microM Carbachol Research Articles

SD‐3212, a new class I and IV antiarrhythmic drug: a potent inhibitor of the muscarinic acetylcholine‐receptor‐operated potassium current in guinea‐pig atrial cells

1. By use of patch-clamp techniques, the effects of SD-3212, a novel antiarrhythmic drug, on the calcium current (Ica), the sodium current (INa) and the muscarinic acetylcholine-receptor-operated potassium current (IK.ACh) were examined and compared with those of bepridil in guinea-pig single atrial cells. 2. SD-3212 inhibited ICa and INa in a concentration-dependent manner. The IC50 values of SD-3212 for inhibition of ICa and INa were 1.29 microM and 3.92 microM, respectively. The steady state inactivation curves of ICa and INa were shifted in the hyperpolarizing direction in the presence of 1 microM SD-3212. Similar inhibition of ICa and INa was also observed with bepridil. The IC50 values of bepridil for depression of ICa and INa were 1.55 microM and 4.43 microM, respectively. 3. The muscarinic acetylcholine-receptor-operated potassium current (IK.ACh) was activated by the extracellular application of 1 microM carbachol in the GTP-loaded cells or by the intracellular loading of GTP gamma S, a nonhydrolysable GTP analogue. SD-3212 potently inhibited the carbachol- and GTP gamma S-induced IK.ACh and the IC50 values were 0.38 microM and 0.20 microM, respectively. These IC50 values were very close and about 10 times lower than those for inhibiting ICa and INa. Bepridil also suppressed the carbachol- and GTP gamma S-induced IK.ACh with the IC50 values of 0.69 microM and 0.84 microM, respectively. 4. In guinea-pig atrial cells stimulated at 0.2 Hz, carbachol at a concentration of 1 microM markedly shortened action potential duration. Both SD-3212 (0.1-1 microM) and bepridil (1-10 microM) reversed the action potential shortening in a concentration-dependent manner. The antagonizing effect of SD-3212 on the carbachol-induced action potential shortening was more potent than that of bepridil. 5. These results suggest that SD-3212 inhibits IK.ACh by depressing the function of the potassium channel itself and/or associated GTP-binding proteins. SD-3212 is a unique antiarrhythmic drug, which potently inhibits IK.Ach in addition to its class I and IV effects. SD-3212 and bepridil may be useful for the termination and prevention of vagally-induced atrial flutter and fibrillation.

G-protein activation enhances Ca+2-dependent lipid secretion of the rat Harderian gland

We studied the secretory mechanism of the Harderian gland of rats. After perfusion with HEPES-buffered Ringer's solution containing NaF (10 mM) with AlCl3 (10 microM), a G-protein activator, the glandular cells of the Harderian gland showed massive exocytosis and apocrine-like protrusions on the luminal surface. Some of the secretory vacuoles aggregated within the cytoplasm, and large vacuoles were formed. Contraction of the myoepithelial cells covering the glandular endpieces caused a narrowing of the glandular lumina, which contained cytoplasmic fragments, and deformation of the basal contour of the glandular end-pieces. The basal regions of the glandular cells also bulged between the myoepithelial cells. Secretory vacuoles were also discharged to the lateral cell surface, and the intercellular spaces were dilated. The enhanced secretory activities of the glandular cells and the contraction of the myoepithelial cells were similar to those in rats stimulated with 10 microM carbachol (CCh). However, dilatation of the endoplasmic reticulum in glandular cells (type A cells), which leads to the formation of small vesicles, was observed in those glands stimulated by NaF+AlCl3, but not in those stimulated by CCh. Removal of Ca2+ from the perfusing HR or addition of EDTA (0.5 mM) diminished and inhibited NaF+AlCl3- or CCh-enhanced secretory activity of the glandular cells and also allayed the deformation of glandular cells caused by myoepithelial cell contraction. The present results demonstrate the involvement of G-proteins and Ca(2+)-influx in the lipid secretion of glandular cells and in the contraction of myoepithelial cells of the Harderian gland in rats.

Corticosteroid-mediated modulation of carbachol responsiveness in CA1 pyramidal neurons: a voltage clamp analysis.

Pyramidal neurons in the rat hippocampal CA1 area contain mineralocorticoid (MRs) and glucocorticoid (GRs) receptors for corticosterone. Previous current clamp experiments showed that depolarizations evoked by carbachol (1-3 microM) depend on relative MR/GR occupation: carbachol responses are small with predominant MR-activation and larger when both receptor types are occupied. Multiple K-conductances underlie the carbachol-induced depolarization. In the present study we used the single electrode voltage clamp technique to examine which K-conductances modulated by carbachol are sensitive to corticosteroid treatment in vitro. We observed that 1 microM carbachol significantly reduced the IK,Leak while the IM was hardly affected; carbachol effects on the IK,Leak were significantly reduced under conditions of predominant MR activation compared to simultaneous activation of MRs and GRs. With a higher (10 microM) carbachol dose, steroid modulation of the IK,Leak showed a similar tendency. The amplitude of the IM was largely reduced by 10 microM carbachol but appeared to be not affected by steroid treatment. We conclude that the previously described suppression of the carbachol-induced depolarization with predominant activation MRs is caused by an attenuation of the carbachol action on the IK,Leak.

