Presence Of ACh Research Articles

Angiotensin II-induced modulation of endothelium-dependent relaxation in rabbit mesenteric resistance arteries.

The role of local endogenous angiotensin II (Ang II) in endothelial function in resistance arteries was investigated using rabbit mesenteric resistance arteries. First, the presence of immunoreactive Ang II together with Ang II type-1 receptor (AT1R) and angiotensin converting enzyme (ACE) was confirmed in these arteries. In endothelium-intact strips, the AT1R-blocker olmesartan (1 microM) and the ACE-inhibitor temocaprilat (1 microM) each enhanced the ACh (0.03 microM)-induced relaxation during the contraction induced by noradrenaline (NA, 10 microM). Similar effects were obtained using CV-11974 (another AT1R blocker) and enalaprilat (another ACE inhibitor). The nitric-oxide-synthase inhibitor NG-nitro-L-arginine (L-NNA) abolished the above effect of olmesartan. In endothelium-denuded strips, olmesartan enhanced the relaxation induced by the NO donor NOC-7 (10 nM). Olmesartan had no effect on cGMP production (1) in endothelium-intact strips (in the absence or presence of ACh) or (2) in endothelium-denuded strips (in the absence or presence of NOC-7). In beta-escin-skinned strips, 8-bromoguanosine 3',5' cyclic monophosphate (8-Br-cGMP, 0.01-1 microM) concentration dependently inhibited the contractions induced (a) by 0.3 microM Ca2+ in the presence of NA+GTP and (b) by 0.2 microM Ca2++GTPgammaS. Olmesartan significantly enhanced, while Ang II (0.1 nM) significantly inhibited, the 8-Br-cGMP-induced relaxation. We propose the novel hypothesis that in these arteries, Ang II localized within smooth muscle cells activates AT1Rs and inhibits ACh-induced, endothelium-dependent relaxation at least partly by inhibiting the action of cGMP on these cells.

Liquid secretion inhibitors reduce mucociliary transport in glandular airways

Because of its possible importance in cystic fibrosis (CF) pulmonary pathogenesis, the effect of anion and liquid secretion inhibitors on airway mucociliary transport was examined. When excised porcine tracheas were treated with ACh to induce gland liquid secretion, the rate of mucociliary transport was increased nearly threefold from 2.5 +/- 0.5 to 6.8 +/- 0.8 mm/min. Pretreatment with both bumetanide and dimethylamiloride (DMA), to respectively inhibit Cl(-) and HCO secretion, significantly reduced mucociliary transport in the presence of ACh by 92%. Pretreatment with the anion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid similarly reduced mucociliary transport in ACh-treated airways by 97%. These agents did not, however, reduce ciliary beat frequency. Luminal application of benzamil to block liquid absorption significantly attenuated the inhibitory effects of bumetanide and DMA on mucociliary transport. We conclude that anion and liquid secretion is essential for normal mucociliary transport in glandular airways. Because the CF transmembrane conductance regulator protein likely mediates Cl(-), HCO, and liquid secretion in normal glands, we speculate that impairment of gland liquid secretion significantly contributes to defective mucociliary transport in CF.

Matrix Photochemistry of CH3C(O)SX Molecules with X = H, CH3, and C(O)CH3: Formation of Ketene in Another Decomposition Channel of Sulfenyl Carbonyl Compounds

CH 3 C(O)SC(O)CH 3 isolated in solid Ar, N 2 , or 5% CO-doped Ar at 15 K and CH 3 C(O)SH and CH 3 C(O)-SCH 3 isolated in solid Ar at 15 K were irradiated with broad-band UV-visible light (200 ≤ λ ≤ 800 nm). On the basis of the IR spectra of the matrixes, hydrogen abstraction from the CH 3 group to give ketene, H 2 C=C=O, with the elimination of HX [X = SC(O)CH 3 , SH, or SCH 3 ] was identified in each case as the main channel of photodecomposition within the confines of a solid matrix environment. The presence of a CH 3 ligand attached to the carbonyl function thus introduces a third type of elimination reaction for matrix-isolated sulfenyl carbonyl compounds of the general type XC(O)SY. Additionally, the conformational properties of the CH 3 C(O)SX molecules have been investigated. In the case of thioacetic acid (X = H), the matrix experiments indicate that the vapor at ambient temperatures consists of a mixture of the syn and anti forms, with the syn form predominating (ca. 85%). Broad-band UV-visible irradiation then results in interconversion of the conformers prior to decomposition. The identification of the different molecules has been underpinned by the results of ab initio and density functional theory (DFT) calculations.

