Concentrations Of Carbachol Research Articles

Leukotriene C(4) enhances the contraction of porcine tracheal smooth muscle through the activation of Y-27632, a rho kinase inhibitor, sensitive pathway.

1. An unsaturated fatty acid, leukotriene C(4) (LTC(4)), has a potent contractile effect on human airway smooth muscle, and has been implicated in the pathogenesis of human asthma. Using front-surface fluorometry with fura-PE3, the effect of LTC(4) on the intracellular Ca(2+) concentration ([Ca(2+)](i)) and tension were investigated in porcine tracheal smooth muscle strips. 2. The application of LTC(4) induced little or no contraction despite a small and transient increase in [Ca(2+)](i). In the presence of LTC(4), however, the contractions evoked by high K(+) depolarization or a low concentration of carbachol (CCh) were markedly enhanced without inducing any changes in the [Ca(2+)](i) levels, thus indicating that LTC(4) increases the Ca(2+) responsiveness of the contractile apparatus. This LTC(4)-induced increase in Ca(2+) responsiveness could partly be reproduced in the permeabilized preparation of tracheal smooth muscle strips. 3. The LTC(4)-induced enhancement of contraction was accompanied by an increase in myosin light chain (MLC) phosphorylation and was blocked by a rho kinase inhibitor (Y-27632), but not by either a PKC inhibitor (calphostin C) or a tyrosine kinase inhibitor (genistein). 4. These results indicated that, in porcine tracheal smooth muscle, LTC(4) enhances the contraction by increasing the Ca(2+) responsiveness of the contractile apparatus in a MLC phosphorylation dependent manner, possibly through the activation of the rho-rho kinase pathway.

Identification and characterization of muscarinic acetylcholine receptor subtypes expressed in human skin melanocytes.

The present study was designed to identify and characterize muscarinic acetylcholine receptors in normal human melanocytes. We used subtype-specific oligonucleotide primers to localize the five genetically defined mAChR mRNAs (ml through m5) by reverse transcription-polymerase chain reaction. These experiments showed that all five mAChR subtype mRNAs are expressed in melanocytes. The PCR products were verified by restriction analysis and Southern blotting. Receptors were visualized in cultures of normal human melanocytes and specimens of normal human skin by subtype-specific rabbit anti-receptor polyclonal antibodies. Radioligand binding assays with the lipophilic drug [3H]quinuclidinyl benzilate demonstrated approximately 9,000 high affinity binding sites/cell. Micromolar concentrations of muscarine or carbachol transiently increased intracellular Ca2+, which could be attenuated by atropine, demonstrating coupling of the receptors to mobilization of intracellular free Ca2+. Lower concentrations of muscarine induced spontaneous repetitive spike-like increases of intracellular Ca2+ which is characteristic for the activation of muscarinic receptors. These results indicate that normal human skin melanocytes express the ml, m2, m3, m4, and m5 subtypes of classic muscarinic acetylcholine receptors on their cell membrane and that these receptors regulate the concentration of intracellular free Ca2+, which may play an important physiologic role in melanocyte behavior and skin pigmentation.

Anti-muscarinic actions of mitoxantrone in isolated heart muscles of guinea pigs

A hypotheses that mitoxantrone is a competitive antagonist at muscarinic cholinergic receptors was examined in guinea-pig hearts. In isolated left atrial muscle preparations, electrically paced at 2 Hz, the muscarinic agonist, carbachol, caused a concentration-dependent decrease in developed tension. Mitoxantrone caused a parallel right-ward shift of the concentration–response curve for carbachol. Schild plots for the effect of mitoxantrone on the carbachol concentration–response relationship were linear with a slope of 0.88 which was not significantly different from the unity. The right-ward shift of the carbachol concentration–response relationship by mitoxantrone significantly reversed after an additional incubation with a mitoxantrone-free solution, although the reversal was incomplete after a 2-h incubation in the mitoxantrone-free solution. Mitoxantrone caused a concentration-dependent displacement of specific [ 3H]quinuclidinyl benzilate binding to membrane preparations obtained from ventricular muscles of guinea-pig hearts. These results indicate that mitoxantrone acts as a competitive antagonist for the muscarinic receptors.

