Absence Of Acetylcholine Research Articles

Role of the Outer β-Sheet in Divalent Cation Modulation of α7 Nicotinic Receptors

alpha-7 Nicotinic acetylcholine receptors (AChRs) exhibit a positive modulation by divalent cations similar to that observed in other AChRs. In the chick alpha7 AChR, this modulation involves a conserved glutamate in loop 9 (Glu172) that undergoes agonist-dependent movements during activation. From these observations, we hypothesized that movements of the nearby beta-sheet formed by the beta7, beta9, and beta10 strands may be involved in agonist activation and/or divalent modulation. To test this hypothesis, we examined functional properties of cysteine mutations of the beta7 and beta10 strands, alone or in pairs. We postulated that reduced flexibility or mobility of the beta7/beta9/beta10-sheet as a result of introduction of a disulfide bond between the beta strands would alter activation by agonists. Using a nondesensitizing alpha7 mutant background (L247T), we identified one mutant pair, K144C + T198C, that exhibited a unique characteristic: it was fully activated by divalent cations (Ca2+, Ba2+, or Sr2+) in the absence of acetylcholine (ACh). Divalent-evoked currents were blocked by the alpha7 antagonist methyllycaconitine and were abolished when Glu172 was mutated to glutamine. When the K144C + T198C pair was expressed in wild-type alpha7 receptors, activation required both ACh and divalent cations. We conclude that the introduction of a disulfide bond into beta7/beta9/beta10 lowers the energetic barrier between open and closed conformations, probably by reducing the torsional flexibility of the beta-sheet. In this setting, divalent cations, acting at the conserved glutamate in loop 9, act as full agonists or requisite coagonists.

The modulation of GIRK channels by halothane is essentially determined by its C terminus

G protein-activated K+ channels (GIRK1 or Kir3) are targets for volatile anaesthetics. The modulation by halothane of channels coexpressed with the m2 acetylcholine (ACh) receptor in Xenopus oocytes is characterized by inhibition of the agonist activated current through GIRK1F137S and GIRK2 channels. In contrast, in the absence of ACh, halothane is able to induce GIRK1F137S mediated currents. Such an increase in background currents by halothane was never observed with GIRK2 channels. Deleting the C terminus of GIRK2 (GIRK2Δ356) rendered the channel insensitive against the inhibition by halothane; in contrast, like GIRK1F137S channels GIRK2Δ356 channels were activated by halothane in the absence of an agonist. Because the C terminus is crucial for these effects, an interaction of halothane with the channel seems to be involved in the mechanism of GIRK current modulation.

Disruption and Recovery of Patterned Retinal Activity in the Absence of Acetylcholine

Many developing neural circuits generate synchronized bursting activity among neighboring neurons, a pattern thought to be important for sculpting precise neural connectivity. Network output remains relatively constant as the cellular and synaptic components of these immature circuits change during development, suggesting the presence of homeostatic mechanisms. In the retina, spontaneous waves of activity are present even before chemical synapse formation, needing gap junctions to propagate. However, as synaptogenesis proceeds, retinal waves become dependent on cholinergic neurotransmission, no longer requiring gap junctions. Later still in development, waves are driven by glutamatergic rather than cholinergic synapses. Here, we asked how retinal activity evolves in the absence of cholinergic transmission by using a conditional mutant in which the gene encoding choline acetyltransferase (ChAT), the sole synthetic enzyme for acetylcholine (ACh), was deleted from large retinal regions. ChAT-negative regions lacked retinal waves for the first few days after birth, but by postnatal day 5 (P5), ACh-independent waves propagated across these regions. Pharmacological analysis of the waves in ChAT knock-out regions revealed a requirement for gap junctions but not glutamate, suggesting that patterned activity may have emerged via restoration of previous gap-junctional networks. Similarly, in P5 wild-type retinas, spontaneous activity recovered after a few hours in nicotinic receptor antagonists, often as local patches of coactive cells but not waves. The rapid recovery of rhythmic spontaneous activity in the presence of cholinergic antagonists and the eventual emergence of waves in ChAT knock-out regions suggest that homeostatic mechanisms regulate retinal output during development.

