PubMed · 4319513
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B Csillik. 1969. Acetylcholine.. https://doi.org/10.1007/978-3-662-25519-3_8
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1. Intracellular pH (pHi) was measured by spectrofluorometry in perfused mandibular salivary glands isolated from the rat and loaded with the pH-sensitive fluoroprobe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Cell volume changes were estimated from changes in intracellular water content measured by proton NMR spectroscopy. 2. Stimulation with 1 microM acetylcholine (ACh) led to a 15 +/- 2% decrease in cell volume. A transient decrease in pHi was followed by a sustained increase (0.17 +/- 0.03 pH units) that has previously been attributed to the upregulation of the Na(+)-H+ exchanger. 3. Increasing perfusate osmolarity by addition of 60 mM sucrose caused a 19 +/- 2% decrease in cell volume and a sustained increase in pHi (0.12 +/- 0.01 pH units) that was abolished by 1 mM amiloride. Acid loading experiments indicated that the increase in pHi was due to an alkaline shift in the pH dependence of the Na(+)-H+ exchanger. 4. A 20% reduction in perfusate osmolarity prevented the cell shrinkage normally associated with ACh stimulation and largely abolished the ACh-induced increase in pHi. 5. Steady-state Na(+)-H+ exchanger activity, estimated from the initial rate of change in pHi following addition of amiloride, increased 9-fold during stimulation with ACh. When cell shrinkage was prevented by simultaneous exposure to the hypotonic solution, the activity of the exchanger still increased 7-fold in response to ACh. 6. We conclude that, although cell shrinkage leads to upregulation of the Na(+)-H+ exchanger, this factor alone is insufficient to account for the marked increase in exchanger activity that follows muscarinic stimulation.
1. Whole-cell patch-clamp technique was used to study the beta-adrenergic and cholinergic regulation of the inwardly rectifying K+ conductance (gK1) in isolated guinea-pig ventricular myocytes. 2. In Cl(-)-free solutions or in the presence of 9-anthracenecarboxylic acid or Co2+, bath-applied isoprenaline (Iso) partially inhibited the steady-state whole-cell conductance (gss) calculated from the steady-state current (Iss)-voltage (Iss-V) curve at membrane voltages (Vm) negative to the equilibrium potential for potassium (EK). Iss was also inhibited at Vm positive to EK when the extracellular [K+] was 20 mM. The Iso-sensitive component of gss exhibited the characteristics of the inwardly rectifying K+ conductance (gK1). 3. The Iso-induced inhibition of gK1 was reversible, concentration dependent, blocked by propranolol, mimicked by both forskolin and dibutyryl cAMP, and prevented by including a cAMP-dependent protein kinase (PKA) inhibitor in the pipette solution. These findings suggest that PKA mediates the Iso-induced inhibition of gK1. 4. The apparent dissociation constant (KD) for the concentration dependence of Iso-induced inhibition was 0.035 microM and the Hill coefficient was approximately 1.0. A maximal Iso concentration (1 microM) inhibited gK1 by 40 +/- 4.1% (mean +/- S.E.M.; n = 13). 5. Bath application of acetylcholine (ACh, 0.1 microM or more) antagonized the Iso-induced (1 microM) inhibition of gK1; [ACh] > 1.0 microM antagonized 88 +/- 2.1% (n = 10) of the inhibition. ACh increased the KD for Iso to inhibit Iso-sensitive gK1 and also reduced the maximal Iso-induced inhibition. 6. ACh-induced antagonism could be abolished by pre-incubating myocytes with pertussis toxin (PTX), suggesting that a muscarinic receptor-coupled, PTX-sensitive G protein, Gi, is involved. 7. ACh (10 microM) also antagonized approximately 70% of the dibutyryl cyclic AMP (1 mM)-induced inhibition of gK1 (n = 3), suggesting that the ACh-induced antagonism involves more than simply inhibiting the Iso-mediated activation of adenylyl cyclase via the activated Gi. 8. Intracellularly applied okadaic acid (OkA, 1 microM) did not alter gK1 (control = 134 +/- 5.1 nS vs. OkA = 136 +/- 6.1 nS), but the Iso-induced decrease in gK1 was less (P < 0.001) with OkA present (42.1 +/- 2.4 nS, n = 5) than when absent (54.0 +/- 2.2 nS, n = 10). However, ACh (10 microM) failed to antagonize Iso-induced inhibition with OkA present, suggesting involvement of a protein phosphatase.
1. To clarify the nature of the inhibition of whole-cell inwardly rectifying K+ current (IK1) by isoprenaline (Iso) and its antagonism by acetylcholine (ACh), we studied the effects of Iso and ACh and their surrogates on single channel currents (iK1) carried by inwardly rectifying K+ channels in cell-attached and excised inside-out patches obtained from guinea-pig ventricular myocytes. 2. Bath application of Iso suppressed iK1 channel activity in cell-attached patches. This was inhibited by propranolol. Bath-applied forskolin or dibutyryl cAMP mimicked the effect of bath-applied Iso. 3. Exposure of the cytosolic face of inside-out patches to purified catalytic subunit of the cAMP-dependent protein kinase (PKA) also suppressed iK1 channel activity, mimicking the effect of bath-applied Iso on iK1 recorded from cell-attached patches. 4. When applied directly to cell-attached patches via the patch pipette solution, ACh antagonized Iso-induced (1 microM applied via the bath) suppression of iK1 channels. In contrast, bath-applied ACh (10 microM) partially antagonized the effect of low concentrations of Iso (e.g. < 50 nM) on iK1 channels in cell-attached patches but had no detectable effect when 1 microM or more Iso was used. 5. In myocytes pretreated with pertussis toxin (PTX), ACh failed to antagonize Iso-induced suppression of iK1 channels. When inside-out patches were used, bath-applied preactivated exogenous inhibitory G protein subunit, G1 alpha, antagonized the suppression of iK1 channels induced by bath-applied catalytic subunit of PKA (PKA-CS), suggesting that a PTX-sensitive G1 alpha mediates ACh-induced antagonism of Iso-induced suppression of iK1. 6. Neither GTP gamma S nor G1 alpha antagonized the suppression of iK1 produced by bath-applied PKA-CS in inside-out patches when okadaic acid was present in the bath. In addition, bath application of alkaline phosphatase also reactivated iK1 channels suppressed by PKA-CS. 7. Findings in guinea-pig ventricular myocytes suggest that iK1 can be suppressed by a PKA-mediated phosphorylation of the iK1 channel occurring in response to Iso-induced beta-adrenergic receptor activation and that ACh can antagonize the suppression by mechanisms that involve both intracellular and membrane-delimited pathways. The membrane-delimited pathway appears to involve M2-cholinergic receptors, their associated G protein, G1, and a protein phosphatase, all located in the sarcolemma in close proximity to the involved iK1 channels.