A dose-response study of anticholinesterase drugs on contractile and phosphatidylinositol responses of rat trachea

We investigated whether anticholinesterase drugs in large doses inhibit muscarinic receptors of airway smooth muscle. In vitro measurements of isometric tension and [(3)H]inositol monophosphate (IP(1)) that formed were conducted by using rat tracheal rings or slices. Neostigmine and pyridostigmine caused muscular contraction and IP(1) accumulation in small doses (10 microM and < or = 100 microM, respectively), but they attenuated muscular contraction and IP(1) accumulation in larger doses (1000 microM). Edrophonium did not affect the smooth muscle tone and IP(1) levels. Neostigmine, pyridostigmine, and edrophonium attenuated the carbachol (5.5 microM)-induced smooth muscle contraction and IP(1) accumulation, when administered in large doses (1000 microM). The attenuation of contraction by neostigmine at large doses was not affected by methoctramine, an M(2) muscarinic receptor antagonist, but was reversed by washing with fresh Krebs-Henseleit solution. The results suggest that anticholinesterase drugs have dual effects on the tension and phosphatidylinositol responses of rat trachea. Large doses of anticholinesterase drugs cause airway smooth muscle relaxation, which may be seen in patients with myasthenia gravis who have received excessive anticholinesterase therapy. Implications: Neostigmine and pyridostigmine, but not edrophonium, have dual effects on the tension and phosphatidylinositol responses of rat trachea. Large doses of anticholinesterase drugs cause airway smooth muscle relaxation, which may be seen in patients with myasthenia gravis who have received excessive anticholinesterase therapy.     

Interactions of edrophonium with neostigmine in the rat trachea

The muscarinic M(3) receptor of airway smooth muscle has both an orthosteric binding site and an allosteric binding site. Edrophonium may bind to the allosteric site, resulting in the inhibition of the action of the orthosteric site. Therefore, we examined the effects of edrophonium on neostigmine-induced contractile and phosphatidylinositol responses of rat trachea. Neostigmine (100 micro M in final concentration) was added, and ring tension was examined by the addition of edrophonium. After the completion of the experiment, Krebs-Henseleit (K-H) solution containing both edrophonium and neostigmine was changed three times with fresh K-H solution, and the tension was recorded. Tracheal slices were incubated with [(3)H]myo-inositol and 100 micro M neostigmine in the presence or absence of edrophonium. The [(3)H]inositol monophosphate (IP(1)) was measured. Data were expressed as mean +/- SE. Statistical significance (P < 0.05) was determined with analysis of variance. Neostigmine-induced tension and IP(1) accumulation were attenuated by edrophonium at concentrations of 100 micro M or more. This attenuation was reversed to more than 80% of control levels by washing with fresh K-H solution. The results suggest that edrophonium would bind to the allosteric site, resulting in the inhibition of the action of the orthosteric site of muscarinic M(3) receptors of rat trachea. IMPLICATIONS: We examined the effects of edrophonium on neostigmine-induced contractile and phosphatidylinositol responses of rat trachea. Neostigmine-induced tension and inositol monophosphate accumulation were attenuated by edrophonium. This attenuation was reversed by washing. The results suggest that edrophonium would bind to the allosteric site.     

The Antiallodynic Effects of Intrathecal Cholinesterase Inhibitors in a Rat Model of Neuropathic Pain

Background: This study determined the effect of intrathecally administered cholinesterase inhibitors, edrophonium and neostigmine, on nerve injury-induced, touch-evoked allodynia and identified the pharmacologic characteristics of this action.