NO donors potentiate the beta-adrenergic stimulation of I(Ca,L) and the muscarinic activation of I(K,ACh) in rat cardiac myocytes.

The effects of nitric oxide (NO) donors on the L-type Ca(2+) current (I(Ca,L)) and the muscarinic activated K(+) current (I(K,ACh)) were studied in isolated rat cardiac myocytes. The nitrosothiol S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 1 pM-1 microM) strongly potentiated the stimulation of the I(Ca,L) elicited by subthreshold concentrations of isoprenaline (Iso, 0.1-0.5 nM) in ventricular myocytes. The effect of SNAP was mimicked by 2-(N,N-diethylamino)-diazenolate-2-oxide (DEANO, 1 pM-1 nM), a NONOate that spontaneously releases NO in a pH-controlled manner, and was blunted by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (100 microM), a NO trap. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxaline-1-one (10 microM), a guanylyl cyclase inhibitor, did not alter the effect of SNAP. SNAP (1 pM-1 microM) did not modify the effect of L858051 (0.1-0.3 microM), a forskolin analogue that activates adenylyl cyclase, on I(Ca,L) and did not enhance the basal I(Ca,L) in the presence of rolipram (1 microM), a phosphodiesterase type 4 inhibitor. Superfusion with Rp-CPT-cAMPS (500 microM), or internal dialysis with cAMP-dependent protein kinase (cA-PK) inhibitory peptide (PKI; 20 microM), inhibitors of the cA-PK, blunted the effect of SNAP (1 nM and 1 microM) on the Iso-stimulated (1-100 pM) I(Ca,L). SNAP (1 nM and 1 microM) potentiated the threshold stimulation of I(Ca,L) elicited by internal GTP-gammaS (10 microM), a non-hydrolysable analogue of GTP. SNAP (1 pM-1 microM) and DEANO (1 microM) potentiated the stimulation of I(K,ACh) elicited by low concentrations of ACh (1-2 nM) in rat atrial myocytes. The threshold stimulation of I(K,ACh) elicited by internal 5'-guanylylimidodiphosphate (10 microM) was also potentiated by NO donors. SNAP (1 microM) did not modify I(K,ACh) reconstituted in human embryonic kidney 293 cells, in the absence or in the presence of ACh (1 or 10 nM). Taken together, these data suggest that NO is a cGMP-independent modulator of G-protein-coupled muscarinic and beta-adrenergic receptor actions on cardiac ion channels. Although this action of NO seemed to occur at the level of G proteins, it appeared to require a component distinct from receptors, G proteins or their effectors.

Calcium sensitization produced by G protein activation in airway smooth muscle.

We determined whether activation of G proteins can affect the force developed for a given intracellular Ca(2+) concentration ([Ca(2+)]; i.e., the Ca(2+) sensitivity) by mechanisms in addition to changes in regulatory myosin light chain (rMLC) phosphorylation. Responses in alpha-toxin-permeabilized canine tracheal smooth muscle were determined with Ca(2+) alone or in the presence of ACh, endothelin-1 (ET-1), or aluminum fluoride (AlF; acute or 1-h exposure). Acute exposure to each compound increased Ca(2+) sensitivity without changing the response to high [Ca(2+)] (maximal force). However, chronic exposure to AlF, but not to chronic ACh or ET-1, increased maximal force by increasing the force produced for a given rMLC phosphorylation. Studies employing thiophosphorylation of rMLC showed that the increase in force produced by chronic AlF exposure required Ca(2+) during activation to be manifest. Unlike the acute response to receptor agonists, which is mediated solely by increases in rMLC phosphorylation, chronic direct activation of G proteins further increases Ca(2+) sensitivity in airways by additional mechanisms that are independent of rMLC phosphorylation.