Influence of gender and the oestrous cycle on in vitro contractile responses of the rat urinary bladder to cholinergic stimulation.

1. Experiments were done to determine the influence of gender and the oestrous cycle on rat urinary bladder contractility in response to cholinergic stimulation. 2. Bladder strips from female rats responded to high frequency stimulation with smaller contractile responses than did strips from males, and to low concentrations of carbachol with greater responses. The decreased responsiveness of bladder strips from female rats to electrical field stimulation can be primarily attributed to the rats in the oestrous stage of the oestrous cycle. 3. Bladder strips from female rats in all stages of the oestrous cycle were more sensitive to carbachol than those from males, but there were no differences in sensitivity to electrical field stimulation. 4. The contractile responses of strips from both male and female rats to carbachol were antagonized by muscarinic antagonists with the following rank order of affinity (pA(2)) estimates: 4-DAMP>>pirenzepine>methoctramine, suggesting that the receptor mediating contraction was the M3 subtype. There were no differences in pA(2) values between bladder strips from male and female rats. 5. The data indicate that responsiveness of bladder strips to electrical field stimulation and carbachol is altered in female rats in the oestrous stage of the oestrous cycle. Furthermore, gender influences the sensitivity of rat bladder to muscarinic stimulation.

The actions of metabolic inhibition on human detrusor smooth muscle contractility from stable and unstable bladders.

To determine the important cellular site(s) of action of a brief exposure to NaCN (chosen to reduce mitochondrial respiration and hence mimic cellular hypoxia) on the mechanical properties and regulation of intracellular [Ca2+] in human detrusor smooth muscle. Using muscle samples obtained from patients with stable and unstable bladders, to determine whether the unstable bladder is associated with changes in the functional properties of detrusor muscle under these circumstances. Materials and methods Experiments were conducted in vitro on muscle strips or isolated cells. Isometric tension was recorded in muscle strips during electrical stimulation or exposure to agonists. Intracellular [Ca2+] and [H+] were measured by epifluorescence microscopy, and cell autofluorescence measured as an index of mitochondrial function. There were no differences in the responses to electrical stimulation and varying concentrations of carbachol in muscle strips from stable and unstable bladders. NaCN (2 mmol/L) reduced the contraction induced by carbachol (10 micromol/L) by a mean (SD) of 43 (16)% and 56 (15)% in the two groups; the reduction in the unstable was significantly less than in the stable group. NaCN similarly reduced the response to 10 mmol/L caffeine, but had no effect on the KCl-induced contraction. NaCN significantly increased the resting sarcoplasmic [Ca2+] and attenuated the calcium transients evoked by carbachol and caffeine, but again had no effect on the KCl-induced transient. The reduction of the carbachol calcium transient was also less in cells from unstable bladders than in those from stable bladders. There was no effect of NaCN on intracellular pH, except for a brief, transient alkalosis. NaCN reduces both the contraction and Ca-transient to carbachol by reducing Ca2+ accumulation by intracellular stores, because the carbachol- and caffeine-evoked responses were similar. Any effect on transmembrane Ca2+ flux was minimal because there was no effect on KCl-induced responses. The greater resilience of tissue from unstable bladders to acute cellular hypoxia may reflect some adaptation acquired in vivo.

