a1 Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, Florida, USA.
The addition of acetylcholine (ACh, 100 μl of 10 μM) to the bath in the vicinity of unfertilised sea urchin eggs (Lytechinus variegatus) suspended in sea water (SW) abruptly depolarises the membrane potential (Vm) of the eggs from the resting value of approximately −70 mV. This results in the firing of the egg's action potential, followed by partial repolarisation. Similar addition of ACh to eggs voltage clamped at −70 mV induces an inward current of abrupt onset with peak amplitude of −1.26 ± 0.20 nA (SE, n = 81). When the eggs are clamped at a Vm more positive than −70 mV, the peak amplitude of the ACh-induced inward current decreases, becoming 0 at a clamped Vm of approximately −20 mV. Further positive shift of the Vm fails to cause reversal of the current. Oocytes clamped at −70 mV exhibit similar inward current responses following application of ACh. Since ACh stimulates both nicotinic and muscarinic receptors (nACh-R and mACh-R, respectively), the effects of exposing eggs to the agonists and antagonists for each type of receptor were examined. For unfertilised eggs clamped at −70 mV the application of 100 μM (−)-nicotine hydrogen tartrate, an agonist of the nACh-R, induces an inward current response similar to that elicited by 10 μM ACh, but of smaller peak amplitude. In contrast, the application of (+)-muscarine chloride, an agonist of the mACh-R, fails to induce any response. Antagonists of the nACh-R inhibit either the neuronal type of nACh-R or the skeletal muscle type of nACh-R. The effect of the antagonists on the amplitudes of the ACh-induced inward current response was determined by supervising individual eggs clamped at −70 mV with the desired antagonist dissolved in SW, followed by the addition of 100 μl of 10 μM ACh in the vicinity of the egg. Mecamylamine chloride, an antagonist of the neuronal nACh-R at a concentration of 1 μM, markedly decreases the response to ACh, while at a concentration of 10 μM the response to ACh is abolished. Hexamethonium chloride, another inhibitor of the nACh-R of the neuronal type, also diminishes the ACh-induced response, but at a concentration of 10 μM the response is not completely abolished. Exposure of eggs to α-bungarotoxin, an antagonist of the skeletal muscle nACh-R at concentrations up to 250 nM for periods of 30 min, has no effect on the ACh-induced response. The effects of two antagonists of the mACh-R, atropine sulphate and QNB (R-(−)-3-quinuclidinyl benzilate) were also examined. Exposure of eggs to 1 μM atropine does not affect the ACh-induced response, but at concentrations of 10 μM atropine the amplitude of the ACh-induced inward current is significantly reduced. The exposure of eggs to QNB, a highly specific antagonist of the mACh-R, at concentrations up to 50 nM, has no effect on the ACh-induced response. Consequently, the likely explanation for the inhibitory effect of atropine is that at high concentrations atropine cross-reacts with the nACh-R. These findings reveal the presence in unfertilised sea urchin eggs of an ACh-R resembling the neuronal nACh-R. No evidence could be obtained that these receptors have a role in sperm entry, activation of the egg, or early development.
(Received May 14 1997)
(Accepted June 26 1997)
c1 P.I. Ivonnet, Department of Physiology and Biophysics, University of Miami School of Medicine, PO Box 016430, Miami, FL 33101, USA. Telephone: +1 (305)-243-6322. Fax: +1(305)-243-5931.