Inhibition of endothelium-dependent hyperpolarization by endothelial prostanoids in guinea-pig coronary artery.

1. In smooth muscle of the circumflex coronary artery of guinea-pig, acetylcholine (ACh, 10(-6) M) produced an endothelium-dependent hyperpolarization consisting of two components. An initial component that occurs in the presence of ACh and a slow component that developed after ACh had been withdrawn. Each component of the hyperpolarization was accompanied by an increase in membrane conductance. 2. Indomethacin (5 x 10(-6) M) or diclofenac (10(-6) M), both inhibitors of cyclooxygenase, abolished only the slow hyperpolarization. The initial hyperpolarization was not inhibited by diclofenac nor by nitroarginine, an inhibitor of nitric oxide synthase. 3. Both components of the ACh-induced hyperpolarization were abolished in the presence of atropine (10(-6) M) or high-K solution ([K+]0 = 29.4 mM). 4. The interval between ACh-stimulation required to generate an initial hyperpolarization of reproducible amplitude was 20 min or greater, but it was reduced to less than 5 min after inhibiting cyclooxygenase activity. Conditioning stimulation of the artery with substance P (10(-7) M) also caused a long duration (about 20 min) inhibition of the ACh-response. 5. The amplitude of the hyperpolarization generated by Y-26763, a K+-channel opener, was reproducible within 10 min after withdrawal of ACh. 6. Exogenously applied prostacyclin (PGI2) hyperpolarized the membrane and reduced membrane resistance in concentrations over 2.8 x 10(-9)M. 7. At concentrations below threshold for hyperpolarization and when no alteration of membrane resistance occurred, PGI2 inhibited the initial component of the ACh-induced hyperpolarization. 8. It is concluded that endothelial prostanoids, possibly PGI2, have an inhibitory action on the release of endothelium-derived hyperpolarizing factor.

Acid catalysed reactions of 5-formyluracils with enamines. A facile synthesis of 5-acylvinyluracils

5-Formyl-1,3-dimethyluracil ( 1) reacts with secondary amine derived enamines of ketones and aldehydes to provide 5-(acyvinyl)uracil derivatives. The presence of a CH 3 group at C-6 of 1 induces a competitive annulation reaction. Depending on the bulk of substituents 5-(acylvinyl)-uracils acquire cis -diene and E or Z configurations on the vinyl unit. Enamines derived from 1,3-cyclohexanedione and ethyl acetoacetate react with 1 in 1 : 2 stoichiometric manner to provide 5-(9-xanthenyl)uracil and 5-(6-cycohexadienyl)uracil (X-ray) derivatives.

Accentuated antagonism in canine subendocardium is not altered by chronic exercise.

Acetylcholine often affects cardiac action potential repolarization only during augmented adrenergic tone, i.e., the phenomenon of accentuated antagonism. Since chronic exercise involves repeated changes in autonomic outflow, we determined whether it also influenced adrenergic/cholinergic interactions in isolated canine cardiac tissue. Using standard micro-electrode techniques in thin ventricular subendocardial slices isolated from exercised (EX: 8-10 wk daily exercise) and sedentary (SED): 8-10 wk cage rest) dogs, we examined transmembrane potential responses to isoproterenol (ISO: 10(-8), 10(-7), 10(-6) M) and to ISO in the presence of ACH (10(-5) M). Control transmembrane characteristics at BCL = 500 ms were similar for EX (N = 8 dogs) and SED (N = 9 dogs). ISO (10(-6) M) decreased action potential duration at 50% repolarization (APD50): EX = -29 +/- 15 ms; SED = 11 ms and at 90% repolarization (APD90): EX = -37 +/- 17 ms; and SED = -24 +/- 14 ms (P > 0.05, EX vs SED). ACH alone did not alter APD. With ACH (10(-5) M), delta APD50 with ISO (10(-6) M) was -5 +/- ms and 0 +/- 5 ms for EX and SED, respectively; delta APD90 was -8 +/- 4 ms and -8 +/- 7 ms for EX and SED, respectively (P > 0.05, EX vs SED). Thus, ACH antagonized ISO-mediated acceleration of repolarization equally in both groups. Chronic daily exercise does not influence adrenergic/cholinergic interactions at the cellular level.