Methods: Rats were prepared with tight ligation of the left L5 and L6 spinal nerves and with lumbar intrathecal catheters fitted for long-term monitoring. Edrophonium (3, 10, 30, or 100 [micro sign]g) or neostigmine (0.3, 1, 3, or 10 [micro sign]g) was administered intrathecally. Tactile allodynia and motor weakness were assessed. To evaluate the pharmacologic characteristics of the activity, a muscarinic receptor antagonist or a nicotinic receptor antagonist was administered intrathecally before edrophonium or neostigmine was injected. To compare the action of subtype antagonists, the M1 muscarinic receptor antagonist pirenzepine, the M (2) antagonist methoctramine, the M3 antagonist 4-DAMP (diphenylacetoxy-N-methypiperidine), and the M4 antagonist tropicamide were administered intrathecally before cholinesterase inhibitors were injected.

Results: Intrathecal edrophonium or neostigmine produced a dose-dependent antagonism of the touch-evoked allodynia. Neostigmine resulted in a moderate effect on motor weakness at doses of 3 and 10 [micro sign]g. Pretreated with intrathecal atropine but not mecamylamine yielded a complete antagonism of the effects of the cholinesterase inhibitors. In addition, antiallodynia produced by edrophonium (100 [micro sign]g) was reversed by pretreatment with methoctramine, 4-DAMP, tropicamide, and pirenzepine. In the neostigmine (10 [micro sign]g) group, only M1 antagonist pirenzepine had a moderate effect on reversal of increased allodynic threshold.     

Comparison of anticholinesterases and their effects on acetylcholine-activated ion channels

Single-channel recording techniques have been used to examine interactions between anticholinesterases and ion channels activated by acetylcholine. Single-channel currents activated by 200 nM acetylcholine were recorded from cell-attached patches of BC3H1 mouse tumor cells grown in culture. Channels were recorded in the absence and presence of edrophonium (1-20 microM), neostigmine (2-20 microM), or pyridostigmine (10-200 microM). All three drugs shortened channel open time but did not alter single-channel current amplitude. Effects on channel open time were not secondary to inhibition of cholinesterase but appeared to involve direct interactions between anticholinesterase drugs and acetylcholine-activated channels. Drug concentrations calculated to reduce the time constant of open time distributions by 50% were 3.8 microM edrophonium, 4.6 microM neostigmine, or 97 microM pyridostigmine. Channel open time was decreased by edrophonium at concentrations comparable to those occurring during reversal of neuromuscular block, but it was reduced by neostigmine and pyridostigmine only at levels higher than those encountered clinically. Differences in interactions between anticholinesterases and acetylcholine-activated channels at the end plate may possibly account for some of the clinical differences between these drugs.     

Muscarinic Receptor Subtypes and Receptor-Coupled Phosphatidylinositol Hydrolysis in Rat Bladder Smooth Muscle

Background :
The purpose of this study was to evaluate the muscarinic receptor subtypes expressed in rat bladder smooth muscle and characterize the muscarinic receptor-coupled phosphatidylinositol (PI) hydrolysis in order to clarify the first step of bladder smooth muscle contraction.
Methods :
Expressions of mRNAs of muscarinic receptor subtypes were examined by Northern blot analysis. Changes in the mass of inositol 1,4,5-trisphosphate(IP₃) and the inhibitory effects of muscarinic subtype specific antagonists on PI hydrolysis were determined after carbachol stimulation.
Results :
mRNAs of m2 and m3 genes, encoding M2 and M3 receptors, were expressed in rat bladder smooth muscle. Carbachol produced a rapid increase of IP₃, which returned to the basal level within 30 seconds. 4-Diphenylacetoxyl-N-methylpiperidine methiodide (4-DAMP; M1 and M3 antagonist) strongly inhibited the PI hydrolysis, but methoctramine (M2 antagonist) partially inhibited it at 10^-4 mol/L The 1C₅₀ value for atropine was 9.5 times 10^-9 mol/L, for pirenzepine 6.4 times 10^-6 mol/L, and for 4-DAMP 1.5 times 10^-7 mol/L.
Conclusion :
M2 and M3 receptors are expressed in rat urinary bladder. Only M3 receptor was involved in the production of IP₃, which might induce the initial phase of contractile response in rat bladder smooth muscle after carbachol stimulation.     