Microtubules Regulate Local Ca2+ Spiking in Secretory Epithelial Cells

The role of the cytoskeleton in regulating Ca(2+) release has been explored in epithelial cells. Trains of local Ca(2+) spikes were elicited in pancreatic acinar cells by infusion of inositol trisphosphate through a whole cell patch pipette, and the Ca(2+)-dependent Cl(-) current spikes were recorded. The spikes were only transiently inhibited by cytochalasin B, an agent that acts on microfilaments. In contrast, nocodazole (5-100 micrometer), an agent that disrupts the microtubular network, dose-dependently reduced spike frequency and decreased spike amplitude leading to total blockade of the response. Consistent with an effect of microtubular disruption, colchicine also inhibited spiking but neither Me(2)SO nor beta-lumicolchicine, an inactive analogue of colchicine, had any effect. The microtubule-stabilizing agent, taxol, also inhibited spiking. The nocodazole effects were not due to complete loss of function of the Ca(2+) signaling apparatus, because supramaximal carbachol concentrations were still able to mobilize a Ca(2+) response. Finally, as visualized by 2-photon excitation microscopy of ER-Tracker, nocodazole promoted a loss of the endoplasmic reticulum in the secretory pole region. We conclude that microtubules specifically maintain localized Ca(2+) spikes at least in part because of the local positioning of the endoplasmic reticulum.

The effect of the enantiomers of formoterol on inherent and induced tone in guinea-pig trachea and human bronchus.

The long-acting beta2-adrenoceptor agonist formoterol is, like all other members of this class of drugs, used as a racemate in the clinic. While the effects of the individual enantiomers have been studied on airway smooth muscle from guinea pig, comparable data on human bronchial smooth muscle are scanty or absent. Therefore, we compared the effects of the enantiomers of formoterol on inherent and induced tone in isolated human bronchi with that on guinea-pig trachea in vitro. The human bronchi either were studied under resting tension conditions or were precontracted with 10 microM carbachol or 0.1 mM histamine. The guinea-pig trachea was precontracted with 0.01, 0.1 or 1 microM carbachol. The racemate and (R,R)-formoterol caused a concentration-dependent relaxation of all preparations with an EC50 of about 1 nM. In the guinea-pig trachea, the concentration-effect curve for formoterol was moved to the right in response to an increased concentration of carbachol. In both human bronchus and guinea-pig trachea, (S,S)-formoterol was more than 1,000 times less potent than (R,R)-formoterol. Thus the relaxing effect of formoterol in human airways as well as in guinea-pig trachea was shown to lie with the (R,R)-enantiomer. Notably, (S,S)-formoterol did not exert any contractile effects within the tested concentration range in either airway preparation. Therefore, we conclude that with regard to relaxant effects the pure (R,R)-enantiomer of formoterol does not offer a benefit over the racemate.

Measurement of cellular stimulation through monitoring pH changes by bead injection fluorescence microscopy

This work presents a method for extracellular and intracellular pH measurements in live cells based on a combination of the bead injection (BI) technique and fluorescence microscopy. For extracellular pH measurement, cells are grown on fluorescent beads, packed into a small column by a sequential injection instrument, and fluorescence intensity from the beads stained by the indicator is recorded by a fluorescence microscope. The method is applied to quantifying carbachol stimulation of Chinese hamster ovary (CHO) cells transfected with the m1 muscarinic receptor and is verified by a glucose depletion experiment. The results yield an EC 50 value of 1 μM for carbachol, which is in reasonable agreement with the literature value 3 μM determined by an existing potentiometric technique for measuring acid release. The intracellular measurement utilizes CHO M1 cells growing on non-fluorescent beads. For this method the cells rather than the beads are stained by incubating them in a solution of the fluorescent pH indicator BCECF. The cells are also stimulated with carbachol and the intracellular pH dependent fluorescence from the cells is recorded. The results show dependence between intracellular pH changes and carbachol concentration and yield an EC 50 value of 4 μM.

Mapping of IP(3)-mediated Ca(2+) signals in single human neuroblastoma SH-SY5Y cells: cell volume shaping the Ca(2+) signal.

Fast confocal laser-scanning microscopy was used to study spatiotemporal properties of IP(3)-mediated Ca(2+) release signals in human SH-SY5Y neuroblastoma cells. [Ca(2+)](i) increases were not affected by ryanodine (30 microgM) or caffeine (10 mM) and largely insensitive to removal of external Ca(2+), indicating predominance of IP(3)-induced Ca(2+) release. Ca(2+) signals evoked by high concentration (10 microM) of the muscarinic agonist carbachol appeared as self-propagating waves initiating in cell processes. At low carbachol concentrations (500 nM) Ca(2+) changes in most cells displayed striking spatiotemporal heterogeneity. The Ca(2+) response in the cell body was delayed and had a smaller amplitude and a slower rise time than that in processes. Ca(2+) changes in processes either occurred in a homogeneous manner throughout the whole process or were sometimes confined to hot spots. Regional differences in surface-to-volume ratio appear to be critical clues that determine the spatiotemporal pattern of intracellular Ca(2+) release signals.

