Pharmacological characterization of neurogenic responses of the sheep isolated internal anal sphincter

Abstract

The aim of the study was to establish the nature of the neurogenic responses of the sheep isolated anal sphincter. Isolated strips of sheep internal anal sphincter develop intrinsic contractile tone following the application of stretch tension. On transmural stimulation (1 - 20 Hz, 10 V pulse strength, 0.5 ms pulse width, 1 s every 180 s) transient relaxations were observed. The amplitude of the relaxations were frequency-dependent reaching a maximal response at 10 - 20 Hz and were inhibited by tetrodotoxin (0.3 microM). Neither atropine (0.3 microM) nor phentolamine (1 microM) affected control responses. The nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 100 microM) and the selective inhibitor of soluble guanylyl cyclase ODQ, (1H-[1,2, 4]oxadiazolo[4,3-a]quinoxalin-1-one) (1 microM) completely inhibited the neurogenic relaxations and uncovered contractions that were abolished by 1 microM phentolamine and 0.1 microM prazosin. The effect of L-NAME, but not that of ODQ, was partially reversed by the addition of L-arginine (1 mM). Sodium nitroprusside (10 nM - 10 microM) caused concentration-dependent inhibition of myogenic tone and this effect was significantly reduced by ODQ. Calcium-free Krebs-Henseleit solution also reduced myogenic tone by 85%. Transmural electrical stimulation of the sheep isolated internal anal sphincter causes a transient relaxation of myogenic tone that appears to involve nitric oxide from non-adrenergic, non-cholinergic nerves and, to a lesser degree, noradrenaline from sympathetic nerves. The characteristics of the preparation compares well with that of human tissue and may prove to be a suitable animal based model for further studies.

Figure 1

(a) Representative digitized recording of the effect of increasing frequency of stimulation (1–20 Hz for 1 s every 180 s) on the sheep isolated internal anal sphincter. Two responses were obtained at each frequency of stimulation. (b) Histogram of the frequency response relationship for neurogenic relaxations of the sheep isolated internal anal sphincter. Responses are shown as the mean±s.e.mean of 10 observations. (c) Representative digitized recording showing the occurrence of ‘after-contractions' following the initial rapid relaxation on electrical stimulation (10 Hz, 1 s) which was observed in approximately 50% of preparations.

Figure 2

Histogram of the response of the sheep isolated internal anal sphincter to 10 Hz stimulation for 1 s under control conditions (n=17) and the presence of 0.3 μM atropine (n=5), 1 μM phentolamine (n=5) or 100 μM L-NAME (n=17). Responses are shown as the mean±s.e.mean. ^*Denotes a statistically significant difference from the control value.

Figure 3

(a) Representative digitized recording of neurogenic responses (10 Hz, 1 s stimulation every 180 s) of the sheep isolated internal anal sphincter under control conditions, in the presence of 100 μM L-NAME and subsequent addition of 1 mM L-arginine. (b) A histogram of the neurogenic responses of the sheep isolated internal anal sphincter under control conditions and sequential addition of 100 μM L-NAME, 1 mM L-arginine and 1 μM phentolamine. The responses are shown as the mean±s.e.mean of nine observations. ^*Denotes a statistically significant difference from the control value.

Figure 4

Histogram of the response of the sheep isolated internal anal sphincter to 10 Hz stimulation for 1 s under control conditions and following the sequential addition of 100 μM L-NAME, 1.0 μM RX-811059 and 0.1 μM prazosin (n=10). Responses are shown as the mean±s.e.mean. ^*Denotes a statistically significant difference from the control value.

Figure 5

A histogram of the neurogenic responses of the sheep isolated internal anal sphincter under control conditions and subsequent, sequential addition of 1 μM ODQ, 1 mM L-arginine and 1 μM phentolamine. The responses are shown as the mean±s.e.mean of seven observations. ^*Denotes a statistically significant difference from the control value.

Figure 6

Representative digitized recording of the response of the sheep isolated internal and sphincter to the application of 2 g tension, electrical stimulation (S1; 10 Hz, 1 s every 180 s) and subsequent cumulative addition of sodium nitroprusside (10 nM–10 μM). Following washout (w) the preparation was again stimulated electrically (S2).

Figure 7

A graph of the effect of cumulative addition of sodium nitroprusside on myogenic tone of the sheep isolated internal anal sphincter under control conditions and in the presence of 2 μM ODQ. The contractile tone has been expressed as a percentage of the contraction prior to the addition of sodium nitroprusside and shown as mean±s.e.mean of eight observations.