Agrin promotes synaptic differentiation by counteracting an inhibitory effect of neurotransmitter.

Synaptic organizing molecules and neurotransmission regulate synapse development. Here, we use the skeletal neuromuscular junction to assess the interdependence of effects evoked by an essential synaptic organizing protein, agrin, and the neuromuscular transmitter, acetylcholine (ACh). Mice lacking agrin fail to maintain neuromuscular junctions, whereas neuromuscular synapses differentiate extensively in the absence of ACh. We now demonstrate that agrin's action in vivo depends critically on cholinergic neurotransmission. Using double-mutant mice, we show that synapses do form in the absence of agrin provided that ACh is also absent. We provide evidence that ACh destabilizes nascent postsynaptic sites, and that one major physiological role of agrin is to counteract this "antisynaptogenic" influence. Similar interactions between neurotransmitters and synaptic organizing molecules may operate at synapses in the central nervous system.

Ketamine Attenuates Acetylcholine-induced Contraction by Decreasing Myofilament Ca2+Sensitivity in Pulmonary Veins

The authors investigated the extent and cellular mechanisms by which the intravenous anesthetic ketamine alters acetylcholine-induced contraction in pulmonary veins (PVs). They tested the hypothesis that ketamine inhibits acetylcholine contraction in PVs. Canine PV rings with endothelium (E+) and without endothelium (E-) were isolated for measurement of isometric tension. The effects of ketamine (10(-5) m approximately 10(-3) m) on acetylcholine contraction were assessed in E+ and E- rings. The effects of inhibiting nitric oxide synthase on ketamine-induced changes in acetylcholine contraction were investigated in E+ rings, whereas the effects of Ca2+ influx and Ca2+ release were investigated in E- rings. In fura-2 loaded E- PV strips, the effects of ketamine (10(-4) m) on the intracellular Ca2+ concentration-tension relation (i.e., myofilament Ca sensitivity) were assessed in the presence or absence of acetylcholine. The roles of the protein kinase C and rho-kinase signaling pathways in ketamine-induced changes in myofilament Ca2+ sensitivity were also investigated. Ketamine caused dose-dependent (P < 0.001) inhibition of acetylcholine contraction in E+ and E- PV rings. The ketamine-induced attenuation of acetylcholine contraction was still observed after inhibition of nitric oxide synthase (P = 0.002), Ca2+ influx (P < 0.001), and Ca2+ release (P = 0.021). Ketamine alone had no effect on myofilament Ca2+ sensitivity (P = 0.892) but attenuated (P = 0.038) the acetylcholine-induced increase in myofilament Ca2+ sensitivity. This attenuation was still observed after rho-kinase inhibition (P = 0.039), whereas it was abolished by protein kinase C inhibition (P = 0.798). Ketamine attenuates acetylcholine contraction by inhibiting the acetylcholine-induced increase in myofilament Ca2+ sensitivity, which is mediated by the protein kinase C signaling pathway.

The Sensitivity of G Protein-activated K+ Channels toward Halothane Is Essentially Determined by the C Terminus