Effects of high‐energy phosphates on carbachol‐evoked cationic current in single smooth muscle cells from guinea‐pig ileum.

1. Single smooth muscle cells from the longitudinal muscle layer of guinea-pig small intestine were voltage clamped in the whole-cell recording mode with patch pipettes. The cationic current (Icat) evoked by application of 50 microM carbachol (CCh) was examined when free internal calcium in the cell was 'clamped' at 10(-7) M with 20 mM BAPTA. The effects of varying the composition of the pipette solution were studied. 2. Phosphocreatine (PCr, 6 mM) added to the pipette solution increased Icat by about 7-fold (to near 620 pA); lower concentrations had similar, generally lesser, effects. Na2ATP (3 or 6 mM) with or without 5 mM MgCl2 was much less effective than phosphocreatine alone. Addition of 3 mM Na2ATP reduced Icat, whether or not phosphocreatine was present. 3. Creatine (6 mM) with or without 2 mM Na2ATP was less effective than phosphocreatine in maintaining Icat. 4. GTP (0.1 mM) did not affect Icat evoked by CCh, whether phosphocreatine was present or not. 5. GTP gamma S (0.2 mM) included in pipette solution mimicked the effect of CCH and evoked Icat independently of whether PCr was present or not in the pipette solution. Including 5 mM ATP in the pipette reduced this current, whereas 5'-adenylyl imidodiphosphate (AMP-PNP) and ADP were without effect. 6. The results show that phosphocreatine increases membrane channel responsiveness to receptor activation and that ATP above 2 mM suppresses it.

The effect of barium on [3H]noradrenalin release from the human neuroblastoma SH-SY5Y.

Replacement of Ca2+ with Ba2+ in HEPES-buffered saline stimulated [3H]noradrenalin release in the human neuroblastoma clone SH-SY5Y by up to 20% of the cell content in the absence of other secretory stimuli. The Ba(2+)-evoked release was inhibited by 85% by 3 microM tetrodotoxin and 95% by 5 microM nifedipine. Ba2+ also increased the potency of K(+)-evoked release of [3H]noradrenalin, as maximal release was observed with 60 mM K+ compared with the 100 mM K+ necessary to achieve maximal release in the presence of Ca2+. In contrast, replacing Ca2+ with Ba2+ had little effect on carbachol- and bradykinin-evoked release of [3H]noradrenalin. No evidence was obtained from studies on changes in [Ca2+]i (in response to 100 microM carbachol) using fura-2 that Ba2+ could enter intracellular stores in SH-SY5Y cells. Whole-cell patch-clamp studies showed that Ba2+ depolarizes SH-SY5Y cells as well as enhancing inward Ca2+ channel currents and shifting their voltage dependence to more negative values. These results are discussed in terms of the hypothesis that Ba2+ blocks K+ channels, leading to depolarization followed by opening of voltage-sensitive Na+ channels. This in turn opens voltage-sensitive L-type Ca2+ channels, which are coupled to the release of [3H]noradrenalin in SH-SY5Y cells.

Fluid phase endocytosis by isolated rabbit lacrimal gland acinar cells

It is well known that lacrimal gland acinar cells retrieve secretory vesicle membrane constituents from their apical plasma membranes after stimulated exocytosis of secretory proteins. There have also been indications of a recycling traffic involving the basal-lateral plasma membranes. In an effort to document this traffic, determine how it is regulated, and discern whether it involves more than one intracellular compartment, we studied internalization of the fluid phase marker, Lucifer Yellow, and its relationship to protein release in acinar cells isolated from rabbit lacrimal glands. Loading of intracellular vesicles was apparent with fluoresence microscopy. Stimulation with carbachol increased both the rate of internalization and the intracellular volume equilibrating with extracellular fluid, suggesting the loading of two compartments. A carbachol concentration of 10 microM increased uptake by 80% during 20-min incubations at 37 degrees C. Increasing the carbachol concentration to 1 mM reduced the response by 50%, and it appeared to do so by decreasing the intracellular volume accessible to extracellular fluid, rather than the rate of endocytosis. Carbachol affected protein release differently, increasing it by 50% at 10 microM and 80% at 1 mM. Acceleration of endocytosis by 10 microM carbachol was transient, becoming negligible after 60 min. Vasoactive intestinal peptide (VIP) and isoproterenol increased internalization 35% and 25% respectively; neither reduced uptake at the highest concentrations tested; and only isoproterenol significantly affected protein secretion. Combinations of VIP and carbachol exerted synergistic effects on both fluid phase internalization and protein release. Steady-state uptake at 18 degrees C in the presence of 10 microM carbachol was equal to uptake at 37 degrees C in the absence of carbachol, suggesting a temperature block in the pathway to at least one endocytic compartment. Decreasing the temperature to 18 degrees C eliminated the inhibitory action of excessive carbachol, suggesting that the compartment whose loading was impaired by excessive carbachol was positioned distal to the temperature block. Carbachol accelerated release of marker from preloaded cells, indicating that it stimulated recycling between the plasma membranes and endocytic compartments. This effect was maximal at a concentration of 10 microM and unchanged with increasing concentrations. In accord with the hypothesis that traffic into and out of a certain compartment was particularly dependent on stimulation, a fraction of the marker taken up by optimally stimulated cells at 37 degrees C was retained unless carbachol or VIP was present in the efflux medium.(ABSTRACT TRUNCATED AT 400 WORDS)