Inhibition of the acetylcholine-activated potassium current by endothelin-1 in atrial cardiomyocytes

Endothelin-1 (ET-1), is a 21 amino-acid peptide with potent inotropic and chronotropic actions in the heart. Relatively little is known about the underlying electrophysiological effects of the peptide. In this study the effects of ET-1 on the acetylcholine-activated potassium current (IK(Ach)) were investigated in the absence and presence of the receptor-selective antagonists, PD155080 (ETA receptor-selective) and RES-701 (ETB receptor-selective) in atrial cardiomyocytes. Cells were obtained from New Zealand White rabbits (2·5–3 kg) by enzymatic dissociation using collagenase. Potassium currents were recorded, in the presence of nifedipine (5 μM), using the whole cell ruptured patch-clamp technique. Low resistance electrodes (1–3 MΩ) were filled with potassium gluconate solution (pH 7·2). Cells were superfused with a modified Tyrode's solution. Following stabilization, control recordings were made using standard pulse protocols, and drugs were applied using a gravity fed microperfusion system. Data were expressed as mean±standard error, and comparisons were made using Friedman's ANOVA and Wilcoxon's Signed Ranks tests; p<0·05 was considered significant ET-1 (10 nM) alone did not affect the potassium current. Acetylcholine (Ach, 1 μM) increased (p<0·05) the potassium current to −1321±290 pA, from a control value of −955±191 pA, at a step potential of −100 mV. This change in current is attributed to IK(Ach) as it was blocked by atropine (10 μM). ET-1 (10 nM) inhibited (p<0.05) the IK(Ach) obtained in the presence of Ach (−882±88 compared to a control value of −870±98 pA) Ach also increased the holding current from +80±9 to ±242±38 pA, and this effect was blocked (p<0·05) in the presence of ET-1 (44±13 pA). The ETA receptor-selective antagonist, PD155080 (1 μM), prevented (p<0·05) the ET-1 induced inhibition of IK(Ach) (control vs Ach±ET-1+PD155080: −804±224 vs −1030±210 pA; at −100 mV; n=6). The ETB receptor-selective antagonist, RES-701 (1 μM), also prevented (p<0·05) the inhibitory effect of ET-1 on IK(Ach) (control vs Ach+ET-1+RES-701: −733±116 vs −913±137 pA; at −100 mV; n=6). However, unlike PD155080, RES-701 failed to sustain this inhibitory effect after a further 90 s exposure to Ach and ET-1 (control vs Ach+ET-1+RES-701; −733±116 vs 768±96 pA; at −100mV; n=6). In summary, ET-1 clearly inhibits the effects of Ach on IK(Ach) in rabbit atrial cardiomyocytes. This effect is mediated by an ETA receptor-subtype, and an RES-701 -sensitive ETB receptor subtype.

Electrophysiological Effects of Vasoactive Intestinal Peptide in Rabbit Atrium: a Modulation of Acetylcholine Activity

Vasoactive Intestinal Peptide (VIP) is a 28-amino acid peptide partially co-secreted with acetylcholine (Ach) in the atrial tissue. We studied the electrophysiological effects of VIP and Ach in rabbit isolated right atrium by the microelectrode technique. After a 10-min superfusion with VIP, action potential duration at 90% of repolarization (APD90) was lengthened by 23% (P=0.01) at the concentration of 10−8m(n=10), by 22% (P=0.004) at 10−7m(n=10) and by 33% (P=0.03) at 2×10−7m(n=5). To explain this APD90lengthening, we performed 10 other experiments with VIP 10−7m, including five preparations pretreated with verapamil (10−6m) for 20 min. In the five preparations not pretreated, APD90was increased by 27% (P=0.04) after 10 min but remained unchanged in those previously exposed to verapamil, suggesting that VIP is a calcium current activator. Ach (1.4×10−5m) was superfused in five other experiments and we observed a 31% decrease in APD90(P=0.04) at 10 min. After washout, we simultaneously perfused, on the same preparations, Ach (same concentration) and VIP (10−7m) for 10 min. The decrease in APD90(19%) was no longer significant. VIP (2×10−7m) lengthened cellular effective refractory periods (ERP) by 26% (P=0.04) after 10 min (n=5), whereas Ach (1.4×10−5m) decreased ERP by 33% (P=0.04) at 10 min (n=5). In conclusion, VIP lengthens atrial APD90, which may be the result of calcium current activation. In addition, VIP could modulate Ach activity in limiting APD90shortening in the presence of Ach and because of its opposite effect on atrial ERP. Therefore, VIP could be involved in the control of vagal atrial arrhythmias.