G protein-activated K(+) channels (GIRKs or Kir3.x) are targets for the volatile anesthetic, halothane. When coexpressed with the m(2) acetylcholine (ACh) receptor in Xenopus oocytes, agonist-activated GIRK1(F137S)- and GIRK2-mediated currents are inhibited by halothane, whereas in the absence of ACh, high concentrations of halothane induce GIRK1(F137S)-mediated currents. To elucidate the molecular mechanism of halothane action on GIRK currents of different subunit compositions, we constructed deletion mutants of GIRK1(F137S) (GIRK1(Delta363*)) and GIRK2 (GIRK2(Delta356)) lacking the C-terminal ends, as well as chimeric GIRK channels. Mutated GIRK channels showed normal currents when activated by ACh but exhibited different pharmacological properties toward halothane. GIRK2(Delta356) showed no sensitivity against the inhibitory action of halothane but was activated by halothane in the absence of an agonist. GIRK1(Delta363*) was activated by halothane more efficiently. Currents mediated by chimeric channels were inhibited by anesthetic concentrations that were at least 30-fold lower than those necessary to decrease GIRK2 wild type currents. Glutathione S-transferase pulldown experiments did not show displacement of bound Gbetagamma by halothane, indicating that halothane does not interfere with Gbetagamma binding. Single channel experiments revealed an influence of halothane on the gating of the channels: The agonist-induced currents of GIRK1 and GIRK2, carried mainly by brief openings, were inhibited, whereas higher concentrations of the anesthetic promoted long openings of GIRK1 channels. Because the C terminus is crucial for these effects, an interaction of halothane with the channel seems to be involved in the mechanism of current modulation.

Modulatory Effect of Acetylcholine on Gonadotropin-Stimulated Human Granulosa Cell Steroid Secretion

The aim of this study was to explore the direct action of acetylcholine on gonadotropin-stimulated progesterone (P) and estradiol (E₂) secretion of human granulosa cells (GCs) cultured in serum-free medium. Human GCs were isolated from preovulatory follicular fluid aspirated from 22 women undergoing in vitro fertilization at the University Women’s Hospital of Tübingen. The production of progesterone and E₂ was measured in the presence and absence of acetylcholine, carbachol, atropine, luteinizing hormone (LH) or follicle-stimulating hormone (FSH) using radioimmunoassay. Statistical analysis of the data was performed by ANOVA and Newman-Keuls test. Administration of acetylcholine or carbachol (10^–5M) resulted in a significant increase in P and E₂ secretion. This response was specifically blocked by the muscarinic receptor antagonist atropine. Similarly, carbachol resulted in a significant increase in P and E₂ output, though the response to it was somewhat reduced when compared to that evoked by acetylcholine. Acetylcholine did not show any additive effect on LH-stimulated P secretion, while it augmented the stimulatory effect of FSH on P release. In contrast, carbachol markedly diminished the stimulatory effect of LH on P secretion, while it caused no change in FSH-induced P output. When administered together, acetylcholine did not modify the stimulatory effect of FSH on E₂ secretion, however, it markedly elevated LH-induced E₂ output. Similar to this, carbachol significantly increased LH-induced E₂ release, however it decreased FSH-stimulated E₂ secretion. We suggest that acetylcholine has a direct modulatory effect on gonadotropin-stimulated steroid production of GCs, an effect that is mediated via muscarinic receptors. This effect may have a physiological role in the regulation of GC function during the menstrual cycle.

Effects of halothane on sarcoplasmic reticulum calcium release channels in porcine airway smooth muscle cells.

Volatile anesthetics relax airway smooth muscle (ASM) by altering intracellular Ca2+ concentration ([Ca2+]i). The authors hypothesized that relaxation is produced by decreasing sarcoplasmic reticulum Ca2+ content via increased Ca2+ "leak" through both inositol trisphosphate (IP3) and ryanodine receptor channels. Enzymatically dissociated porcine ASM cells were exposed to acetylcholine in the presence or absence of 2 minimum alveolar concentration (MAC) halothane, and IP3 levels were measured using radioimmunoreceptor assay. Other cells were loaded with the Ca2+ indicator fluo-3 and imaged using real-time confocal microscopy. Halothane increased IP3 concentrations in the presence and absence of acetylcholine. Inhibition of phospholipase C blunted the IP3 response to halothane. Exposure to 2 MAC halothane induced a transient [Ca2+]i response, suggesting depletion of sarcoplasmic reticulum Ca2+. Exposure to 20 microM Xestospongin D, a cell-permeant IP3 receptor antagonist, resulted in a 45+/-13% decrease in the [Ca2+]i response to halothane compared with halothane exposure alone. In permeabilized cells, Xestospongin D or 0.5 mg/ml heparin decreased the [Ca2+]i response to halothane by 65+/-13% and 68+/-22%, respectively, compared with halothane alone. In both intact and permeabilized cells, 20 microM ryanodine blunted the [Ca2+]i response to halothane by 32+/-13% and 39+/-21%, respectively, compared with halothane alone. Simultaneous exposure to Xestospongin D and ryanodine completely inhibited the [Ca2+]i response to halothane. The authors conclude that halothane reduces sarcoplasmic reticulum Ca2+ content in ASM cells via increased Ca2+ leak through both IP3 receptor and ryanodine receptor channels. Effects on IP3 receptor channels are both direct and indirect via elevation of IP3 levels.