Potentiation by endothelin‐1 of cholinergic nerve‐mediated contractions in mouse trachea via activation of ETB receptors

1. We have previously shown that endothelin-1-induced contraction of mouse isolated tracheal smooth muscle was mediated via both ETA and ETB receptors. In the current study, we have investigated endothelin-1-induced potentiation of cholinergic nerve-mediated contractions in mouse isolated trachea and have characterized pharmacologically the endothelin receptors mediating this response. 2. Electrical field stimulation (EFS; 70 V, 0.5 ms duration, 10s train, 0.1-60 Hz) of mouse isolated trachea caused frequency-dependent, monophasic contractions (magnitude of contraction of 60 Hz was 56 +/- 4% Cmax (n = 6), where Cmax is the contractile response to 10 microM carbachol). EFS-induced contractions were abolished by either 0.1 microM atropine or 3 microM tetrodotoxin, but were not affected by 1 microM hexamethonium, indicating that they were induced by stimulation of postganglionic cholinergic nerves. In contrast, contractions induced by exogenously applied acetylcholine were inhibited by atropine, but not by either tetrodotoxin or hexamethonium. 3. The ETB receptor-selective agonist, sarafotoxin S6c, caused marked concentration-dependent potentiation of EFS-induced contractions in mouse isolated tracheal segments. At 0.1 nM, sarafotoxin S6c exerted no direct contractile effect, but significantly increased a standard EFS-induced contraction of 20% Cmax by 8 +/- 2% Cmax (i.e. 1.4 fold, n = 5, P < 0.05). At higher concentrations, 10 nM sarafotoxin S6c induced a large, transient contraction (peak response of 74 +/- 2% Cmax at 10 min; 3 +/- 2% Cmax at 45 min) and enhanced the standard EFS-induced contraction by 30 +/- 4% Cmax (i.e. 2.5 fold, n = 5, P < 0.01). In contrast, 10 nM sarafotoxin S6c did not enhance contractile responses to exogenously applied acetylcholine(n = 6).4. Endothelin-1 also modulated EFS-induced contractions. At 0.1 nM, endothelin-1 exerted no direct contractile effect, but significantly increased the standard EFS-induced contraction of 20%Cmax, by 7 +/- 2%Cma, (i.e. 1.35 fold, n = 5, P<0.05). At 1 nM, endothelin-l induced a small, sustained contraction(16 +/- 3%Cmo) and increased the standard EFS-induced contraction by 19 +/- 2%Cmax (i.e. 1.95 fold,n = 5, P <0.01). Finally, 10 nM endothelin-1 induced a large, sustained contraction (98 +/- 8%Cma), but the EFS-induced contraction was significantly reduced from 20%Cmax to 6 +/- 4%Cmax (n = 6, P <0.05).In contrast, in the presence of 3 microM BQ-123 (ETA receptor-selective antagonist), 1O nM endothelin-1 induced a transient contraction mediated via ETB receptors (peak response of 59 +/- 10%Cmax at 10 min;8 +/- 2%Cmax at 45 min). Under these conditions, the standard EFS-induced contraction was increased by 26+/- l%Cmax (i.e. 2.3 fold, n = 6, P<0.01).5. The potentiation of EFS-induced contractions produced by 1 nM endothelin-1 was not mediated by ETA receptors, since 3 microM BQ-123 did not diminish this effect (n = 6). Furthermore, 1 nM endothelin-1 did not potentiate EFS-induced contractions in preparations in which the function of the ETB receptor effector system had been attenuated by desensitization (n = 6).6. In summary, endothelin-1 potentiates cholinergic nerve-mediated contractions in mouse isolated trachea, apparently by activating prejunctional ETB receptors. This neuronal pathway offers an additional mechanism through which endothelin-1 may elevate bronchomotor tone.

Requirement of a colchicine-sensitive component of the cytoskeleton for acetylcholine receptor recovery.