Metoclopramide causes airway smooth muscle relaxation through inhibition of muscarinic M3 receptor in the rat trachea

Although metoclopramide, often used as an antiemetic, is reported to have an anticholinesterase action, the effect on airway smooth muscle remains unclear. We investigated the effect of metoclopramide on the contraction, phosphatidylinositol response, and binding affinity of muscarinic M(3) receptors in rat trachea preparations. Male Wistar rats were anesthetized and their tracheas excised and chopped into 3-mm-wide rings, 1-mm-wide slices, or frozen 10- microm-thick sections. Contraction was induced with 0.55 microM carbachol (CCh) and, 30 min later, metoclopramide (10 microM to 1 mM) was added. The slices were incubated with (3)[H]myo-inositol, 0.55 microM CCh, and metoclopramide, and the formation of (3)[H] inositol monophosphate was measured. A radioligand binding study was conducted to examine the effects of metoclopramide using [(3)H] 4-diphenylacetoxy-N-methyl-piperidine methobromide (4-DAMP), a muscarinic M(3) receptor antagonist, in sections of the trachea. Metoclopramide concentration dependently attenuated CCh-induced contraction and inositol monophosphate accumulation, and also attenuated the binding affinity of 4-DAMP to muscarinic M(3) receptors. The 50% inhibitory concentration of metoclopramide against the binding affinity of 4-DAMP to muscarinic M(3) receptors of rat trachea was 24 micro M. These findings suggest that the attenuation by metoclopramide of CCh-induced contraction and phosphatidylinositol response may be mediated through the muscarinic M(3) receptors. IMPLICATIONS: We investigated the effect of metoclopramide on the contraction, phosphatidylinositol response, and binding affinity of muscarinic M(3) receptors in rat trachea preparations. Our findings suggest that the attenuation by metoclopramide of carbachol-induced contraction and phosphatidylinositol response may be mediated through the muscarinic M(3) receptors.     

Anticholinesterase drugs stimulate smooth muscle contraction of the rat trachea through the Rho-kinase pathway

We performed this study to determine the effects of Rho-kinase inhibitors, Y-27632 and fasudil, on the anticholinesterase (anti-ChE)-induced contractile and phosphatidylinositol responses of the rat trachea. In vitro measurements of isometric tension and [3H] inositol monophosphate (IP1) that was formed were conducted by using rat tracheal rings or slices. Neostigmine- and pyridostigmine-induced contractions were almost completely inhibited by Y-27632 and fasudil at 30 microM each, whereas acetylcholine-induced contraction was inhibited incompletely, i.e., by 56% by Y-27632 and by 51% by fasudil, at 100 microM for each, respectively. The inhibitory effects of fasudil on neostigmine- and acetylcholine-induced contractions were completely reversed by calyculin-A, a myosin phosphatase inhibitor. Neostigmine-induced IP1 accumulation was attenuated by fasudil at 100 microM. The results suggest that anti-ChEs cause airway smooth muscle contraction, in part, through activation of the Rho-kinase pathway.     

Neostigmine,pyridostigmine and edrophonium as antagonists of deep pancuronium blockade

To compare the ability of equipotent doses of neostigmine, pyridostigmine and edrophonium to antagonize intense pancuronium neuromuscular blockade, one hundred and twenty ASA physical status I or II patients scheduled for elective surgery received 0.06 mg.kg-1 pancuronium during a thiopentone nitrous oxide-enflurane anaesthetic. Train-of-four stimulation was applied every 12 s and the force of contraction of the adductor pollicis muscle was recorded. In the first 60 patients, spontaneous recovery was allowed until ten per cent of initial first twitch height. Then neostigmine (0.005, 0.01, 0.02 or 0.05 mg.kg-1), pyridostigmine (0.02, 0.04, 0.1 or 0.2 mg.kg-1), or edrophonium (0.1, 0.2, 0.4 or 1 mg.kg-1) was injected by random allocation. Dose-response relationships were established from the measurement of first twitch height (T1) ten minutes later. From these, neostigmine, 0.04 and 0.08 mg.kg-1 was found to be equipotent to pyridostigmine, 0.2 and 0.38 mg.kg-1, and edrophonium, 0.54 and 1.15 mg.kg-1, respectively. These doses were given by random allocation to the next 60 patients, but at one per cent spontaneous recovery. Neostigmine, 0.04 mg.kg-1, produced a T1 of 73 +/- 4 per cent (mean +/- SEM), and a train-of-four ratio (TOF) of 39 +/- 3 per cent. This was significantly greater than with pyridostigmine, 0.2 mg.kg-1 (T1 = 50 +/- 6 per cent; TOF = 25 +/- 3 per cent), and edrophonium, 0.54 mg.kg-1 (T1 = 54 +/- 3 per cent; TOF = 17 +/- 2 per cent).(ABSTRACT TRUNCATED AT 250 WORDS)     