Relationship between force and regulatory myosin light chain phosphorylation in airway smooth muscle.

We tested the hypothesis that increases in force at a given cytosolic Ca(2+) concentration (i.e., Ca(2+) sensitization) produced by muscarinic stimulation of canine tracheal smooth muscle (CTSM) are produced in part by mechanisms independent of changes in regulatory myosin light chain (rMLC) phosphorylation. This was accomplished by comparing the relationship between rMLC phosphorylation and force in alpha-toxin-permeabilized CTSM in the absence and presence of acetylcholine (ACh). Forces were normalized to the contraction induced by 10 microM Ca(2+) in each strip, and rMLC phosphorylation is expressed as a percentage of total rMLC. ACh (100 microM) plus GTP (1 microM) significantly shifted the Ca(2+)-force relationship curve to the left (EC(50): 0.39 +/- 0.06 to 0.078 +/- 0.006 microM Ca(2+)) and significantly increased the maximum force (104.4 +/- 4.8 to 120.2 +/- 2.8%; n = 6 observations). The Ca(2+)-rMLC phosphorylation relationship curve was also shifted to the left (EC(50): 1.26 +/- 0.57 to 0.13 +/- 0.04 microM Ca(2+)) and upward (maximum rMLC phosphorylation: 70.9 +/- 7.9 to 88.5 +/- 5. 1%; n = 6 observations). The relationships between rMLC phosphorylation and force constructed from mean values at corresponding Ca(2+) concentrations were not different in the presence and absence of ACh. We find no evidence that muscarinic stimulation increases Ca(2+) sensitivity in CTSM by mechanisms other than increases in rMLC phosphorylation.

Effects of catecholamines and purines on luminescence in the brittlestar Amphipholis squamata (Echinodermata).

The effects of catecholamines (dopamine, adrenaline, noradrenaline and its derivatives), 5-hydroxytryptamine and purines (adenosine, ATP and their derivatives) on the acetylcholine-induced luminescence of isolated arms and dissociated photocytes of the luminescent ophiuroid Amphipholis squamata were tested. The results showed that catecholamines and 5-hydroxytryptamine (10(-)(5) to 10(-)(3 )mol l(-)(1)) had a strong dose-dependent inhibitory effect on acetylcholine-induced luminescence. In contrast, purines (10(-)(4) and 10(-)(3 )mol l(-)(1)) triggered luminescence in the absence of acetylcholine and/or potentiated acetylcholine-induced luminescence. The results with specific purinergic agonists and antagonists indicated the involvement of P(1)- and P(2)-like purinoceptors in the control of luminescence. Our study suggests that, in addition to the previously described cholinergic system in Amphipholis squamata, there may be a purinergic system, acting in synergy with acetylcholine, and an inhibitory neuromodulatory catecholaminergic system, all associated with the control of luminescence.

The intrinsic electrostatic potential and the intermediate ring of charge in the acetylcholine receptor channel.