CAMP-dependent potentiation of the Ca 2+-activated release of the anionic fluorescent dye, calcein, from rat parotid acinar cells

A recent study indicates that elevation of [Ca 2+] i enhances the release of calcein, an anionic fluorescent dye, from isolated exocrine acinar cells, so cytoplasmic calcein is useful for monitoring the secretion of organic anions. In this study, we investigated the effect of cAMP on the calcein release evoked by elevation of [Ca 2+] i. Isoproterenol, forskolin and dibutyryl cyclic AMP (dbcAMP) did not induce the release of calcein from isolated parotid acinar cells, but they potentiated the carbachol-induced release of calcein. Although cytoplasmic calcein is released through an increase in [Ca 2+] i, isoproterenol potentiated the carbachol-induced release of calcein without affecting the increase in [Ca 2+] i evoked by a high concentration of carbachol (10 −6 M). Charybdotoxin, a K + channel blocker, inhibited both the carbachol-induced release and the potentiation by isoproterenol. However, the calcein permeation pathways mediating the carbachol-induced release and the isoproterenol-potentiated release exhibited distinct sensitivities to anion channel blockers. Our results indicate that the calcein release induced by carbachol is potentiated through an increase in intracellular levels of cAMP. Although both the Ca 2+-activated release and the cAMP-potentiated release may be coupled to Ca 2+-activated K + efflux, increases in both [Ca 2+] i and [cAMP] i may activate the calcein conduction pathway which is not activated by an increase in [Ca 2+] i alone.

Caffeine inhibits a low affinity but not a high affinity mechanism for cholecystokinin-evoked Ca2+ signalling and amylase release from guinea pig pancreatic acini.

Caffeine has been found to inhibit the formation and action of Ca2+-mobilizing inositol 1,4,5-trisphosphate (IP3) in pancreatic acinar cells. The aim of the present study was to investigate the effects of caffeine on cytoplasmic Ca2+ concentrations ([Ca2+]i) and amylase release in response to different agonists. [Ca2+]i was determined by cytofluorometry using fura-2 as indicator and amylase release with a substrate reagent. Stimulation with low concentrations of carbachol or cholecystokinin octapeptide (CCK-8) induces [Ca2+]i oscillations whereas higher concentrations cause sustained elevation of [Ca2+]i. The less efficacious agonists pilocarpine and CCK-JMV-180 evoke oscillations only. Caffeine inhibited carbachol-induced elevation of [Ca2+]i and amylase responses in a competitive manner, abolishing the responses to low and incompletely inhibiting the responses to high concentrations of the agonist. Also, the [Ca2+]i elevations by pilocarpine were abolished by caffeine. The effects on CCK-8-induced elevation of [Ca2+]i and amylase secretion were paradoxical, the caffeine inhibition being more pronounced at high than at low concentrations of CCK-8. This enigma was further emphasized by moderate effects of caffeine on the responses to CCK-JMV-180. The results indicate that carbachol, pilocarpine and high concentrations of CCK-8 elicit IP3-mediated responses and that CCK-JMV-180 and low concentrations of CCK-8 elevate [Ca2+]i and stimulate amylase release by another signal transduction mechanism.