1. The effect of colchicine treatment on acetylcholine receptor function was examined in potassium depolarized, voltage-clamped snake twitch fibre endplates. Receptor function was assessed by analysis of miniature endplate currents (m.e.p.c.) as well as acetylcholine (ACh)-induced single channel currents. 2. Pretreatment of snake muscle fibres with colchicine (10 microM to 100 microM) for 16-18 h had no effect on m.e.p.c. amplitude or decay rates. At higher concentrations (1 mM), there was a slight decrease in the average m.e.p.c. amplitude. 3. Colchicine produced a concentration-dependent decrease in the extent of m.e.p.c. amplitude recovery following a 10 min exposure to 540 microM carbachol. Exposure of 100 microM colchicine-treated preparations to 0.5 microM staurosporine further reduced the extent of m.e.p.c. amplitude recovery following carbachol exposure. 4. The decrease in m.e.p.c. amplitude following carbachol exposure was not due to a shift in the m.e.p.c. reversal potential. In addition, the distribution of m.e.p.c. amplitudes remained unimodal in both control and colchicine (100 microM)-treated preparations following carbachol exposure. 5. In addition to the normal, large conductance (approximately 48 pS) ACh-activated channels, a population of small conductance (approximately 29 pS) channels was observed in colchicine-treated preparations following exposure to carbachol. In preparations treated with both colchicine and staurosporine and then exposed to carbachol, the conductance of these small channels was identical to that of colchicine or staurosporine alone. 6. We suggest that prolonged exposure of snake twitch fibre endplates to agonist results in the activation and desensitization of ACh receptors. Furthermore, we propose that for a subpopulation of the inactivated receptors, restoration of function requires both the integrity of a subsynaptic cytoskeletal component and phosphorylation by a staurosporine-sensitive protein kinase. One plausible mechanism is that some receptors become destabilized in the membrane and phosphorylation of a cytoskeletal component, whose distribution may depend on an intact microtubular system, is required to re-anchor these receptors. If this anchoring process is inhibited either by disruption of the cytoskeleton with colchicine, or inhibition of the kinase by staurosporine, these receptors remain activatable, but have a reduced conductance.

Mercuric(II) chloride modulates single-channel properties of carbachol-activated Cl- channels in cultured neurons of Aplysia californica.

1. The effect of mercuric(II) chloride on kinetic parameters of carbachol-activated single chloride channels were studied in cultured neurons of the marine mollusk, Aplysia californica. 2. Single neurons of Aplysia were cultured in L-15 medium containing 1 mM beta-D-xyloside, which improved the success rate for gigaseal formation by 46%. Carbachol-activated single chloride channels were recorded in the cell-attached patch clamp configuration. Recordings with control solution (1 microM carbachol) and with test solution (1 microM carbachol + 1 microM HgCl2) were performed successively on the same neuron. 3. In both the control and the test solution the open and closed time distributions were fitted with a double-exponential function. However, kinetic analysis revealed that Hg2+ caused a significant reduction of the mean closed time (10.37 +/- 1.08 vs. 3.32 +/- 0.02 msec) and of the second time constant tau 2 of the closed time distribution (2.09 +/- 0.05 vs. 0.66 +/- 0.5 msec). The reduction of tau 2, i.e., fewer events in the longer closed state under the action of Hg2+, may be the physical cause for the reduction of the mean closed time and thus underlies the increased open probability p0 (0.13 +/- 0.01 vs. 0.29 +/- 0.01 msec) of carbachol-activated chloride channels. 4. Inorganic Hg2+ affects the acetylcholine receptor at lower concentrations than previously reported.

Variable potency of nitrergic‐nitrovasodilator relaxations of the mouse anococcygeus against different forms of induced tone

1. U46619 (thromboxane A2 receptors; 0.002-1 microM), carbachol (muscarinic M3 receptors; 0.1-100 microM), cyclopiazonic acid (CPA; Ca(2+)-ATPase inhibitor; 0.1-30 microM) and K+ (5-100 mM) produced concentration-dependent contractions of the mouse isolated anococcygeus muscle. Equi-effective, submaximal concentrations of each agent were used in further experiments (40 nM U46619; 5 microM carbachol; 5 microM CPA; 70 mM K+). 2. Nifedipine (1 microM) totally abolished contractile responses to K+; those to U46619, carbachol and CPA were reduced by only 20-30% in the presence of nifedipine, but were greatly reduced (> 90%) by a combination of nifedipine and SKF 96365 (0.1-40 microM). 3. In Ca(2+)-free medium, contractions to K+ and CPA were abolished. Small residual responses remained to both carbachol and U46619; those to carbachol were transient, could not be repeated in the continued absence of Ca2+ and were prevented by pre-incubation with CPA, but unaffected by SKF 96365; those to U46619 were sustained, could be repeated in the absence of Ca2+, and were resistant to CPA and SKF 96365. 4. Tone induced by all four agents could be relaxed by sodium nitroprusside (SNP), but with a clear order of potency. SNP (pIC40) was most effective against U46619 (7.92), less so against carbachol (6.80) and CPA (6.68), and least potent against K+ (5.94). A similar order of potency was observed with 8Br-cyclic GMP (50 microM) and nitrergic field stimulation (1-20 Hz). 5. The relaxant potency of SNP was similar in normal Krebs solution and in high K+ (70 mM) Krebs containing 1 microM nifedipine. 6. Inclusion of SNP (0.01-1 microM) or 8Br-cyclic GMP (50 microM) in the Ca2+-free medium inhibited the transient residual response to carbachol. Inclusion of similar concentrations of SNP or 8Br-cyclic GMP,during Ca2+ re-loading, increased the subsequent residual contraction to carbachol in Ca2+-free medium.7. At higher concentrations, SNP (0.1-10 microM) produced a partial relaxation of the sustained contraction to U46619 in Ca2+-free medium.8. Thus, the relaxant potency of the nitrergic stimuli was dependent on the agent and mechanism used to induce tone in the preparation. Examination of the contractile/relaxant interactions suggests that altered Ca2+ sequestration and inhibition of contractile protein function may underlie nitrergic relaxations of this tissue.