A ring of aligned glutamate residues named the intermediate ring of charge surrounds the intracellular end of the acetylcholine receptor channel and dominates cation conduction (Imoto et al. 1988). Four of the five subunits in mouse-muscle acetylcholine receptor contribute a glutamate to the ring. These glutamates were mutated to glutamine or lysine, and combinations of mutant and native subunits, yielding net ring charges of -1 to -4, were expressed in Xenopus laevis oocytes. In all complexes, the alpha subunit contained a Cys substituted for alphaThr244, three residues away from the ring glutamate alphaGlu241. The rate constants for the reactions of alphaThr244Cys with the neutral 2-hydroxyethyl-methanethiosulfonate, the positively charged 2-ammonioethyl-methanethiosulfonate, and the doubly positively charged 2-ammonioethyl-2'-ammonioethanethiosulfonate were determined from the rates of irreversible inhibition of the responses to acetylcholine. The reagents were added in the presence and absence of acetylcholine and at various transmembrane potentials, and the rate constants were extrapolated to zero transmembrane potential. The intrinsic electrostatic potential in the channel in the vicinity of the ring of charge was estimated from the ratios of the rate constants of differently charged reagents. In the acetylcholine-induced open state, this potential was -230 mV with four glutamates in the ring and increased linearly towards 0 mV by +57 mV for each negative charge removed from the ring. Thus, the intrinsic electrostatic potential in the narrow, intracellular end of the open channel is almost entirely due to the intermediate ring of charge and is strongly correlated with alkali-metal-ion conductance through the channel. The intrinsic electrostatic potential in the closed state of the channel was more positive than in the open state at all values of the ring charge. These electrostatic properties were simulated by theoretical calculations based on a simplified model of the channel.

Delimiting the binding site for quaternary ammonium lidocaine derivatives in the acetylcholine receptor channel.

The triethylammonium QX-314 and the trimethylammonium QX-222 are lidocaine derivatives that act as open-channel blockers of the acetylcholine (ACh) receptor. When bound, these blockers should occlude some of the residues lining the channel. Eight residues in the second membrane-spanning segment (M2) of the mouse-muscle alpha subunit were mutated one at a time to cysteine and expressed together with wild-type beta, gamma, and delta subunits in Xenopus oocytes. The rate constant for the reaction of each substituted cysteine with 2-aminoethyl methanethiosulfonate (MTSEA) was determined from the time course of the irreversible effect of MTSEA on the ACh-induced current. The reactions were carried out in the presence and absence of ACh and in the presence and absence of QX-314 and QX-222. These blockers had no effect on the reactions in the absence of ACh. In the presence of ACh, both blockers retarded the reaction of extracellularly applied MTSEA with cysteine substituted for residues from alphaVal255, one third of the distance in from the extracellular end of M2, to alphaGlu241, flanking the intracellular end of M2, but not with cysteine substituted for alphaLeu258 or alphaGlu262, at the extracellular end of M2. The reactions of MTSEA with cysteines substituted for alphaLeu258 and alphaGlu262 were considerably faster in the presence of ACh than in its absence. That QX-314 and QX-222 did not protect alphaL258C and alphaE262C against reaction with MTSEA in the presence of ACh implies that protection of the other residues was due to occlusion of the channel and not to the promotion of a less reactive state from a remote site. Given the 12-A overall length of the blockers and the alpha-helical conformation of M2 in the open state, the binding site for both blockers extends from alphaVal255 down to alphaSer248.

State-dependent accessibility and electrostatic potential in the channel of the acetylcholine receptor. Inferences from rates of reaction of thiosulfonates with substituted cysteines in the M2 segment of the alpha subunit.