Cholinergic induction of oscillations in the hippocampal slice in the slow (0.5–2 Hz), theta (5–12 Hz), and gamma (35–70 Hz) bands

Carbachol, a muscarinic receptor agonist, produced three distinct spontaneous oscillations in the CA3 region of rat hippocampal slices. Carbachol concentrations in the 4–13 μM range produced regular synchronized CA3 discharges at 0.5–2 Hz (carbachol-delta). Higher concentrations (13–60 μM) produced short episodes of 5–10 Hz (carbachol-theta) oscillations separated by nonsynchronous activity. Concentrations of carbachol ranging from 8–25 μM also produced irregular episodes of high-frequency discharges (carbachol-gamma, 35–70 Hz), in isolation or mixed with carbachol-theta and carbachol-delta. At carbachol concentrations sufficient to induce carbachol-theta, low concentrations of APV reversibly transformed carbachol-theta into carbachol-delta. Higher concentrations of D,L-2-amino-5-phosphonopentanoic acid (APV) reversibly and completely blocked carbachol-theta. A systematic study of the effects of carbachol shows that the frequency of spontaneous oscillations depended nonlinearly on the level of muscarinic activation. Field and intracellular recordings from CA1 and CA3 pyramidal cells and interneurons during carbachol-induced rhythms revealed that the hippocampal circuitry preserved in the slice was capable of spontaneous activity over the range of frequencies observed in vivo and suggests that the presence of these rhythms could be under neuromodulatory control. Hippocampus 10:187–197, 2000 © 2000 Wiley-Liss, Inc.

Cholinergic induction of oscillations in the hippocampal slice in the slow (0.5-2 Hz), theta (5-12 Hz), and gamma (35-70 Hz) bands.

Carbachol, a muscarinic receptor agonist, produced three distinct spontaneous oscillations in the CA3 region of rat hippocampal slices. Carbachol concentrations in the 4-13 microM range produced regular synchronized CA3 discharges at 0.5-2 Hz (carbachol-delta). Higher concentrations (13-60 microM) produced short episodes of 5-10 Hz (carbachol-theta) oscillations separated by nonsynchronous activity. Concentrations of carbachol ranging from 8-25 microM also produced irregular episodes of high-frequency discharges (carbachol-gamma, 35-70 Hz), in isolation or mixed with carbachol-theta and carbachol-delta. At carbachol concentrations sufficient to induce carbachol-theta, low concentrations of APV reversibly transformed carbachol-theta into carbachol-delta. Higher concentrations of D,L-2-amino-5-phosphonopentanoic acid (APV) reversibly and completely blocked carbachol-theta. A systematic study of the effects of carbachol shows that the frequency of spontaneous oscillations depended nonlinearly on the level of muscarinic activation. Field and intracellular recordings from CA1 and CA3 pyramidal cells and interneurons during carbachol-induced rhythms revealed that the hippocampal circuitry preserved in the slice was capable of spontaneous activity over the range of frequencies observed in vivo and suggests that the presence of these rhythms could be under neuromodulatory control.

Airway reactivity measured by barometric whole-body plethysmography in healthy cats

Abstract Objective To assess the validity of barometric whole-body plethysmography (BWBP) as a means of monitoring airway responses to induced bronchoconstriction in healthy cats. Animals 8 healthy cats without history of bronchopulmonary disease or exposure to indoor tobacco smoke. Procedure Cats were placed into a barometric plethysmograph with an internal volume of 38 L, and air flow was recorded at baseline and after carbachol (concentrations 0.005, 0.01, 0.02, 0.05, 0.1, and 0.2%) was introduced into the chamber. A dose-response curve was generated for several flow-derived measurements, and airway reactivity was determined by interpolation of the dose-response curve for enhanced pause. Results Peak inspiratory and expiratory flows increased significantly, but respiratory rate, inspiratory and expiratory times, relaxation time, and tidal volume did not differ significantly from baseline values. Flow-derived measurements (pause, enhanced pause, and end-expiratory pause) increased significantly at carbachol concentrations > 0.02%. Baseline measurements did not correlate with indices of airway reactivity. Conclusions and Clinical Relevance Airway reactivity can be measured by use of BWBP, which is non-invasive. Airway reactivity was highly variable among cats and was not a function of baseline airway caliber, suggesting that other intrinsic mechanisms may be important. (Am J Vet Res 1999;60:1487–1492)

Pharmacological and urodynamic changes in rat urinary bladder function after multiple pregnancies.