High concentrations of landiolol,a β<Subscript>1</Subscript>-adrenoceptor antagonist,stimulate smooth muscle contraction of the rat trachea through the Rho-kinase pathway

Purpose Gradually progressing contraction of airway smooth muscle is suggested to be due to the Rho-kinase signaling pathway. In our preliminary study in rat tracheas, landiolol, a β₁-adrenoceptor antagonist, at high doses caused gradually progressing contraction, and this contraction reached a plateau after 20 min. Therefore, this study was carried out to clarify whether landiolol could stimulate the Rho-kinase pathway or the phosphatidylinositol (PI) response in the rat trachea. Methods Seventy-eight male Wistar rats weighing 250–350 g were used for the experiments. Their tracheas were cut into 3-mm-wide ring segments or 1-mm-wide slices. Measurements of isometric tension and [³H] inositol monophosphate (IP₁) production were conducted, using these tracheal rings or slices. Data values are expressed as means ± SD, and statistical significance (P < 0.05) was determined using analysis of variance (ANOVA). Results Landiolol (700 μM)-induced contraction was completely inhibited by fasudil at 30 μM, while the landiolol-induced contraction was not inhibited by 4-diphenylacetoxy-N-methyl-piperidine methobromide (4-DAMP), ketanserin, or nicardipine. Landiolol did not stimulate IP₁ production. Conclusion These results suggest that high concentrations of landiolol could cause airway smooth muscle contraction through the Rho-kinase pathway, but not through the PI response coupled with muscarinic M₃ receptors, 5-HT receptors or the activation of L-type Ca²⁺ channels.     

Antinociception of intrathecal cholinesterase inhibitors and cholinergic receptors in rats

BACKGROUND: Intrathecal cholinesterase inhibitors have been shown to have an antinociceptive effect which is mediated through the spinal cholinergic receptors, mainly muscarinic receptor. Spinal nicotinic receptor also has been involved in the control of nociception. Authors characterized the respective role of muscarinic or nicotinic receptor for the antinociception of cholinesterase inhibitors and further determined the antinociceptive potency of them. METHODS: Rats were prepared with intrathecal catheters. Formalin-induced flinching response was regarded as a nociceptive behavior. RESULTS: Intrathecal neostigmine, physostigmine and edrophonium produced a dose-dependent suppression of flinching in both phases. Atropine and the M1 selective antagonist attenuated the effect of them, while the M2 selective antagonist did not affect. M3, M4 selective, and nicotinic receptor antagonists reversed the antinociception induced by edrophonium, but by neither neostigmine nor physostigmine. The ordering of potency was neostigmine > physostigmine > > edrophonium. CONCLUSION: These data indicate that the nicotinic receptor may be involved, at least in part, in the antinociceptive action of cholinesterase inhibitor at the spinal level, and M1 receptor subtype may be a common pharmacologic site of action. Moreover, neostigmine is more potent than physostigmine and edrophonium.     

