Duffy, Stephen Mark; (1994) Pharmacological control of membrane currents generating the after-hyperpolarization of rat sympathetic neurones. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Sympathetic neurones show a calcium-dependent after-hyperpolarization (a.h.p.) following action potentials. This study has investigated the currents responsible for generating the a.h.p. in the rat superior cervical ganglion (s.c.g.), and their inhibition by pharmacological agents. The calcium current (ICa) was recorded from voltage-clamped dissociated s.c.g. neurones using Cs+ filled electrodes. ICa was abolished by removal of external Ca2+, and blocked by inorganic divalent cations (IC50):- Cd2+(3.3μM), Cu2+(87μM), Zn2+(190μM), Ni2+(520μM), Mn2+(580μM) and Co2+ (1.1mM) and trivalent cations:- Gd3+(2.6μM), Lu3+(72μM) and La3+(3.0μM). Noradrenaline suppressed ICa by approximately 50% (EC50; 87nM) and slowed the rising phase of the current. ICa was also reduced by the a2-adrenergic agonists, UK14304, clonidine and oxymetazoline (EC50; 37,57 and 27nM respectively) whilst α1- and β-adrenergic agonists had no effect in low concentrations. The inhibition by noradrenaline was antagonised by phentolamine, yohimbine and prazosin with pKB values of 8.0, 7.5 and 5.4 respectively, indicating that adrenergic suppression of ICa is mediated by an a2-receptor. ICa was also reduced by the muscarinic agonist methacholine. The current underlying the a.h.p. (IAHP) was investigated in intact and dissociated neurones using both intracellular and whole-cell electrodes. The current was characterised as a small slowly decaying outward current following depolarizing voltage commands. A 37mV shift in reversal potential was observed as extracellular potassium was raised from 4.8 to 20mM confirming that IAHP was carried by potassium ions. Calcium dependence was shown by removal of extracellular calcium and block by Cd2+. The current was blocked by d-tubocurarine (dTC) and apamin but was insensitive to tetraethylammonium ions, distinguishing it from the fast calcium-activated potassium current, IC. Noradrenaline and methacholine suppressed IAHP. Control of IAHP by calcium was investigated further. Reducing the calcium load (by reducing extracellular calcium, blocking Ca2+ entry with Cd2+ or decreasing the duration of the voltage commands) increased the rate of decay. dTC (which blocked IAHP without effect on ICa) had no effect upon the rate of IAHP. These observations suggest that the rate of decay of IAHP is dependent upon the size of calcium load. Inhibition of IAHP by both noradrenaline and methacholine showed, in addition to reduction of peak amplitude, an increase in rate of decay suggesting that the inhibition of IAHP is due to suppression of ICa. It is concluded that (1) The adrenergic suppression of ICa is mediated by a2-receptors. (2) The a.h.p. is generated by a calcium-activated potassium current that is similar to the IAHP of bullfrog sympathetic neurones. (3) The kinetics of IAHP decay by are determined by the calcium load. (4) Noradrenaline and methacholine inhibit IAHP by reduction of the preceding calcium entry.

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