Acetylcholine receptor and calcium leakage activity in nondystrophic and dystrophic myotubes (MDX)

To determine whether the lack of dystrophin alters the occurrence of calcium leakage activity (CLA) and acetylcholine receptor (AChR) activity, the frequency of each event class was determined from several cell attached patches on nondystrophic and dystrophic (mdx) myotubes. The frequency of CLA observed in the presence of ACh was significantly (P < 0.05) elevated in mdx myotubes, an effect which was partly due to a significant (P < 0.05) increase in the proportion of cell attached patches that exhibited 100% CLA with no AChR activity. Areas of mdx and nondystrophic membrane that exhibited reduced or absent AChR activity had significantly (P < 0.01) and substantially elevated calcium leakage event frequencies. This inverse and discontinuous relationship between CLA and AChR activity provides further evidence that some CLA in dystrophic muscle is produced by clusters of AChRs that form unusual physical associations with the dystrophic cytoskeleton during the processes associated with receptor localization and stabilization.

Downregulation of muscarinic M2 receptors linked to K+ current in cultured guinea-pig atrial myocytes.

1. Desensitization of muscarinic K+ current (IK(ACh)) was studied in cultured atrial myocytes from guinea-pig hearts using whole-cell voltage clamp. 2. Three different types of desensitization could be identified. A fast component which upon rapid superfusion with ACh resulted in a partial relaxation of IK(ACh) within a few seconds to a plateau which was maintained in the presence of ACh. Recovery from this type of desensitization paralleled the decay of IK(ACh) after washout of the agonist. A second type of desensitization was observed within minutes. This was reversed around 10 min after washout of ACh. Both types were heterologous with regard to the A1 receptor and the novel phospholipid (Pl) receptor, both of which activate IK(ACh) via the same signalling pathway. 3. A third type of desensitization (downregulation) occurred upon exposure of the cultures for 24-48 h to the muscarinic agonist carbachol (CCh). The level of downregulation depended on the concentration of CCh (0.1 microM < or = [CCh] < or = 10 microM). No recovery was observed within 5 h after washout of CCh. Thereafter sensitivity to ACh slowly returned (half-time (t1/2), approximately 20 h). 4. Downregulation by CCh (0.1-5 microM) was characterized by an increase in EC50 for ACh with no reduction in maximum IK(ACh). With 5 microM CCh, EC50 was increased from 0.1 to 3.7 microM. At 10 microM CCh EC50 was increased to 15 microM and maximal current that could be evoked by ACh was reduced to 15%. 5. Downregulation by CCh was homologous with regard to A1 and Pl receptors. Maximum IK(ACh), assayed by a saturating concentration of Pl, was not reduced in downregulated cells, suggesting a mechanism localized at the M2 receptor. 6. The changes in the concentration-response curves can be accounted for by assuming an excess of M2 receptors relative to the subsequent component of the signalling pathway. 7. As the intact heart is under tonic vagal control, downregulation is likely to contribute to controlling the sensitivity of the heart to vagal activity in situ.

Differentiated effects of vasodilators on myogenic reactivity during partial inhibition of myogenic tone in pressurized skeletal muscle small arteries of the rat