Hg2+ increases the open probability of carbachol-activated Cl- channels in Aplysia neurons.

Carbachol and acetylcholine (ACh)-activated whole-cell chloride currents were recorded in identified neurons of Aplysia californica. Application of 0.1-10 microM HgCl2 potentiated the chloride response and reduced the rate of desensitization. To investigate the underlying mechanism we recorded single chloride channels in a cell-attached patch configuration. Recordings were performed successively on the same neuron with 1 microM carbachol and with 1 microM carbachol + 1 microM HgCl2, respectively. The slope conductance did not change significantly, but there was a manyfold increase in the open probability, Po. This may underlie the Hg(2+)-induced enhancement of carbachol- and ACh-evoked chloride currents in Aplysia neurons and may serve as a model for the neurotoxic effect of Hg2+ on the mammalian ACh receptor.

Muscarinic (M1) mediation of carbachol-induced theta in the cat entorhinal cortex in vitro.

Entorhinal cortex slice preparations obtained from the cat exhibited theta rhythm during perfusion with 50 microM carbachol. The effect of carbachol was antagonized by the muscarinic blocker atropine sulphate, but not by hexamethonium and mecamylamine, which are antagonists of the nicotinic receptor. Further analysis of the pharmacological profile of these carbachol-induced theta oscillations showed that M1 receptor subtype to be involved in mediation of this EEG activity: the theta rhythm was antagonized by the M1 receptor blocker pirenzepine, but was unaffected by gallamine, an antagonist of the M2 receptor subtype.

Receptor crosstalk: effects of prolonged carbachol exposure on ?1-adrenoceptors and adenylyl cyclase activity in neonatal rat ventricular myocytes

Supersensitivity of adenylyl cyclase after exposure to inhibitory agonists is a general means of cellular adaptation. We hypothesized that such "crosstalk" between muscarinic cholinergic agonists, beta 1-adrenoceptors, and adenylyl cyclase may be an important mechanism of cardiac adaptation to interventions that enhance vagal activity. We used primary cultures of neonatal rat ventricular myocytes and measured beta-adrenoceptors by radioligand binding and adenylyl cyclase activity by a single column method. Carbachol induced a time- and dose-dependent reversible decrease in cell surface beta 1-adrenoceptors. The peak effect occurred after 20 h of exposure to 100 microM carbachol which caused a decrease in the maximum number of binding sites for the beta-adrenoceptor antagonist 3H-CGP-12177 from 42.3 +/- 3.4 to 33.0 +/- 2.6 fmol/mg protein (n = 12, P < 0.03) without a change in antagonist affinity. Loss of cell surface receptors was prevented by atropine and by the protein kinase C inhibitor H7. The decrease in cell surface receptors was not accompanied by receptor internalization as assessed by equilibrium binding experiments in a cytosolic fraction using 125I-iodocyanopindolol. In contrast to the well-known acute inhibitory effects of carbachol on adenylyl cyclase activation, prolonged carbachol exposure preserved (-)-isoprenaline-stimulated adenylyl cyclase activity and enhanced postreceptor stimulated adenylyl cyclase activity. Carbachol did not further enhance adenylyl cyclase activity after pretreatment with pertussis toxin. The protein kinase C inhibitor chelerythrine prevented the carbachol induced enhancement of forskolin-stimulated adenylyl cyclase activity. We conclude that prolonged incubation with carbachol in rat neonatal ventricular myocytes causes a reduction in cell surface beta 1-Adrenoceptor density. beta 1-Adrenoceptor-mediated adenylyl cyclase activity is preserved and postreceptor-mediated adenylyl cyclase activity is augmented. Our data suggest that carbachol-stimulated protein kinase C activity may play a key role in the prolonged muscarinic regulation of adenylyl cyclase activity.