To investigate the changes in bladder function after multiple pregnancies and parturition in rats, and to establish links between the changes in voiding profiles and in vitro pharmacological responses. Cystometry was used in conscious virgin and multiparous female Wistar rats (2 weeks after the last parturition) with chronically implanted lines to continuously record bladder pressure during five reproducible voiding cycles. In vitro, detrusor muscle contractile responses induced by cumulative concentrations of KCl, carbachol, noradrenaline and alpha, beta-methylene-ATP (mATP) were compared. In multiparous rats, there was a significant increase in the amplitude of voiding pressure (+31%), bladder capacity (+83%) and residual volume (about threefold); 60% of the multiparous rats but only 10% of the virgin rats showed bladder instability during the filling phase. Cumulative concentration-response curves to KCl, expressed as the tension developed vs tissue weight, were identical in the two groups of rats. Contractile responses induced by carbachol (0.1-30 micromol/L) were significantly larger in multiparous than in virgin rats. Similarly, noradrenaline-induced contractions (0.3-10 micromol/L) were significantly higher for multiparous animals. However, the sensitivity of the detrusor muscle to mATP was not modified by multiple pregnancies. After multiple gestations, female rats develop bladder hypertrophy, bladder instabilities and a higher amplitude of voiding pressure associated with an increased residual volume. These altered patterns are similar to those found in rats after chronic infravesical outlet obstruction. We propose that pregnancies and parturition modify urinary bladder function, leading to a dysfunction similar to that induced by obstruction, and involving an increased sensitivity to adrenergic and cholinergic stimulation.

Mechanical strain modulates maximal phosphatidylinositol turnover in airway smooth muscle.

Mechanical strain regulates the maximal level of myosin light chain phosphorylation mediated by muscarinic activation in airway smooth muscle. Accordingly, we tested the hypothesis that mechanical strain regulates maximal phosphatidylinositol (PI) turnover (V(max)) coupled to muscarinic receptors in bovine tracheal smooth muscle. We found that PI turnover was not significantly length dependent in unstimulated tissues. However, carbachol-induced PI turnover was linearly dependent on muscle length at both 1 and 100 microM. The observed linear length dependence of PI turnover at maximal carbachol concentration (100 microM) suggests that mechanical strain regulates V(max). When carbachol concentration-PI turnover relationships were measured at optimal length and at 20% optimal length, the results could be explained by changes in V(max) alone. To determine whether the length-dependent step is upstream from heterotrimeric G proteins, we investigated the length dependence of fluoroaluminate-induced PI turnover. The results indicate that fluoroaluminate-induced PI turnover remained significantly length dependent at maximal concentration. These findings together suggest that regulating functional units of G proteins and/or phospholipase C enzymes may be the primary mechanism of mechanosensitive modulation in airway smooth muscle.

Development of Intracellular Calcium Measurement by Time‐resolved Photon‐counting Fluorescence

Calcium green I, a ratiometric probe based on fluorescence lifetime measurements, was used to monitor intracellular calcium activity ([Ca2+]i) in RINm5F cells using a time-resolved fluorescence confocal microscope. The probe affinity constant has been recalibrated in single cells using ionomycin as a calcium ionophore and ethylenebis(oxyethylenenitrilo)tetraacetic acid as a calcium buffer; Kd was found to equal 150 nmol/L. The kinetics of ionomycin equilibration showed that the calcium release from calcium stores occurs before equilibration with extracellular calcium. The response to the muscarinic agonist carbachol, measured on 17 cells receiving three consecutive applications was characterized both by a [Ca2+]i peak lasting 50 s without any trailing plateau and by desensitization with a 30% decrease in the response. The dose-dependent response was obtained for a carbachol concentration from 5 mumol/L to 0.5 mmol/L. The ability of our set-up to obtain a value every 10 ms enabled us to record asynchronous spikes of [Ca2+]i in the RINm5F cells. The spikes, lasting less than 1 s, are significantly bigger than the noise, and they are not observed in the colonic HT29 cells.