Twitch depression and train-of-four ratio after antagonism of pancuronium with edrophonium, neostigmine, or pyridostigmine

During N2O-O2-halothane anesthesia pancuronium (3 mg/70 kg) was antagonized with neostigmine (2.5 or 5 mg/70 kg), pyridostigmine (10 or 20 mg/70 kg), or edrophonium (50 or 100 mg/70 kg) in 36 human subjects (6 in each group). Reversal was attempted at 10% spontaneous recovery of muscle twitch, which was measured using train-of-four stimulation. When first twitch tension was less than 70% of the control it was found that for the same tension, the train-of-four ratio was greater with edrophonium than with neostigmine, and greater with neostigmine than with pyridostigmine. It was concluded that the three antagonists have different mechanisms of action. In comparison with neostigmine, edrophonium is more and pyridostigmine is less effective at presynaptic (or fade) receptors.     

Sepsis attenuates the intensity of the neuromuscular blocking effect of d-tubocurarine and the antagonistic actions of neostigmine and edrophonium accompanying depression of muscle contractility of the diaphragm

BACKGROUND: Prolonged effects of non-depolarizing muscle relaxants in septic patients have been reported, although the influence of sepsis on neuromuscular transmission has not yet been clarified satisfactorily. These studies were intended to elucidate the influence of sepsis on neuromuscular transmission and on the action of drugs being utilized for regulation of muscle tone (a neuromuscular blocker and anti-cholinesterase (anti-ChE) drugs). METHODS: The effect of d-tubocurarine (dTc) on neuromuscular transmission and the antagonistic action of anti-ChE drugs (neostigmine and edrophonium) on dTc-induced twitch depression were estimated using sham control and septic rat nerve-hemidiaphragm preparations in vitro. Isometric twitch tension elicited by indirect (phrenic nerve) or direct (muscle) stimulation at 0.1 Hz was evaluated. RESULTS: Sepsis induced by panperitonitis attenuated the twitch tension elicited by indirect or direct stimulation (P < 0.01) without obvious morphological inflammatory damage to the diaphragm. dTc dose-dependently decreased twitch tension elicited by indirect stimulation (P < 0.01) less intensely in the septic group than in the sham group (P < 0.01). The antagonistic actions of the anti-ChE drugs on dTc (1 microM)-induced twitch depression were less intense in the septic group (P < 0.01 or 0.05). CONCLUSION: These results demonstrate that sepsis in the acute phase attenuates the effects of a neuromuscular blocker and anti-ChE drugs and depresses muscle contractility simultaneously.     

Antagonism of pancuronium- and pipecuronium-induced neuromuscular block

We have compared the antagonism of neuro muscular block producedby pipecuronium with pancuronium in 80 anaesthetized surgicalpatients using mechanomyography and electromyography. Pancuronium0.1 mg kg or pipecuronium 0.07 mg kg^–1 was given afterinduction of anaesthesia and neuromuscular block was adjustedto 75% twitch depression at the time of antagonism. The followingregimens were used: edrophonium 0.5 and 1.0 mg kg^–1, neostigmine0.04 mg kg^–1 pyridostigmine 0.3 mg kg^–1 and edrophonium0.25 mg kg^–1 with pyridostigmine 0.15 mg kg^–1. Antagonismwas evaluated also by the head lift test. There was no differencebetween the reversibility of neuromuscular block produced bypancuronium or pipecuronium. Edrophonium produced a significantlyfaster antagonism than neostigmine or pyridostigmine but onsetof action was not significantly faster than that of edrophoniumwith pyridostigmine. All regimens produced 100% (or near 100%)antagonism of twitch response within 15 min. However, TOF fadeantagonism was more complete with pyridostigmine, neostigmineand edrophonium 1.0 mg kg^–1 than with edrophonium 0.5mg kg^–1. The head lift test indicated somewhat less antagonismwith edrophonium 0.5 and 1.0 mg kg^–1. Using five monitoringmethods, the rank order of reversal potency was: pyridostigmine neostigmine > edrophonium 1.0 mg kg^–1 edrophonium+ pyridostigmine > edrophonium 0.5 mg kg^–1.     