ETB but not ETA receptor-mediated contractions to endothelin-1 attenuated by respiratory tract viral infection in mouse airways.

1. The current study investigated the effects of respiratory tract viral infection on the density of ETA and ETB receptors in murine tracheal smooth muscle and on the contractile response to endothelin-1 mediated by these receptors. 2. Quantitative autoradiographic studies using [125I]-endothelin-1 revealed that tracheal smooth muscle from control mice contained ETA and ETB receptors in the ratio of 42%:58% (+/- 4%, n = 10 mice), respectively. In contrast, tracheal smooth muscle obtained from mice 2 days post-inoculation with Influenza A/PR-8/34 virus contained 23 +/- 2% fewer receptors for [125I]-endothelin-1 (n = 10, P < 0.01). This reflected a selective reduction in ETB receptor density and a change in the ratio of ETA and ETB receptors to 77%:23% (+/- 5%, n = 10 mice), respectively. 3. The ETB receptor-selective agonist, sarafotoxin S6c, was a potent spasmogen of murine isolated tracheal smooth muscle and the EC50 for contraction was similar in preparations from control (3.6 nM [95% confidence limits, 2.7-4.8 nM], n = 16 preparations from 8 mice) and virus-inoculated mice (3.0 nM [2.4-3.7 nM], n = 16 preparations from 8 mice). However, the maximum contractions induced by sarafotoxin S6c (100 nM) in the preparations from virus-inoculated mice (37 +/- 5% Cmax, where 100% Cmax was the response to 10 microM carbachol) were significantly smaller than those from control mice (85 +/- 4% Cmax, P < 0.01). 4. Contractions induced by endothelin-1 in tracheal smooth muscle preparations obtained from virus inoculated mice (EC50 for contraction, 6.5 nM [95% confidence limits, 2.7-16 nM]; maximum contraction,112 +/- 5% Cm.; n = 4) were similar to those induced by endothelin-1 in control preparations (ECm9.3 nM (4.2-21); maximum contraction, 110 +/- 3% Cmax; n = 4). Endothelin-1-induced contractions in control preparations were only marginally inhibited by the ETA receptor-selective antagonist BQ-123 (in the presence of 3 micro M BQ-123; EC50 for contraction, 5.9 nM [4.1-8.5]; maximum contraction, 82 +/- 4%Cmax; n = 4). In contrast, 3 microM BQ-123 caused a 50 fold rightward shift (17-160, n =4) of the concentration-effect curve to endothlin-1 in preparations obtained from virus-inoculated mice (measured at the 30% Cmax level of contraction).5. Tracheal smooth muscle preparations exposed to 100 nM sarafotoxin S6c for 30 min (followed by a 30 min washout period) did not contract to subsequently administered sarafotoxin S6c (1-100 nM;n = 8), but contracted normally in response to endothelin-1 (EC50 6.5 nM (2.3-18); maximum contraction,109 +/- 2% Cmax; n = 4). Endothelin-l-induced contractions in these ETB receptor desensitized preparations were markedly inhibited by 3 microM BQ-123, irrespective of whether the preparations were obtained from control (63 fold shift (10-400) at the 30% Cma. level of contraction, n = 4) or virus inoculated mice (46 fold shift (18-120), n = 4).6. In summary, tracheal smooth muscle obtained from mice infected with a respiratory tract virus,Influenza A/PR-8/34 had a reduced density of ETB receptors and an attenuated ETB receptor-mediated contractile response to sarafotoxin S6c and endothelin-1. Virus-inoculation was also associated with a modest increase in tracheal smooth muscle ETA receptor density, although no significant change in ETA receptor-mediated contractile activity was seen. Thus, virus infection in murine airways produced profound alterations in endothelin receptor density, some of which were associated with changes in receptor-mediated contractile activity.

Modulation of the input/output function of rat piriform cortex pyramidal cells.