Myogenic tone and reactivity were studied in isolated, cannulated and pressurized small branches of the femoral artery of the rat. Myogenic tone developed spontaneously under control conditions (1.5 mM Ca2+), reducing the diameter at 80 mmHg to 64 +/- 6% of the 'max 80', i.e. the diameter in Ca2+ free solution, which was 221 +/- 23 microns (mean +/- SD, n = 18). The calcium channel blockers verapamil and diltiazem, dose-dependently, decreased this myogenic tone with pIC50 values of 6.9 +/- 0.2 (n = 7) and 6.6 +/- 0.1 (n = 6), respectively. Myogenic reactivity was demonstrated under control conditions as a sustained decrease in diameter, by 5 +/- 3% of max 80 (after an initial, transient distension), in response to a step increase in transmural pressure from 80 to 140 mmHg. This response to the pressure increase was markedly inhibited when myogenic tone had been reduced by 50% with verapamil or diltiazem resulting, in fact, in an increased steady state diameter by 2 +/- 1 and 1 +/- 1% at 140 compared with 80 mmHg. However, if myogenic tone was reduced to the same extent by low extracellular Ca2+ (approximately 0.3 mM) the vessels constricted by 6 +/- 1% in response to the pressure increase, an effect comparable to that in control Ca2+. Moreover, 50% reductions in myogenic tone by ACh (approximately 0.1 microM) or pinacidil (approximately 0.3 microM) were associated with significantly enhanced reactivity; steady state diameter decreased by as much as 11 +/- 4 and 15 +/- 5% of max 80. These results suggest that voltage dependent L-type Ca2+ channels are involved both in myogenic tone and in the myogenic response to a rise in vascular transmural pressure in skeletal muscle arteries. Partial inhibition of myogenic tone by other pharmacological routes do not necessarily interfere with myogenic reactivity since the response was, in fact, enhanced in the presence of ACh or pinacidil.

Cholinergic modulation of activity-dependent synaptic plasticity in the piriform cortex and associative memory function in a network biophysical simulation

The effect of activation of cholinergic receptors on long-term potentiation (LTP) in rat piriform cortex pyramidal cells was studied using extracellular and intracellular recordings in brain slice preparations. The functional role of this modulation was studied in a realistic network biophysical stimulation. Repetitive stimuli were applied in two paradigms: one in which the recorded cell was held at its resting potential and one in which synaptic activity was superimposed on a depolarizing pulse strong enough to evoke four action potentials. In the absence of cholinergic modulation, stimulation at 5 Hz induced LTP primarily in the second condition (13.7%, n = 6 out of 9, measured at 10 min after tetanus). When stimuli were applied in the presence of the muscarinic agonist carbachol (20 microM), LTP of greater amplitude was induced in both paradigms (resting: 41.5%, n = 11 out of 16, depolarized: 36%, n = 5 out of 7, measured at 10 min after tetanus). Increases in excitatory postsynaptic potential (EPSP) amplitudes in the presence of carbachol were gradual, starting at the time 5 Hz stimuli were applied and continuing until an action potential was evoked synaptically. In the presence of the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV), LTP could not be induced. The muscarinic antagonist atropine also prevented LTP induction in the presence of carbachol. Cholinergic modulation of synaptic plasticity was examined in a previously developed realistic biophysical network simulation. In simulations, use of a gradual rate of synaptic modification prevented excessive strengthening of synapses, which could cause interference between stored patterns. The effect of excess synaptic strengthening can be avoided by introducing activity dependent depression of synaptic strength. Coactivation of learning and depression rules results in a stable system where no interference occurs, at any rate of learning. Implementing the depression rule only during recall does not improve the network's performance. This implies that reduction in the strength of synaptic connections should occur in the presence of ACh, more than in normal conditions. We propose that two effects of ACh--enhancement of LTP and enhancement of LTD--should act together to increase the stability of the cortical network in the process of acquiring information.

On the mechanism of basal and agonist-induced activation of the G protein-gated muscarinic K+ channel in atrial myocytes of guinea pig heart.