Pharmacokinetics and pharmacodynamics of edrophonium in elderly surgical patients

The effect of age (over 70 yr) on the pharmacokinetics and pharmacodynamics of edrophonium was evaluated in seven patients aged 76-87 yr and in seven patients aged 27-57 yr. When elderly patients were compared with younger controls, the elderly exhibited a statistically significant decreased plasma clearance (5.9 +/- 2 versus 12.1 +/- 4 mL.kg-1.min-1) and a prolonged elimination half-life (84.2 +/- 17 versus 56.6 +/- 16 min). Pharmacodynamically, a higher concentration of edrophonium is required in elderly patients to produce the same effect as in the younger controls. This observation may be explained in part by changes in neuromuscular transmission that are a function of the aging process. In addition, even though plasma concentrations were significantly greater at every sampling point in the elderly than the younger group, there was no difference between either the maximum duration or the total duration of action of edrophonium in the two groups. The maximum duration of action of edrophonium in both groups was very brief (1.3-2.2 min). These results contrast with a previous study of the anticholinesterases neostigmine and pyridostigmine, in which the action of these drugs was significantly prolonged in elderly patients. Explanations for the observed differences between edrophonium and other anticholinesterases may relate to differences in chemical structure and the possibility that edrophonium produces antagonism of neuromuscular blockade by a different mechanism than neostigmine or pyridostigmine.     

Effects of ketamine on neostigmine-induced contractile and phosphatidylinositol responses of the rat trachea

Purpose. Neostigmine causes airway smooth muscle contraction through the direct stimulation of muscarinic receptors and the activation of phosphatidylinositol (PI) responses. Ketamine attenuates airway smooth muscle contraction. It is not clear whether ketamine attenuates neostigmine-induced airway smooth muscle contraction by inhibiting the PI response. This study was designed to examine the effects of ketamine on neostigmine-induced contractile and PI responses of the rat trachea. Methods. Thirty male Wistar rats weighing 250–350 g were used. In the experiment on the contractile response, active contraction was induced with 1 μM neostigmine in the presence or absence of ketamine. In the experiment on the phosphatidylinositol response, the trachea slices were incubated with [³H]myo-inositol, 1 μM neostigmine, or 100 μM aluminum fluoride, and ketamine. The formation of [³H]inositol monophosphate (IP₁), a degradation product of the phosphatidylinositol response, was measured with a liquid scintillation counter. Statistical significance (P < 0.05) was determined by analysis of variance. Results. Neostigmine 1 μM caused tracheal ring contraction. This contraction was attenuated by ketamine dose-dependently and reached resting tension at 100 μM. Neostigmine- and aluminum fluoride-induced IP₁ accumulation was also attenuated by ketamine. Conclusion. The results suggest that ketamine attenuates neostigmine-induced contractile responses, at least in part, through the inhibition of phospholipase C coupled with G protein in the PI response. Received: December 12, 2002 / Accepted: February 8, 2003 Acknowledgments. This study was supported in part by Grant-in-Aid for Scientific Research C, No. 10671421, from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. Address correspondence to: M. Saito     

Characterization of cholinergic receptors in Madin-Darby canine kidney cells.