1. In transverse brain slice preparations of rat piriform cortex, we characterized the repetitive firing properties of layer II pyramidal cells in control conditions (n = 78) and during perfusion of the cholinergic agonist carbachol (n = 26), with the ultimate goal of developing realistic computational simulations of the cholinergic modulation of the input/output function of these neurons. The response of neurons to prolonged (1 s) intracellular current injections was examined at a full range of current injection amplitudes, providing three-dimensional plots of firing frequency versus current amplitude versus time. 2. All neurons showed adaptation in response to intracellular current injection, with repetitive generation of action potentials at frequencies that were highest at the onset of the pulse and that decreased considerably thereafter. Substantial differences were observed between cells with regard to their rates of adaptation and the maximal number of action potentials they could generate during the current pulse. 3. The adaptation characteristics of each neuron were quantified by plotting the number of action potentials generated in 1 s as a function of the normalized current injection amplitude and measuring the area beneath this plot of the number of spikes versus current injection amplitude (S-I plot). This value was termed S-I value and allowed neurons to be plotted on a continuum including neurons showing strong adaptation (S-I value < 8.0) and neurons showing weak adaptation (S-I value > 8.0). The group showing weak adaptation contained 36% of the cells in control solution and 93.8% of the cells in 20 microM carbachol. 4. Neurons showing strong adaptation did not differ significantly from neurons showing weak adaptation in control conditions in measurements of resting potential, input resistance, threshold, and spike amplitude. Only a small difference was found in frequencies of firing measured soon after pulse onset (after 100 ms). This implies that differences in S-I values are primarily due to different rates of adaptation in later parts of the response. 5. Perfusion with solution containing the cholinergic agonist carbachol (2-100 microM) or 0 Ca2+ and 200 microM cadmium resulted in a substantial increase in the S-I values of neurons showing strong adaptation but had only a small effect on their initial firing rates. The effect on weakly adapting cells was smaller. In the presence of 20 microM carbachol, neurons showed a distribution shifted predominantly toward weak adaptation (n = 26).(ABSTRACT TRUNCATED AT 400 WORDS)

Low concentrations of protein kinase C-activating agonists suppress cholecystokinin-OPE-evoked Ca2+ mobilization in rat pancreatic acini.

The phenethyl ester analogues of cholecystokinin, OPE and JMV-180, are fully efficacious rat pancreatic secretagogues which, unlike cholecystokinin (CCK), do not elicit supramaximal inhibition of secretion, and stimulate a sustained rise of cytosolic calcium ([Ca2+]i) above basal levels. We have recently shown that low-level protein kinase C (PKC) activation by preincubation of acini with 1 nM 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or minimally secreting concentrations of PKC-activating receptor agonists (1 pM CCK-8, 0.1 microM carbachol or 10 pM bombesin) cause supramaximal inhibition of OPE-stimulated enzyme secretion. We now show that treatment of acini under these conditions also suppresses the sustained rise of [Ca2+]i stimulated by OPE to basal levels in these cells, without changing the initial OPE-stimulated [Ca2+]i peak. The resultant pattern of calcium signalling is similar to that evoked by supramaximal concentrations of native CCK. This suggests that even low concentrations of PKC-activating agonists have the potential to induce inhibitory effects on Ca2+ mobilization and that this kinase is important in generating the supramaximal inhibition observed in response to CCK.

Activity-dependent expression and distribution of M1 muscarinic ACh receptors in visual cortex neuronal cultures

The fluorescently labeled muscarinic M1 receptor-selective antagonist BODIPY FL pirenzepine has been employed to study the activity-dependent distribution and expression of muscarinic M1 ACh receptors (M1AChRs) in cultured neurons derived from rat visual cortex. Displacement experiments showed that like pirenzepine, binding of BODIPY FL pirenzepine was specific to M1 receptors and its K(i) was similar to that of unlabeled pirenzepine. Using confocal laser scanning microscopy, M1 receptors were predominantly localized to cell bodies early in development in the culture environment. After 2 weeks in culture, the receptors showed labeling not only in cell bodies but also in neuritic processes, especially on the initial segments of the processes. Chronic membrane depolarization with 40 mM potassium chloride caused a dramatic increase in M1 receptor expression on these neurons. Conversely, blockade of neuronal activity with 0.1 microM TTX decreased expression of the receptors. Receptor expression increased after cells were treated chronically with 50 nM pirenzepine, whereas it decreased after exposure to 10 microM carbachol. The results demonstrate for the first time the exact location of muscarinic receptors in living cultured neurons and also the activity-dependent expression of M1 receptors on these neurons. Both chronic membrane depolarization and antagonist application upregulate receptor expression, whereas blocking bioelectrical activity or chronic agonist application downregulates expression.

Caffeine inhibits cytoplasmic Ca2+ oscillations induced by carbachol and guanosine 5′-O-(3-thiotriphosphate) in hyperpolarized pancreatic ß-cells

The effects of caffeine on cytoplasmic Ca2+ oscillations induced by carbachol and guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) were studied in individual mouse pancreatic beta-cells clamped at a hyperpolarized potential. Addition of 10 mM caffeine did not affect the cytoplasmic Ca2+ concentration ([Ca2+]i) in beta-cells exposed to 20 mM glucose and hyperpolarized with diazoxide. Under similar conditions 100 microM carbachol induced a typical response with a marked [Ca2+]i peak followed by a lower sustained elevation. Irrespective of whether 10 mM caffeine was present, there were [Ca2+]i transients with frequencies of 1-5/min superimposed on the sustained phase in 50-60% of the cells. In previously non-exposed cells the introduction of 10 mM caffeine caused temporary lowering of the sustained phase with disappearance of the transients. Subsequent omission of caffeine in the continued presence of carbachol caused a marked [Ca2+]i peak followed by reappearance of the [Ca2+]i transients. However, in cells oscillating in the presence of caffeine its omission caused disappearance of the transients. In this case reintroduction of caffeine restored the transients. In cells kept at -70 mV by a patch pipette containing 100 microM GTP-gamma-S and 3 mM Mg-ATP there were [Ca2+]i transients with frequencies of 0.5-2.5/min. These transients were sufficiently pronounced to activate repetitively a K+ current. Addition of 10 mM caffeine caused disappearance of the [Ca2+]i transients or reduction of their amplitudes and frequencies.(ABSTRACT TRUNCATED AT 250 WORDS)

Relaxation of guinea-pig trachea by cyclic AMP phosphodiesterase inhibitors and their enhancement by sodium nitroprusside.

