applies only to the endogenous cholinergic response. Neutrophils isolated from control and COPD-affected horses were activated by zymosan, producing 18.1 +/- 2.3 and 25.0 +/- 2.3 nmol superoxide. 10(6) cells-1. 30 min-1, respectively. However, in contrast to the profound effect of mediators, incubation of SA for over 1 h in a suspension of up to 30 x 10(6) zymosan-treated neutrophils/ml did not significantly affect EFS responses of SA isolated from either control or COPD-affected horses. We conclude that in equine SA 1) the endogenous cholinergic responses are subject to strong facilitation by inflammatory mediators; 2) activated neutrophils do not affect cholinergic responses in SA; and 3) in acute bouts of equine COPD, histamine, LTD4, and serotonin (mediators primarily associated with type I allergic reaction) rather than mediators derived from neutrophils most likely contribute to increased cholinergic airway tone.

Non-monotonic contrast behavior in directionally selective ganglion cells and evidence for its dependence on their GABAergic input.

We serendipitously discovered that the preferred-direction responses of ON-OFF directionally selective (DS) ganglion cells in the rabbit retina fall as a function of contrast when the contrast of a moving bar exceeds about 100%. Null-direction responses did not fall for contrasts up to 400%. Because the non-monotonic (rise-then-fall) behavior as a function of contrast occurred only for preferred-direction responses, it must depend on the mechanism of directional selectivity. It became thus of interest to investigate how this non-monotonicity depends on the major synapses involved in directional selectivity. Blockades of nicotinic acetylcholine (ACh) and NMDA glutamate receptors reduced responses without eliminating preferred-response non-monotonicity. Blocking GABAergic inhibition, however, did eliminate non-monotonicity. These results pose a difficult puzzle, since in the accompanying paper (Grzywacz et al., 1998), we showed that residual responses under combined nicotinic and NMDA blockades are not statistically significantly directionally selective. How is it possible that null-direction GABAergic inhibition affects non-nicotinic-non-NMDA residual responses without generating directional selectivity? This may happen if there exists an asymmetric GABAergic input to distal dendrites of the DS cell while the excitatory, non-nicotinic-non-NMDA input is to proximal dendrites. In support of this hypothesis, bath-applied GABA reduces responses to exogenous ACh under synaptic block, providing for the first time in the rabbit's retina, direct evidence of GABA receptors on DS cells.

The central respiratory effects of acetylcholine vary with CSF pH

Hydrogen ion concentration [H +] centrally is a major determinant of ventilation. Its action involves central cholinergic mechanisms. The point(s) where increased [H +] induces its changes in the cholinergic system is unclear. If H + acts presynaptically by increasing endogenous ACh synthesis and release, its effect should be absent when ACh is supplied exogenously. If H + acts postsynaptically by changing ACh degradation or ACh receptor sensitivity, its effect should persist in the presence of exogenous ACh. We perfused the brain ventricular system in spontaneously breathing anesthetized dogs with progressively higher concentrations of ACh (0–52.8 mM) in cerebrospinal fluid (CSF) at pH 7.4 and CSF pH 7.1. Increasing concentrations of ACh increased ventilation >4-fold in a linear manner in the presence of non-acidic and acidic CSF. With acidic CSF the ACh ventilatory response line was shifted to a higher y-intercept, resulting in a higher ventilation at any [ACh]. These findings are consistent with the hypothesis that central acidosis augments ventilation by postsynaptic cholinergic events.

Effects of α2-adrenergic receptor agonist and antagonist drugs on cholinergic contraction in bovine tracheal smooth muscle in vitro

SUMMARY Effects of α2-adrenergic receptor stimulation on the cholinergic contractile response of bovine tracheal smooth muscle were studied. To determine the presence and function of α2-adrenergic receptors on cholinergic nerves innervating bovine tracheal muscle, effects of 2 α2-adrenoceptor agonists and an antagonist were determined. Muscular contractions were elicited by either electrical field stimulation (efs) or exogenous acetylcholine (ach). The contractile response to efs and exogenous ach was examined for each tissue. Electrical field stimulation of bovine tracheal smooth muscle caused contractions that were completely abolished by atropine, indicating the predominant excitatory innervation of bovine trachea is cholinergic. The α2-adrenoceptor agonists clonidine and medetomidine (10-6M to 10-4M) concentration-dependently inhibited the contractile response to efs but not the response to exogenous ach. Contractions induced by efs were significantly (P &lt; 0.05) inhibited in clonidine (10-4M)-treated tissues at low frequencies (0.1 to 10 Hz), whereas medetomidine (10-4M, 10-4M) inhibited contractions at all frequencies (0.1 to 30 Hz). Inhibitory effects of the α2-adrenoceptor agonists clonidine and medetomidine were attenuated by the (α2-adrenoceptor antagonist tolazoline. The α2-agonists used in this study appear to cause prejunctional inhibition of cholinergic nerves, because the smooth muscle contractions elicited by efs, but not exogenous ach, were inhibited, compared with controls.

Spontaneous quantal transmitter secretion from myocytes and fibroblasts: comparison with neuronal secretion

When exogenous ACh is loaded into the cytoplasm of cultured amphibian myocytes and fibroblasts, the cells undergo spontaneous quantal ACh secretion, as detected by the appearance of pulsatile membrane currents in Xenopus myocytes which are manipulated into contact with the cells. These currents resemble in many ways the miniature endplate currents (MEPCs) observed at developing neuromuscular synapses formed on these Xenopus myocytes. Analyses of the frequency, amplitude, and time course of these currents suggests similarity in the cellular mechanisms involved in the packaging and secretion of ACh quanta in fibroblasts, myocytes, and developing neurons. The size of the ACh packets released by the non-neuronal cells were found to be very similar to the size of the neuronal ACh quanta, which are thought to result from the exocytotic release of synaptic vesicles. Moreover, the kinetics with which the ACh packets are discharged from all three cell types are comparable, although the speed of secretion in non-neuronal cells is somewhat slower and more irregular. The spontaneous quantal ACh secretion from neurons and myocytes was decreased by reducing cytosolic Ca2+ level and enhanced by activation of protein kinase C with phorbol ester, but secretion from fibroblasts was unaffected by both treatments. The spontaneous secretion from fibroblasts did show some sensitivity to a rise in cytosolic Ca2+ after treatment with a Ca2+ ionophore. These observations support the hypothesis that the basic machinery for transmitter secretion operating in neurons derive from a more ubiquitous mechanism used for constitutive secretion and membrane trafficking in non-neuronal cells, and neuronal differentiation involves expression of additional unique components for the regulation of the spontaneous quantal secretion.