Previous studies on the pharmacological characteristics of neurogenic relaxations in sheep internal anal sphincter have suggested the presence of a non-adrenergic, non-nitrergic transmitter (Mundey et al., 2000; Nisar et al., this meeting). ATP and PACAP, acting on apamin-sensitive K channels, have been suggested as inhibitor transmitters in gastrointestinal smooth muscle (De Luca et al., 1999; Lenard et al., 2000). We have examined the effect of apamin on neurogenic responses of the sheep isolated internal anal sphincter.

Segments of sheep isolated internal anal sphincter were prepared for isometric tension recording (Mundey et al., 2000). Neurogenic responses to 1 and 30 Hz stimulation (300 mA, 0.3 ms, 30 s duration) were examined in the absence and presence of 300 nM apamin. The magnitude of responses is in gram weight and shown as the mean ± s.e.mean of n observations. The time course of neurogenic relaxations is expressed as the time taken from the cessation of field stimulation for a 50% decline in maximal relaxation (t₅₀).

Under control conditions, electrical field stimulation caused sustained neurogenic relaxations at 1Hz (1.14 ± 0.26 g wt; t₅₀ - 4.8 ± 0.7 sec, n=7) but transient responses at 30 Hz (1.33 ± 0.19 g wt, n=7; t₅₀ -12.0 ± 3.5 sec, n=7). Following exposure to 300nM apamin the magnitude of responses at 1 Hz (0.95 ± 0.17g wt, n=7) and 30 Hz (0.88 ± 0.32 g wt, n=7) were not significantly different from control values (Student t-test p > 0.05).

Figure 1. Digitized recording of the responses to 1Hz (Left) and 30 Hz (Right) stimulation (30s) in the presence and absence of 300nM Apamin.

Apamin (300nM) significantly (p < 0.05) reduced the time course of relaxations at 1Hz (t₅₀ 0.2 ± 2.9 sec, n=7) and 30 Hz (t₅₀ -21.6 ± 1.2 sec, n=7) (see: Figure 1). In the presence of 100µM L-NAME and 3µM phentolamine, apamin abolished the response to 1Hz (0.27 ± 0.1 g wt, n=13) and converted the 30Hz relaxation (0.43 ± 2.1 g wt, n=13) into a contraction (0.14 ± 0.27 g wt, n=13). These findings indicate that the non-adrenergic, non-nitrergic transmitter in the sheep anal sphincter has a relatively minor role, but at high frequencies of stimulation acts via apamin-sensitive channels to enhance the effect of nitric oxide.

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Lenard, L., et al., (2000). N-S Arch. Pharmacol 361, 492-497.
Mundey M.K. et al., (2000) Br. J. Pharmacol. 130(3):489-94.