1. The effects of agents that elevate either cyclic AMP (the phosphodiesterase (PDE) III inhibitor siguazodan, salbutamol) or cyclic GMP (sodium nitroprusside (SNP)) on the relaxant activity of the PDE IV inhibitor, rolipram, were investigated in carbachol (0.1 microM) precontracted guinea-pig tracheal sheets. 2. Rolipram, siguazodan and SNP caused concentration-related reductions in tone of tissues precontracted with 0.1 microM carbachol (EC50 values 12.5; 2.73 and 0.35 microM respectively). Whilst the concentration-response relationship for the PDE III inhibitor, siguazodan, was monophasic that of the PDE IV inhibitor, rolipram, was biphasic. 3. The relaxant activity of rolipram was markedly enhanced in the presence of 10 microM siguazodan (EC50 < 0.01 microM), 0.1 microM salbutamol (EC50 0.03 microM) and 0.3 microM SNP (EC50 0.03 microM). In contrast, the relaxant activity of siguazodan was unaffected by SNP and only modestly enhanced by rolipram (10 microM) and salbutamol (0.1 microM). 4. The relaxant activity of SNP was enhanced by the PDE V inhibitor SK&F 96231 (30 microM: EC50 0.06 microM) and rolipram (30 microM, EC50 0.08 microM) but was unaffected by 30 microM siguazodan. 5. At concentrations up to 10 microM, neither siguazodan nor rolipram elevated tracheal cyclic AMP levels. However, the combination of 10 microM rolipram and siguazodan caused a two fold increase in the cyclic AMP content (from 2.19 to 4.36 pmol cyclic AMP mg-1 protein). SNP (0.1-10 microM) failed to produce a significant increase in tracheal cyclic AMP levels. At 0.1 microM the effect of SNP on tracheal cyclic AMP levels was significantly (P < 0.05) increased in the presence of rolipram but not siguadozan. 6. The results indicate that the relaxant effects of rolipram are markedly enhanced by agents that inhibit PDE III activity or elevate cyclic GMP. They support the hypothesis that SNP potentiates the effects of rolipram via the inhibitory action of cyclic GMP on hydrolysis of cyclic AMP by PDE III. The findings also suggest that whilst PDE III may be more significant in regulating basal smooth muscle tone in the absence of any exogenous stimulus to cyclic AMP accumulation, PDE IV activity may be more tightly coupled to the pool of adenylyl cyclase stimulated by beta2-adrenoceptor agonists.

Luminal perfusion of isolated gastric glands

We have extended to rabbit gastric glands the technique for perfusing single isolated renal tubules. We isolated glands by hand dissection and used concentric glass pipettes to hold them and perfuse their lumina. Parietal cells (PCs), which tended to be located toward the gland opening, were identified by their pyramidal shape, large size, and autofluorescence. Chief cells (CCs) were identified by their round shape and smaller size. In some experiments, we perfused the lumen with hydroxypyrenetrisulfonate, a pH-sensitive fluorophore, at pH 7.4 and used digital image processing to monitor luminal pH (pH1). Solutions were buffered with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid to pH 7.4 at 37 degrees C. With fast perfusion, we found no evidence of decreased pH1, even with stimulation by 10 microM carbachol. With slow perfusion, pH1 often fell below the dye's sensitive range (pH < 5), especially at low perfusate buffering power. In other experiments, we loaded cells with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and monitored intracellular pH (pHi) in multiple individual PCs and CCs in a single gland. Mean pHi was 7.21 +/- 0.02 (n = 136 cells) for PCs and 7.27 +/- 0.03 (n = 103) for CCs. To examine the response to decreased pH1 and basolateral pH (pHb), we lowered pHb to 6.4 or lowered pH1 to 3.4 or 1.4. Lowering pHb to 6.4 for approximately 1 min caused pHi to fall reversibly by 0.39 +/- 0.05 (n = 53) in PCs and 0.58 +/- 0.03 (n = 50) in CCs. Lowering pH1 to 3.4 or 1.4 caused no significant pHi changes in PCs (n = 38 and 82) or in CCs (n = 44 and 77). Carbachol did not affect the response to changes in pH1 or pHb. We conclude that the apical surfaces of PCs and CCs are unusually resistant to extreme pH gradients.