Methodologic aspects of acetylcholine-evoked relaxation of rabbit aorta

The acetylcholine-evoked relaxation of rabbit isolated thoracic aorta precontracted by phenylephrine was studied. Phenylephrine caused a steady contraction that was maintained for 6 h. In the presence of calcium disodium ethylenediaminetetraacetate (EDTA) and ascorbic acid the contraction decreased with time. N G-Nitro- l-arginine abolished the inhibitory effect of EDTA and ascorbic acid. Acetylcholine evoked a rapid concentration-dependent relaxation that recovered spontaneously and slowly, but fully, with time. Relaxation evoked by equieffective concentrations of carbachol and acetylcholine had the same time course. Cumulative addition of acetylcholine (10 −7 − 3 × 10 −5 M) caused a marked relaxation that was reverted slightly at high concentrations. The relaxation was the same with rings derived from the upper, middle, and lower part of the thoracic aorta. Two consecutive concentration-response curves for acetylcholine obtained at a 2-h interval demonstrated a slight development of tachyphylaxis. The relaxation was inversely related to precontractile tension evoked by phenylephrine when expressed as a percentage, but independent when expressed as g tension. Storage of aorta in cold salt solution for 24 h did not alter the relaxation. EDTA and ascorbic acid did not alter the relaxation. It is concluded that (1) EDTA and ascorbic acid can not be used with impunity to stabilize catecholamines used as preconstriction agents; (2) the reversal of the acetylcholine-evoked relaxation is not due to hydrolysis of acetylcholine; (3) the relaxation is uniform in all segments of thoracic aorta; (4) cold storage of aorta does not alter the relaxation; and (5) acetylcholine releases the same amount of relaxing factor, irrespective of the precontractile tension.

Non‐Muscarinic and Non‐Nicotinic Inhibition by the Acetylcholine Analogue Carbachol of the Delayed Rectifier Potassium Current, ik, in Rabbit Isolated Sino‐Atrial Node Cells

The effect of carbachol, an analogue of acetylcholine, on the delayed rectifier potassium current, iK, was investigated in rabbit isolated sino-atrial node cells using the whole cell patch clamp technique with amphotericin-permeabilized patches. In the presence of 500 nM atropine and 500 nM hexamethonium to block muscarinic and nicotinic receptors, respectively, 500 nM carbachol decreased the amplitude and rate of deactivation of iK without, however, affecting the slope of the iK activation curve. The same concentration of carbachol decreased the pacemaking rate of spontaneously active sino-atrial node cells by more than 13%. Thus, there is a non-muscarinic and non-nicotinic pathway for cholinergically induced reduction in the amplitude and rate of deactivation of iK that would appear to contribute to negative chronotropy in rabbit sinoatrial node pacemaker cells.

In vitro investigation of the bladder-vascular selectivity of levcromakalim and YM934 in human tissues.

To examine the potencies of the potassium-channel openers (KCOs) levcromakalim and YM934 as relaxing agents on human detrusor and human mesenteric artery smooth muscle, and to determine their bladder-vascular selectivity in vitro. Strips of human detrusor muscle and mesenteric artery (with the endothelium removed) were set up in physiological salt solution and the tension developed by the tissues recorded. Tissues were precontracted with a concentration of carbachol (detrusor) or phenylephrine (artery) which caused 80% of maximal contraction, and relaxation responses to levcromakalim and YM934 were then obtained. Both KCOs caused relaxation of the bladder detrusor muscle and the mesenteric artery. Maximal responses, when plotted as a percentage of the precontraction, were greater for both KCOs in the bladder muscle than the artery, but the differences were small and not statistically significant. The sensitivity (drug potency) of the detrusor muscle to the KCOs was more than twice that of the artery but this selectivity was only statistically significant for YM934. Only minor bladder-vascular selectivity for levcromakalim and YM934 could be detected in vitro. This suggests that neither drug would be tolerated clinically, although the results suggest that further development of bladder-selective KCO agents appears feasible.