Ion channel function depends on the chemical and physical properties and spatial arrangement of the residues that line the channel lumen and on the electrostatic potential within the lumen. We have used small, sulfhydryl-specific thiosulfonate reagents, both positively charged and neutral, to probe the environment within the acetylcholine (ACh) receptor channel. Rate constants were determined for their reactions with cysteines substituted for nine exposed residues in the second membrane-spanning segment (M2) of the alpha subunit. The largest rate constants, both in the presence and absence of ACh, were for the reactions with the cysteine substituted for alpha Thr244, near the intracellular end of the channel. In the open state of the channel, but not in the closed state, the rate constants for the reactions of the charged reagents with several substituted cysteines depended on the transmembrane electrostatic potential, and the electrical distance of these cysteines increased from the extracellular to the intracellular end of M2. Even at zero transmembrane potential, the ratios of the rate constants for the reactions of three positively charged reagents with alpha T244C, alpha L251C, and alpha L258C to the rate constant for the reaction of an uncharged reagent were much greater in the open than in the closed state. This dependence of the rate constants on reagent charge is consistent with an intrinsic electrostatic potential in the channel that is considerably more negative in the open state than in the closed state. The effects of ACh on the rate constants for the reactions of substituted Cys along the length of alpha M2, on the dependence of the rate constants on the transmembrane potential, and on the intrinsic potential support a location of a gate more intracellular than alpha Thr244.

Contribution of the beta subunit M2 segment to the ion-conducting pathway of the acetylcholine receptor.

We have applied the substituted-cysteine-accessibility method (SCAM) to the M2 segment and the M1-M2 loop of the acetylcholine (ACh) receptor beta subunit. Each residue from beta P248 to beta D273 was mutated one at a time to Cys, and the mutant beta subunits were expressed together with wild-type alpha, beta, and delta subunits in Xenopus oocytes. For each of the mutants, the ACh-induced current was near wild-type. The accessibility of the substituted Cys was inferred from the irreversible inhibition or potentiation of ACh-induced current by methanethiosulfonate (MTS) derivatives added extracellularly. Inhibition by MTSethylammonium of beta G255C, in the narrow part of the channel, was mainly due to a reduction in the single-channel conductance. Conversely, potentiation by MTSethylammonium of beta V266C, in a wider part of the channel, was mainly due to an increase in channel open-time. Two substituted Cys at the intracellular end of M2 and three at the extracellular end were accessible to MTSethylammonium in the absence of ACh. Three additional Cys in the middle of M2 and three in the M1-M2 loop were accessible in the presence of ACh. In the presence of ACh, the secondary structure of beta M2 is alpha-helical from beta G255 to beta V266 and extended from beta L268 to beta D273. The accessible residues in beta M2 are remarkably hydrophobic, while the accessible residues in the M1-M2 loop are charged. beta M2, like alpha M2, alpha M1, and beta M1, undergoes widespread structural changes concomitant with gating, but the gate itself is close to the intracellular end of the channel. Many aligned residues in the M2 segments of alpha and beta are not identically accessible, indicating that the two subunits contribute differently to the channel lining.

Inhibitory effects of large Ca2+-activated K+ channel blockers on beta-adrenergic- and NO-donor-mediated relaxations of human and guinea-pig airway smooth muscles.

In human bronchi, relaxations to salbutamol and sodium nitroprusside were performed in the presence or absence of blockers of the large Ca2+-activated K+-channels (BKCa): charybdotoxin (Chtx), iberiotoxin (Ibtx) or tetraethylammonium (TEA). In bronchi under basal tone in presence of indomethacin (1 microM) or precontracted with acetylcholine (in presence or absence of indomethacin), the relaxations to salbutamol or sodium nitroprusside were unaffected or weakly inhibited by pretreatment with the BKca blockers (Chtx (100 nM), Ibtx (100 and 300 nM) and TEA (1 mM)). Significant inhibitions were mainly observed with TEA (1 mM) and iberiotoxin at high concentration (300 nM). These results contrasts with the potent inhibitory effects exerted by Chtx (100 nM) or Ibtx (100 nM) in guinea-pig trachea precontracted with acetylcholine in absence or presence of indomethacin indicating that human airways are less susceptible to BKCa blockade than guinea-pig airways. In addition, the BKCa blockers induced slowly developing contractions of human bronchi at basal tone. The contraction induced by TEA (1 mM) was abolished by verapamil (10 microM) suggesting that BKca blockade promotes an increase in membrane Ca2+-conductance through activation of voltage-gated Ca2+-channels. Verapamil also reversed the effects of TEA on salbutamol-induced relaxations in human bronchi as well as the effects of Ibtx on salbutamol- or sodium nitroprusside-induced relaxations in guinea-pig trachea. These data suggest that BKCa blockers induce activation of voltage-gated Ca2+-channels and therefore influx of Ca2+ which in turn cause a functional antagonism of beta2-adrenoceptor-agonist- and NO-donor-induced relaxations. Moreover, the BKCa opener, NS-1619, induced weak relaxations in human bronchi and guinea-pig trachea which were not blocked by TEA or Ibtx suggesting that BKCa opening is of minor significance for the relaxation of human airway smooth muscles. In conclusion, although a wealth of studies have demonstrated that beta-adrenoceptor agonists or NO-donors activate BKCa, the present study provides evidence that in human bronchi, as recently suggested in guinea-pig trachea, opening of BKCa does not appear to functionally participate in the relaxation to these relaxant agents.