Muscarinic-type cholinergic receptors coupled to the phosphoinositide (PI) second messenger system are reported to be present in the inner medullary collecting duct cells. Madin-Darby canine kidney (MDCK) cells have several characteristics of collecting duct cells and have been shown to respond to muscarinic agonists. To determine if MDCK cells have PI-coupled muscarinic receptors, the radioligand binding and the effects of cholinergic agonists and antagonists on PI hydrolysis in MDCK cells were studied. The specific binding of [3H]1-quinuclidinyl benzilate ([3H]QNB), a muscarinic antagonist, to MDCK cell membranes had a Kd = 88 +/- 7 pM and a Bmax = 1464 +/- 88 fmol/mg of protein. The displacement of [3H]QNB from MDCK cell membranes by various cholinergic antagonists and agonists showed the order of potency: atropine greater than 4-diphenylacetoxy N-methylpiperidine (4-DAMP) greater than p-fluorohexahydrosiladifenidol greater than pirenzepine greater than metoctramine greater than arecoline greater than carbachol. The cholinergic agonists carbachol and arecoline stimulated PI hydrolysis in a concentration-dependent manner with an EC50 of 3.7 and 1.3 microM, respectively. Muscarinic antagonists abolished carbachol-stimulated PI hydrolysis in the following order of potency: atropine greater than 4-DAMP greater than pirenzepine much greater than methoctramine. The order of potency of muscarinic antagonists is consistent with the characteristics of the M3 subtype of muscarinic receptors. It is concluded that: (1) muscarinic receptor density in MDCK cells is 50 times higher than that in inner medullary collecting duct cells; (2) muscarinic receptors in MDCK cells are putative M3 subtype; and (3) muscarinic receptors in MDCK cells are functionally coupled to the PI second messenger system. This intracellular messenger system may, at least, be partially responsible for the action of cholinergic agonists in these cells and in the kidney.     

Effects of vasopressors on contractile and phosphatidylinositol responses of rat trachea

Purpose. Vasopressors, such as dopamine (DA), norepinephrine (NE), and phenylephrine (Phe), are commonly used during anesthesia to increase blood pressure through α₁-adrenoceptors. The present study was designed to examine the effects of DA, NE, and Phe on the contractile and phosphatidylinositol (PI) responses of the rat trachea induced by a muscarinic agonist, carbachol (CCh). Methods. A rat tracheal ring was suspended between two stainless-steel hooks in Krebs-Henseleit (K-H) solution. Contraction was induced with 0.55 μM CCh, and 30 min later DA, NE, or Phe was added. The tracheal slices were incubated in K-H solution containing LiCl, ³[H]myo-inositol, and CCh in the presence or absence of DA, NE, or Phe. The ³[H]inositol monophosphate (IP₁) formed was measured. Results. CCh caused tracheal ring contraction. NE attenuated CCh-induced contraction at a dose of 1 μM or greater and had a maximal effect at 3 μM. DA and Phe did not affect CCh-induced contraction. CCh-induced IP₁ accumulation was potentiated significantly by NE and Phe, but not by DA. Conclusion. Although NE and Phe potentiated CCh-induced IP₁ accumulation, they could not potentiate CCh-induced contraction, suggesting that in clinical settings, vasopressors such as NE, DA, and Phe might be safely used in patients with asthma. Received: February 25, 2002 / Accepted: July 2, 2002 Acknowledgments. This study was supported in part by a Grant-in-Aid for Scientific Research C (no. 10671421), from the Ministry of Education, Science, and Culture, Japan. Address correspondence to: O. Shibata     

The role of M2 muscarinic receptor subtypes mediating contraction of the circular and longitudinal smooth muscle of the pig proximal urethra

PURPOSE: We investigated the characterization of muscarinic receptor subtypes in the female pig urethra. MATERIALS AND METHODS: The affinities of muscarinic antagonists against carbachol responses were calculated in normal and cyclic adenosine monophosphate (cAMP) elevated tissues (contraction with KCl and relaxation with isoprenaline) using longitudinal and transverse strips of urethra. RESULTS: In displacement experiments with 1-quinuclidinyl [phenyl-4-3H] benzilate inhibitory constant (pKi) values of 4-diphenylacetoxyl-N-methyl-piperidine methiodide (DAMP) M3 selective antagonist) and methoctramine (M2 antagonist) indicated the presence of the M2 receptor. In functional studies contraction responses to carbachol were greater in longitudinal than in circular muscle. After cAMP elevation the contraction response increased in circular muscles to the level close to that of longitudinal muscles but did not change significantly in cAMP elevated longitudinal muscle. On normal circular tissues 4-DAMP had high mean affinity (mean apparent pKB value 9.3) but with a Schild slope of less than unity (0.76). Methoctramine competitively antagonized carbachol responses (mean affinity [pA2] value 6.9). On normal longitudinal tissues 4-DAMP and methoctramine competitively antagonized carbachol responses (mean pA2 9.0 and 6.2, respectively). After cAMP elevation in circular tissues mean pA2 values for 4-DAMP (8.7) were significantly lower (p = 0.0015), and those for methoctramine (7.3) were significantly higher (p = 0.0193) than in normal tissues. In longitudinal tissues the mean pA2 value for methoctramine (6.9) was significantly greater than in normal tissues (p <0.0001) but the value for 4-DAMP (8.8) did not alter. CONCLUSIONS: Pig urethra appears to have predominantly M2 muscarinic receptors. Contraction of the normal urethra appears to be predominantly mediated by M2 and M3 receptors in circular muscle but by M3 receptors in longitudinal muscle. After cAMP elevation a contribution to contraction of M2 receptors appeared to be demonstrated in the 2 tissues but the involvement of M2 receptors appeared greater in circular muscle.     

