Mechanisms involved in the regulation of mRNA for M2 muscarinic acetylcholine receptors and endothelial and neuronal NO synthases in rat atria

Abstract

Background and purpose: Agonists of the M(2) muscarinic acetylcholine receptor (mAChR) increase mRNA for this receptor and mRNA for endothelial and neuronal isoforms of NO synthase (eNOS or nNOS). Here we examine the different signalling pathways involved in such events in rat cardiac atria.

Experimental approach: In isolated atria, the effects of carbachol on mRNA for M(2) receptors, eNOS and nNOS were measured along with changes in phosphoinositide (PI) turnover, translocation of protein kinase C (PKC), NOS activity and atrial contractility.

Key results: Carbachol increased mRNA for M(2) receptors, activation of PI turnover, translocation of PKC and NOS activity and decreased atrial contractility. Inhibitors of phospholipase C (PLC), calcium/calmodulin (CaM), NOS and PKC prevented the carbachol-dependent increase in mRNA for M(2) receptors. These inhibitors also attenuated the carbachol induced increase in nNOS- and eNOS-mRNA levels. Inhibition of nNOS shifted the dose response curve of carbachol on contractility to the right, whereas inhibition of eNOS shifted it to the left.

Conclusions and implications: From our results, activation of M(2) receptors induced nNOS and eNOS expression and activation of NOS up-regulated M(2) receptor gene expression. The signalling pathways involved included stimulation of PI turnover via PLC activation, CaM and PKC. nNOS and eNOS mediated opposing effects on the negative inotropic effect in atria, induced by stimulation of M(2) receptors. These results may contribute to a better understanding of the effects and side effects of cholinomimetic treatment in patients with cardiac neuromyopathy.

Figure 1

Analysis of target mRNA levels by competitive PCR. The upper panel shows a quantitative analysis of the competitive PCR experiment. Ordinate, ratio of the target to MIMIC products as determined by band densitometry. Abscissa, inverse amount of MIMIC added to the amplification. When the resultant target and MIMIC densitometric values are equal (y=1), extrapolation can be performed to yield the target amount.The lower panel shows an autoradiogram of neuronal nNOS in digoxigenin-labeled competitive PCR. The same applies for Figures 2, 5, 6 and 7.

Figure 2

Effects of carbachol on semi-quantitative RT-PCR analysis of mRNA for M₂ receptors from rat atria incubated for 2 h with each concentration of carbachol. (a) Dose–response curve of carbachol in absence or in presence of 1 × 10⁻⁷ M AF-DX116. Basal values correspond to mRNA levels after 2-h incubation without carbachol. Values are mean±s.e.m. of six experiments in each group. The RT-PCR products obtained from carbachol dose–response curve in absence (b) and in the presence (c) of AF-DX 116 are shown. P<0.001 between carbachol and carbachol+AF-DX 116 at all concentrations used.

Figure 3

Concentration–response curve of carbachol-induced NOS activity (a) PI production (b) and membrane PKC translocation (c) in rat atria, with or without 1 × 10⁻⁷ M AF-DX 116. (c) PKC translocation from cytosol (full line) to membrane (dotted line) is shown. Basal values correspond to data before addition of carbachol. Values are mean±s.e.m. of six experiments in each group.

Figure 4

(a) Correlation between the stimulatory effects of carbachol (1 × 10⁻⁹ to 1 × 10⁻⁶ M) on NOS, membrane PKC activities and PI accumulation. NOS was plotted as a function of both PI (r²=0.9556) and PKC (r²=0.9405). Values are mean±s.e.m. of five experiments on each group. (b) Correlation between the stimulatory effect of carbachol (1 × 10⁻⁹–1 × 10⁻⁶ M) on M₂ receptor mRNA and NOS activity. M₂ receptor mRNA was plotted as a function of NOS activity (r²=0.9704). Values are mean±s.e.m. of six experiments in each group. For other details see legend of Figure 3.

Figure 5

Carbachol-induced increase in mRNA for M₂ receptors in rat atria. Preparations were incubated for 2 h with 1 × 10⁻⁷ M carbachol in the absence or in the presence of 5 × 10⁻⁶ M U-73122 or 5 × 10⁻⁶ M trifluoperazine or 1 × 10⁻⁵ M L-NMMA or 5 × 10⁻⁹ M calphostin. The effect of 5 × 10⁻⁵ M SNP is also shown. Values are mean±s.e.m. of six experiments in each group. ^*P<0.001 versus carbachol alone. An example of the RT-PCR products obtained from this analysis is shown in the lower panel.

Figure 6

(a) Concentration–response curve of NOS activity induced by carbachol, in the absence or in the presence of 5 × 10⁻⁶ M L-NIO or 5 × 10⁻⁶ M NZ. Values are mean±s.e.m. of six experiments in each group. (b) Carbachol effects on semi-quantitative RT-PCR analysis for nNOS and eNOS mRNA expression. Rat atria were incubated for 2 h in the absence (basal) or in the presence of different carbachol concentrations. Values are mean±s.e.m. of seven experiments in each group. (c) RT-PCR products obtained from carbachol dose–response curves are illustrated for eNOS (360 bp) and nNOS (240 bp).

Figure 7

Effects of carbachol on semi-quantitative RT-PCR analysis for eNOS (a), nNOS (b) mRNA levels from rat atria incubated for 2 h with 1 × 10⁻⁷ M carbachol in the absence or in the presence of 1 × 10⁻⁷ M AF-DX116 or 5 × 10⁻⁶ M U-73122 or 5 × 10⁻⁶ M trifluoperazine or 5 × 10⁻⁹ M calphostin. Basal values correspond to mRNA levels after 2-h incubation without carbachol. Values are mean±s.e.m. of six experiments in each group. The RT-PCR products obtained from carbachol alone or in presence of different blockers are shown in the lower panel. ^*P<0.001 between carbachol and carbachol+inhibitors.

Figure 8

Carbachol-induced decrease of contractility (expressed as dF/dt) in rat-isolated atria. Preparations were incubated for 30 min with 5 × 10⁻⁶ M NZ, 1−10⁻⁶ M L-NIO or vehicle (shown as carbachol) the concentration–response curves of carbachol were plotted out. Values are mean±s.e.m. of six experiments in each group.

Figure 9

Effects of carbachol on atrial contractility (dF/dt) (a) and NOS activity (b) from WT mice or eNOS−/− mice, in absence (full line) or in presence of 5 × 10⁻⁶ M NZ (dotted line). Contractility (dF/dt) is expressed in % of basal values (before addition of the first concentration of the agonists) taken as 100%. Actual basal values were: 4.2±0.4, WT mice (n=5); and 3.9±0.4, eNOS−/− mice (n=5); in the presence of NZ, 4.6±0.5 WT mice (n=5) and 4.2±0.5 eNOS−/− mice (n=5). In (b), the NOS activity triggered by 1 × 10⁻⁷ M carbachol alone or in the presence of 5 × 10⁻⁶ M NZ and 5 × 10⁻⁶ M L-NIO in atria from WT mice or eNOS−/− mice is also shown. Data shown are the means±s.e.m. of five experiments in each group.