Worm Breeder's Gazette 13(1): 44 (October 1, 1993)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
In order to make the pharynx more directly accessible to drugs and electrophysiological probes, I have developed a medium that supports normal pumping of dissected pharynxes. Leon Avery and David Raizen showed that when one decapitates a worm by cutting just behind the terminal bulb (TB) with a scalpel, the pharynx protrudes from under the remaining cuticle and body muscle, directly exposing the pharynx to the medium. When dissected in Ascaris Saline (AS) and 1 µM serotonin, the pharynxes pump regularly and their electropharyngeograms (EPGs) can be measured. AS was chosen because it almost exactly approximates the ionic composition of Ascaris pseudocoelomic fluid as determined by Brading & Caldwell (J. Physiol. 173.36P) and has been used successfully for electrophysiological studies of Ascaris. However, dissected C. elegans pharynxes in AS have brief pumps with little corpus motion and a correspondingly short p-phase in the EPG. By varying the ion concentrations in the medium I found that: 1) Using chloride instead of an organic anion (acetate in the case of AS) did not affect the pharynx adversely, contrary the idea that a high chloride concentration is bad for (all) nematode muscles, 2) For unknown reasons, the usually inert organic cations choline and methylglucamine cause hypercontraction of the pharynx at low (10 mM) concentrations, 3) Pump length is dependent on the external calcium concentration.
The effect of calcium concentration on pump length is remarkable. By measuring the length of the P-phase on an EPG, the precise length of the pump (as defined by the period of depolarization) can be determined even when the calcium concentration is so low that the muscle stops contracting. Basically one can vary the length of a pump at will by changing the calcium concentration - high [Ca], short pump, low [Ca], long pump. By contrast, equivalent changes in the sodium or potassium concentrations mostly just reduced the amplitude of the EPG and at extremes caused pumping to cease. All effects were reversed by changing [Ca] back to normal (about 3 mM). To see if this could be an effect of calcium flux across the membrane, I treated the pharynxes with the calcium channel blockers verapamil, diltiazem, and nifedipine at concentrations of 10-20 µM. All extended the pump length significantly. Increasing concentrations of magnesium also lengthened the P-phase, presumably by competitively blocking calcium channels. The effects of calcium channel blockers could be rescued by raising the [Ca]. Unfortunately Bay K, a calcium channel activator, had no effect at a concentration of 5 µM.
The reason for the calcium effect is not clear. If changing the external calcium concentration were affecting the ability of internal calcium to maintain the depolarization, then decreasing [Ca] should decrease the pump length, not increase it. This is not an effect on the nervous system because it occurs in a worm in which the pharyngeal nervous system was ablated. Changing external [Ca] could change the resting potential of the muscle, making the membrane more or less excitable. Alternatively, calcium could be a second messenger that is necessary for repolarization of the membrane, for example by interacting with a calcium-activated K channel; this explanation would also account for the effect of the calcium channel blockers.
· Improved pharynx pumping medium:
138 mM NaCl
6 mM KCI
1 mM Mg Cl(2) mM phosphate buffer pH 7.4
1 µM serotonin