The overall aim of the experiments proposed is an increased understanding of the actions of opioids at the cellular and molecular level; this understanding is sought so that the full therapeutic potential of this class of drugs can be safely exploited, and so that the adverse effects of the drugs such as tolerance and dependence can be managed and even prevented. The work is predicated on prior knowledge that opioids affect nerve cells by acting on three or more distinct receptor types;
the specific aim i s to determine the consequences for individual nerve cells of activation of these receptors by endogenous and exogenous opioids and how these consequences change with long term exposure. The methods used will be primarily electrophysiological - intracellular recording of membrane ion currents using single electrode voltage clamp and patch clamp techniques. The currents of single cells will be separated by their voltage and time dependence, and by the effects of channel blockers. The cells are chosen for study (guinea-pig myenteric and submucous plexus neurons) because earlier work has shown them to express mu, delta, and kappa receptors. Agonists and antagonists will be applied to the cells in known concentrations under steady-state conditions, or instantaneously so as to measure the time course of opioid action. The nerve cells will be maintained in vitro, with experimental recording carried out during the first few hours after removal from the animal, or after up to three weeks in cell culture. Hypotheses to be tested are that (i) mu and delta receptors activate a potassium conductance by a direct mechanism not involving changes in intracellular calcium, (ii) that kappa agonists depress a single class of calcium conductance, (iii) that cell processes from which transmitter is released express opioid receptors, and that consequences of their activation can be assessed by Fura-two measurements of intracellular calcium, and that (iv) chronic treatment of guinea-pigs with morphine results in tolerance to actions at mu receptors, but not at delta or kappa receptors.
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