Intracellular recordings from the guinea pig internal anal sphincter (IAS) have demonstrated that a neurogenic relaxation of the smooth muscle is often associated with a membrane hyperpolarisation. (Baird and Muir 1990). In our previous studies we have described the pharmacology of the isolated sheep IAS using organ bath studies (Mundey et al 2000, Jonas-Obichere 2002). Here we describe our initial findings using intracellular microelectrodes to measure the membrane potential in the sheep IAS. A segment (5x2x1 mm) of the distal internal anal sphincter (IAS) was cut and pinned out under tension in a 3ml sylgard-coated petri dish. The tissue was superfused continuously (3 ml /min) with Krebs-Henseleit solution (pH 7.4), gassed (95% O₂ /5% CO₂) at 37 °C. Cells were impaled with sharp electrodes (tip resistance 55-190M) filled with 3M KCl. Signals were amplified and the output displayed on an oscilloscope. A successful intracellular impalement was indicated by a rapid drop in voltage to a negative value, the resting membrane potential (Vm). If the Vm was stable for > 3 mins, drugs were superfused in a volume of 10-20 ml of Krebs-Henseleit. To facilitate stable recording, either nifedipine, phentolamine or atropine (1µM) were added to the perfusate to prevent spontaneous activity from cholinergic and adrenergic nerves in any residual circular and longitudinal muscle fibres (Baird and Muir 1990, Rae and Muir 1996). A students t-test was used to test for statistical significance between mean values (p<0.05). The mean resting membrane potential recorded from stable cells was -42.2 ± 1.8 mV (n=28 cells from n=14 IAS). Tetraethylammonium chloride (TEA, 2-10 mM) a non-selective inhibitor of K⁺ channels significantly depolarised Vm from -32.8 ± 3.7 mV to -11.7 ± 5.3 mV (n=7, p<0.05).

Alpha-adrenoceptor agonist phenylephrine (1-10 µM) depolarised Vm from -34.4 ± 2.6 mV to -14.0 ± 6.4 mV (n=5, p<0.05)*.
Sodium nitroprusside (SNP) (30-100 µM) hyperpolarised Vm from -35.6 ± 3.5 mV to -47.0 ± 4.8 mV. (n=5, p<0.05).

*(Phentolamine was omitted from the perfusate). Sodium orthovanadate (inhibits tyrosine phosphatase, 1-3 mM) depolarised Vm from -38.5 ± 3.6 mV to -12.3 ± 4.8 mV (n=6, p<0.05). SNP is a NO donor and inhibits the myogenic tone of IAS in the organ bath (Mundey et al., 2000). The current findings suggest that this relaxation may be attributable to a membrane hyperpolarisation mediated via an increase in K+ conductance. Previous organ bath studies have also demonstrated that phenylephrine (10nM-10µM) produced a concentration-dependent increase in myogenic tone and that sodium orthovanadate (30nM-3mM) caused a slow-developing contraction of the IAS (Jonas-Obichere 2002). The electrophysiological findings thus correlate a membrane depolarisation with contractile events. This initial study compares well with previous studies.

Jonas-Obichere, M (2002). Thesis for Doctorate of Medicine (DM).
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