Inhibition of muscarinic receptor-operated Ca 2+ sensitization by short-term desensitization of α-toxin-permeabilized smooth muscle cells from guinea pig stomach

1. 1. Isolated single smooth muscle cells from the guinea pig stomach were permeabilized with Staphylococcus aureus α-toxin. 2. 2. The permeabilized single cells showed a shortening in response to Ca 2+ in an all-or-none manner. Moreover, the addition of acetylcholine (ACh) or guanosine 5′-triphosphate (GTP) resulted in a decrease in concentration of Ca 2+ required to trigger a threshold response, suggesting that Ca 2+ sensitization is induced by the stimulation of muscarinic acetylcholine receptors (mAChRs) or GTP-binding protein(s). 3. 3. Short-term desensitization was induced by incubating the permeabilized cells with 100 μM ACh for 10 min. 4. 4. In desensitized cells, the concentration of Ca 2+ required to trigger a threshold response in the presence of ACh was increased, however, the cell shortening in response to Ca 2+ in the absence of ACh and GTP-induced Ca 2+ sensitization was not affected by short-term desensitization. 5. 5. These results suggest that the receptor-operated augmentation of Ca 2+ sensitivity is inhibited by short-term desensitization and that the development of short-term desensitization is due to an uncoupling of mAChR/GTP-binding protein(s).

Absence of acetylcholine- and ionomycin-activated Cl- currents in submandibular cells of early postnatal rats.

Using the whole-cell patch-clamp technique, we investigated developmental changes in the expression of an acetylcholine- (Ach-) activated Cl- conductance in rat submandibular acinar cells. ACh induced an oscillatory inward current in cells isolated from animals older than 5 weeks, but not in animals less than 2-3 weeks of age. The current/voltage (I/V) relationship of the ACh-induced current was that of an outward rectifier, and the current was inhibited by intracellular BAPTA, a Ca2+ buffer, indicating the current was Ca2+ activated. The ACh-induced current was also blocked in the presence of DPC and SITS, two Cl- current inhibitors in other tissues. Ionomycin mimicked the effect of ACh but in a nonoscillatory fashion. The appearance of the ionomycin-induced currents was also age related, as the current was not observed to occur in animals less than 2-3 weeks old. Since both ACh and ionomycin significantly increase cytosolic [Ca2+] in the acinar cells of young animals, the correlation between the age dependence of the ACh-activated Cl- current and the ionomycin-activated Cl- current responses suggests that the lack of responsiveness observed in the young animals is due to the absence of Ca2+-activated Cl- channels, rather than to a deficiency of a cellular mediator.