Effects of cholinesterase inhibitors on the neuromuscular blocking action of suxamethonium

Prolonged neuromuscular block occurs when suxamethonium is givenafter neostigmine or pyridostigmine; however, studies of edrophoniumand suxamethonium have yielded conflicting results. We havestudied, therefore, interactions between suxamethonium and allthree anticholinesterases in rats anaesthetized with pentobarbitone.After recovery from an initial bolus of suxamethonium, saline,edrophonium, pyridostigmine or neostigmine was administeredand a second dose of suxamethonium was then given. All threeanticholinesterases prolonged the duration of neuromuscularblock (90% suppression to 50% twitch recovery) to 127(SEM 9)%,127(10)% and 138 (11)% of baseline for edrophonium, pyridostigmineand neostigmine, respectively. Recovery index (25% to 75% twitchrecovery) was increased also to 125 (9)%; 149 (10%) and 185(15)% of baseline, respectively for the three drugs. Presented in part at the 1992 annual meetings of the AmericanSociety of Anesthesiologists and the California Society of Anesthesiologists.     

Interaction of edrophonium with muscarinic acetylcholine M2 and M3 receptors

BACKGROUND: It has been reported that edrophonium can antagonize the negative chronotropic effect of carbachol. This study was undertaken to evaluate in detail the interaction of edrophonium with muscarinic Mz and M3 receptors. METHODS: A functional study was conducted to evaluate the effects of edrophonium on the concentration-response curves for the negative chronotropic effect and the bronchoconstricting effect of carbachol in spontaneously beating right atria and tracheas of guinea pigs. An electrophysiologic study was conducted to compare the effects of edrophonium on carbachol-, guanosine triphosphate (GTP)gama S-, and adenosine-induced outward K+ currents in guinea pig atrial cells by whole cell voltage clamp technique. A radioligand binding study was conducted to examine the effects of edrophonium on specific [3HIN-methylscopolamine (NMS) binding to guinea pig atrial (M2) and submandibular gland (M3) membrane preparations, and on atropine-induced dissociation of [3H]NMS. RESULTS: Edrophonium shifted rightward the concentration-response curves for the negative chronotropic and bronchoconstricting effects of carbachol in a competitive manner. The pA2 values for cardiac and tracheal muscarinic receptors were 4.61 and 4.03, respectively. Edrophonium abolished the carbachol-induced outward current without affecting the GTPgamma S- and adenosine-induced currents in the atrial cells. Edrophonium inhibited [3H]NMS binding to M2 and M3 receptors in a concentration-dependent manner. The pseudo-Hill coefficient values and apparent dissociation constants of edrophonium for M2 and M3 receptors were 1.02 and 1.07 and 21 and 34 microM, respectively. Edrophonium also changed dissociation constant values of [3H]NMS without affecting its maximum binding capacities. CONCLUSION: Edrophonium binds to muscarinic M2 and M3 receptors nonselectively, and acts as a competitive antagonist.