Using the patch clamp technique, we examined the agonist-free, basal interaction between the muscarinic acetylcholine (m-ACh) receptor and the G protein (GK)-gated muscarinic K+ channel (IK.ACh), and the modification of this interaction by ACh binding to the receptor in single atrial myocytes of guinea pig heart. In the whole cell clamp mode, guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma S) gradually increased the IK.ACh current in the absence of agonists (e.g., acetylcholine). This increase was inhibited in cells that were pretreated with islet-activating protein (IAP, pertussis toxin) or N-ethylmaleimide (NEM). In inside-out patches, even in the absence of agonists, intracellular GTP caused openings of IK.ACh in a concentration-dependent manner in approximately 80% of the patches. Channel activation by GTP in the absence of agonist was much less than that caused by GTP-gamma S. The agonist-independent, GTP-induced activation of IK.ACh was inhibited by the A promoter of IAP (with nicotinamide adenine dinucleotide) or NEM. As the ACh concentration was increased, the GTP-induced maximal open probability of IK.ACh was increased and the GTP concentration for the half-maximal activation of IK.ACh was decreased. Intracellular GDP inhibited the GTP-induced openings of IK.ACh in a concentration-dependent fashion. The half-inhibition of IK.ACh openings occurred at a much lower concentration of GDP in the absence of agonists than in the presence of ACh. From these results, we concluded (a) that the interaction between the m-ACh receptor and GK is essential for basal stimulation of IK.ACh, and (b) that ACh binding to the receptor accelerates the turnover of GK and increases GK's affinity to GTP analogues over GDP.

β-Agonists modulate ACh-inhibition of a K current in intestinal smooth muscle cells

ACh causes a long-lasting inhibition of STOCs via G proteins in intestinal smooth muscle cells. We examined the effects of isoproterenol (Iso) on the ACh-induced inhibition of STOCs in isolated ileal smooth muscle cells using the GΩ-seal whole cell clamp technique. In control, ACh (1 μM) completely suppressed STOCs, which did not desensitize over a period lasting 20 minutes. When Iso (10 μM) was added to the bath in the presence of ACh, the ACh-induced inhibition of STOCs was gradually removed. This effect of Iso was prevented by propranolot (10 μM). Application of Db-cAMP (500 μM) mimicked the Iso effects. Intracellulary applied GTP-γS (100 μM) gradually suppressed STOCs in the absence of ACh, which could not be removed by either Iso or Db-cAMP. These results suggest that β-adrenergic stimulation causes a removal of the muscarinic inhibition of STOCs via a cAMP-dependent process.

Cholinergically-induced changes: outward currents in hair cells isolated from the semicircular canal of the frog

Two cholinergically-induced modulations of membrane conductances have been identified in hair cells isolated from the crista ampullaris of the leopard frog (Rana pipiens), using the whole cell recording configuration of the patch clamp technique. Of 56 crista hair cells tested, 28 showed drug-induced changes in membrane current or membrane potential which were repeatable and could be reversed with washout of drug.The predominant effect (observed in 20 hair cells) of acetylcholine (Arch, 100μM to 1mM) or carbachol (1μM to 50μM) applied to these hair cells was the reduction of an outward current corresponding to a change in conductance of approximately −0.22 nS. This action by Ach on hair cells has been inferred from previous studies of afferent fiber discharge which reported an increase in firing rate with stimulation of efferent fibers or exogenous application of cholinomimetics (Bernard et al., 1985; Valli et al., 1986; Guth et al., 1986; Norris et al., 1988). The Ach-induced reduction in outward current was associated with a depolarization of the zero-current membrane potential by approximately +2.5 mV.In a total of 8 hair cells, an Ach-induced reversible increase in outward current was recorded. Changes in conductance were approximately +0.13 nS and were associated with a hyperpolarization of the zero-current membrane potential by approximately −2.2 mV. This current increase is likely to be responsible for the inhibitory post-synaptic potentials (IPSPs) which have previously been recorded intracellularly from acoustico-lateralis hair cells during stimulation of the efferent innervation (Flock and Russell, 1976; Ashmore and Russell, 1982; Art et al., 1984, 1985).Of the remaining 28 hair cells, six cells failed to exhibit any change in membrane conductance or membrane potential in the presence of cholinomimetics while an additional 15 cells exhibited decreases, and 7 cells exhibited increases in outward conductance, during application of Ach or carbachol, which were neither reversible with washout nor repeatable.The Ach-induced decrease in outward current could be reversible blocked by removal of Ca2+ from the external solution.The antagonism of the Ach-induced decrease on outward current by atropine (10−5 M) suggests that this current may correspond to a facilitatory become-preferring Ach receptor mediated response previously identified in the isolated semicircular canal (Norris et al., 1988).The enhanced conductance observed in the presence of Ach in some cells may be related to the suppressive actions of Ach in the isolated semicircular canal also reported by us (Norris et al. 1988) and to the inhibitory actions of efferent stimulation and Ach application previously investigated in turtle cochlear hair cells (Art et al., 1984).The possible identities of these conductances are discussed in relation to ionic currents previously identified in hair cells.