Halothane counteracts acetylcholine-induced increase in Ca 2+ sensitivity of the contractile apparatus in airway smooth muscle

The direct relaxing effect of halothane on airway smooth muscle has been reported to involve the reduction of the cytosolic Ca 2+ concentration ([Ca 2+] i) and the [Ca 2+] i-independent inhibitory mechanism. To clarify the extent of the contribution of these mechanisms, the effect of halothane on the [Ca 2+] i-tension relationship in porcine tracheal smooth muscle strips was evaluated, using fura-2 fluorometry. The control [Ca 2+] i-tension relationship was constructed from data of [Ca 2+] i and tension during the contractions induced by the stepwise increment of extracellular Ca 2+ concentration under high K + depolarization. In the presence of acetylcholine (1 μM), the [Ca 2+] i-tension relationship shifted upward, which indicated the acetylcholine-induced increase in the Ca 2+ sensitivity of the contractile apparatus. Halothane (0.034 mM), in the absence of acetylcholine, did not alter the increases in either [Ca 2+] i or tension, hence no change in the [Ca 2+] i-tension relationship. However, in the presence of acetylcholine, halothane did attenuate the acetylcholine-induced upward shift of the [Ca 2+] i-tension relationship. Halothane proved to have a potent attenuating effect on the acetylcholine-induced increase in Ca 2+ sensitivity of the contractile apparatus with little influence on [Ca 2+] i. This desensitization of the contractile apparatus to [Ca 2+] i may play a major role in the direct airway relaxing effect of halothane.

Single channel evidence for a cytoskeletal defect involving acetylcholine receptors and calcium influx in cultured dystrophic (mdx) myotubes.

Single channel events that exhibited the conductance, event duration, and ion selectivity characteristics of calcium leakage activity (CLA) were recorded in association with acetylcholine receptor (AChR) activity in cultured nondystrophic myotubes. The CLA was observed in the presence or absence of acetylcholine (ACh), and at normal or elevated concentrations of calcium. In contrast to results from nondystrophic myotubes, cell-attached patches from several cultured dystrophic (mdx) myotubes exhibited 100% CLA with no AChR activity, even though ACh was present in the pipette solution. Acquisition of an inside-out patch from these membrane areas produced a profound decrease in CLA and the appearance of AChR events exhibiting typical conductance and event duration characteristics. These results suggest that CLA in dystrophic muscle is produced, in part, by unusual physical interactions between AChRs and the dystrophic cytoskeleton that are mediated by the action of intracellular modulators responsible for aggregating and stabilizing AChRs.

Prostanoid Production in Rabbit Corpus Cavernosum: I. Regulation by Oxygen Tension

Purpose To investigate the effects of oxygen tension on prostanoid synthesis in rabbit penile corpus cavernosum tissue (RCC) in organ culture. Materials and Methods Strips of rabbit corpus cavernosum were incubated in organ culture media under varying oxygen conditions (0 percent, 12 percent and 21 percent oxygen), in the presence or absence of acetylcholine and arachidonate stimulation. Prostanoids were measured in collected media by radioimmunoassay. Prostaglandin H synthase (PGHS) protein levels and mRNA PGHS expression were measured under both 0 percent and 21 percent oxygen conditions. Results Basal and acetylcholine-stimulated PGI 2 release was progressively diminished as a function of diminishing oxygen tension (pO 2 from approximately 165 to 25 mm.Hg). The basal and stimulated production of other prostanoids, thromboxane A 2, PGF 2 alpha and PGE 2, was also significantly inhibited under 0 percent oxygen (approximately 25 mm.Hg) conditions. However, incubation under 0 percent oxygen did not alter PGHS protein levels nor mRNA PGHS expression. Cavernosal strips incubated under 0 percent oxygen but supplemented with exogenous arachidonate (10 micromolar) maintained significantly lower PGI 2 production than tissues exposed to 21 percent oxygen (approximately 165 mm.Hg). Conclusions These data demonstrate that oxygen tension regulates prostaglandin production in corporal tissue. The reduction in prostanoid production during hypoxia can be attributed to inhibition of PGHS activity rather than the expression of the enzyme. In view of the role of PGI 2 as an inhibitor of platelet aggregation and white cell-endothelial adhesion, our findings may provide mechanistic insight into the alteration in corporal blood homeostasis during ischemic-hypoxic priapism.