Mechanisms underlying ACh induced modulation of neurogenic and applied ATP constrictions in the submucosal arterioles of the guinea-pig small intestine

Abstract

1. Role of the vascular endothelium in acetylcholine (ACh) induced modulation of neurogenic and applied ATP (adenosine 5'-triphosphate) constrictions of intestinal submucosal arterioles was investigated. 2. Arteriole constrictions, induced either by exogenous ATP or evoked by perivascular nerve stimulation, were attenuated in the presence of ACh. 100 nM ACh almost completely abolished neurogenic constrictions whereas up to 10 microM ACh reduced constrictions to exogenous ATP by only about 60%. 3. Treatment of the arterioles with 100 microM Nomega-nitro-L-arginine (NOLA) and 5 microM indomethacin, to block respectively nitric oxide (NO) and prostanoid release from the endothelium, had no effect on the ACh induced inhibition of neurogenic constrictions but significantly attenuated the inhibitory effects of ACh on constrictions to exogenous ATP. 4. Disruption of the vascular endothelium had no effect on the ACh induced inhibition of neurogenic constrictions but attenuated the inhibitory effects of ACh on applied ATP constrictions to the same extent as after treatment with NOLA and indomethacin. In comparison, endothelial disruption completely abolished the inhibitory effect of substance P (SP) on exogenously applied ATP constrictions. 5. 50 nM ACh significantly attenuated the amplitude of neurally evoked excitatory junction potentials (ejps) recorded from the vascular smooth muscle without altering the time constant of decay (taudecay) of the ejps. 6. It is concluded that ACh inhibits neurogenic constrictions by prejunctional modulation of transmitter release from the perivascular sympathetic nerves with no major role for endothelial paracrine factors. 7. Endothelial NO and/or prostanoids mediate some of the ACh induced inhibition of constrictions to exogenous ATP whereas the endothelium independent inhibitory effects of ACh are attributed to a direct action of ACh on the vascular smooth muscle. However, an indirect effect resulting from activation of vasodilator nerves cannot be ruled out.

Figure 1

Protocol used to assess the inhibitory effect of ACh on neurogenic and exogenous ATP constrictions. After obtaining consistent constrictions to either neurogenic or exogenous ATP, ACh was superfused in the organ bath in increasing concentrations, from 2 nM to 10 μM, and then washed out. 100 nM ACh completely abolished the neurogenic constrictions whereas up to 10  μM failed to completely inhibit the constrictions to exogenous application of ATP.

Figure 2

Summary of the inhibition produced by ACh on the constrictions to perivascular nerve stimulation and iontophoretically applied ATP in the guinea-pig submucosal arteriole. Relationship between the change in the size of constrictions (plotted as per cent inhibition) of neurogenic ATP (maximum inhibition: 100%; EC₅₀: 12.2±4.0 nM: sigmoid curve in solid line) and exogenous ATP (maximum inhibition: 61.6±6.3%; EC₅₀: 25.7±7.9 nM: sigmoid curve in dashed line) and the concentration of ACh. Each point is the mean±s.e.mean of six observations and the sigmoid curves were obtained from an average of individual sigmoid curves fitted to each set of data.

Figure 3

Summary of the inhibition produced by ACh on the constrictions to perivascular nerve stimulation in control solution and after treatment with NOLA and indomethacin or disruption of the vascular endothelium. Comparison between the inhibitory effect of ACh on the amplitude of constrictions to neurogenic ATP before (maximum inhibition: 100%; EC₅₀: 12.2±4.0 nM: sigmoid curve in solid line) and after treatment with 100 μM NOLA+5 μM indomethacin (maximum inhibition: 100%; EC₅₀: 9.9±3.3 nM: sigmoid curve in short dashed line). NOLA+indomethacin did not have any significant effect on the ACh induced inhibition of neurogenic constrictions. Disruption of the vascular endothelium also failed to change the concentration-response relationship of ACh on the neurogenic constrictions (maximum inhibition: 100%; EC₅₀: 10.1±0.7 nM: sigmoid curve in long dashed line). Each point is the mean±s.e.mean of six observations and the sigmoid curves were obtained from an average of individual sigmoid curves fitted to each set of data.

Figure 4

Summary of the inhibition produced by ACh on the constrictions to iontophoretic ATP in control solution and after treatment with NOLA and indomethacin or disruption of the endothelium. Comparison between the inhibitory effect of ACh on the amplitude of constrictions to exogenous ATP before (maximum inhibition: 61.6±6.3%; EC₅₀: 25.9±7.9 nM: sigmoid curve in solid line) and after treatment with 100 μM NOLA+5 μM indomethacin (maximum inhibition: 33.9±6.9%; EC₅₀: 103.2±31.3 nM: sigmoid curve in short dashed line). NOLA+indomethacin significantly attenuated the effect on the ACh induced inhibition of applied ATP constrictions. Disruption of the vascular endothelium attenuated the inhibitory effect of ACh on applied ATP constrictions (maximum inhibition: 28.4±3.4%; EC₅₀: 113±22.4 nM: sigmoid curve in long dashed line) to the same extent as that observed after treatment with NOLA+indomethacin. Each point is the mean±s.e.mean of six observations and the sigmoid curves were obtained from an average of individual sigmoid curves fitted to each set of data.

Figure 5

Picture showing evidence of endothelium and muscle damage in arterioles perfused with CHAPS. (A) Picture showing successful disruption of vascular endothelium. Nuclei of damaged endothelial cells (e) are stained with the fluorescent dye, ethidium bromide. In (B), the arterioles were overexposed to CHAPS causing extensive muscle damage as shown by staining nuclei of muscle cells (m) with ethidium bromide. Calibration bar: 100 μM.

Figure 6

Summary of the inhibition produced by SP on the constrictions to iontophoretic ATP in control solution and after disruption of the endothelium. The inhibitory effect of SP on ATP induced constriction was not dose-dependent for the three chosen concentrations (62±7.2% at 10 nM; 67±5.9% at 20 nM and 66±2.4% at 50 nM). Disruption of the endothelium significantly attenuated the inhibitory effect of SP (3±2.7% at 10 nM; 4±2.3% at 20 nM and −2±4.1% at 50 nM). The inhibition produced by SP after endothelial disruption was not significantly different from 0%. Each point is the mean±s.e.mean of three observations.

Figure 7

Effect of ACh on the summation of ejps evoked by a train of perivascular nerve stimulation. Perivascular nerves were stimulated at 10 Hz, 1 s to give ejps which summed to give a peak value of 26 mVs. Each ejp is preceded with a stimulus artefact. In the same experiment, presence of 50 nM ACh reduced the peak amplitude of the summed ejps to 14 mVs. For measurement of change in ejp amplitude and ‘τ_decay' the effect of ACh on single ejps was observed.

Figure 8

Effect of ACh on the amplitude and time constant of decay of ejp evoked by perivascular nerve stimulation. For measurement of change in ejp amplitude and ‘τ_decay' the effect of ACh on single ejps was observed. The ejps were evoked by stimulation of the perivascular sympathetic nerves with single pulses. The control ejp had an amplitude of 11.5 mV and ‘τ_decay' of 322 ms. In the same experiment, presence of 50 nM ACh reduced the amplitude of the ejp to 7.1 mV with a ‘τ_decay' of 327 ms.