AH5183 and Cetiedil: Two Potent Inhibitors of Acetylcholine Uptake into Isolated Synaptic Vesicles from Torpedo marmorata

Synaptic vesicles purified on a sucrose-KCl sedimentation gradient were tested for their ability to accumulate [1-14C]acetylcholine ([1-14C]ACh) in the absence and in the presence of AH5183 and cetiedil. Kinetic studies of ACh transport showed that it was time dependent and saturable as a function of ACh concentration, with a KT of 1.2 mM. The protein-modifying agents N-ethylmaleimide and 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole were powerful inhibitors of ACh uptake. In agreement with other studies, AH5183 was found to be a potent inhibitor of ACh uptake by synaptic vesicles. Inhibition was of the mixed noncompetitive type, and the inhibition constant was 45.2 +/- 3.4 nM. Cetiedil, a drug that resembles ACh, was previously shown on intact nerve endings to inhibit the translocation of newly synthesized ACh into the synaptic vesicle compartment, and we demonstrate here that cetiedil is indeed an efficient blocker of ACh uptake by isolated synaptic vesicles. It acted as a competitive inhibitor, with a Ki of 118.5 +/- 9.5 nM. Neither ATP-dependent calcium uptake nor Mg2+-ATPase activity was affected by the drugs, a finding showing their specificity toward the ACh uptake process. The binding of L-[3H]AH5183 to intact vesicles was characterized in the absence or the presence of ACh or cetiedil. Saturation experiments showed a total binding capacity of approximately 126 pmol/mg of vesicular protein and a dissociation constant of 19.9 +/- 4.1 nM under control conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Desensitization of the isolated bovine iris sphincter muscles by pilocarpine.

We investigated the effect of pilocarpine on the bovine iris sphincter. This muscle contracted in a dose-dependent manner from 2 x 10(-6) M, with a maximal response at 3 x 10(-3) M. The ED50 values was (1.6 +/- 0.3) x 10(-4) M. Pilocarpine generated a desensitization while desensitization was not significant in the case of other cholinergic agents such as acetylcholine (Ach) and carbachol. Desensitization was profoundly increased in the presence of Ach, neostigmine or eserine: the responses to the second and third trials of pilocarpine were reduced to approximately 8-10% or 30% of the corresponding first responses. Pilocarpine reportedly releases transmitters and alters choline-acetyltransferase activity. These results taken together suggest that either variable Ach synthesis, inhibitory transmitter release or possible toxic action in high concentrations may be involved in the pilocarpine-induced responses of the bovine iris sphincter muscle. The desensitization and partial agonist-antagonist action of pilocarpine could not explain the characteristics of the bovine iris sphincter muscle.

Acetylcholinesterase in prenatal rat heart: a marker for the early development of the cardiac conductive tissue?

In rat embryos, acetylcholinesterase (AChE, EC 3.1.1.7) activity is present in a continuous sleeve of myocytes that extends from the myocardium that is adjacent to the atrioventricular endocardial cushions via the ventricular trabeculae to the outflow tract. No activity is found in the atrial roof, in the ventricular walls and in the interventricular septum except for its subendocardial surface. AChE-positive cells are first identified in 11-day rat embryos, while the prototypical distribution is best demonstrable in 13-day embryos. Part of the AChE-positive cell system is identifiable as a precursor of the adult conduction system by topographical criteria in 16-day fetuses and by morphological criteria in 20-day fetuses. At birth (2 days later), AChE activity has disappeared from the cardiac myocytes except for a ring of tissue at the atrial side of the atrioventricular junction. These findings suggest that the embryonic heart can be divided into an upstream myocardium that has no AChE activity and a downstream myocardium that is characterized by the presence of AChE. Furthermore they suggest that an acetylcholine-dependent mechanism may be responsible for the retardation of the depolarization wave in the downstream parts of the heart. Finally they show that the adult conduction system is formed by a transdifferentiation of part of a far more extensive embryonic precursor system.