The recently discovered kappa2 receptor is the most abundant known opiate receptor subtype in both rat and guinea pig brain. It is found in many regions of the central nervous system, including the spinal tract of the trigeminal nerve, making it an important target area in orofacial pain management. Since compounds that interact with the opiate system are used clinically for the management of pain, this high capacity site could be expected to play an important physiological role. However, little is known about its receptor binding or activation properties. The overall goal of this project is to develop an in vitro system to determine recognition and activation through the kappa2-receptor(s) in an effort to determine which opiate ligands can produce their effects through this site. This information will be very useful in determining which compounds can be used to target this high capacity opiate site. To this end, a two step process is required. The first step involves the development of a receptor binding system which utilizes a nonselective radioligand with mu- ,delta-and kappa1-opiate receptors masked by antagonists or covalently bound blocking agents. The use of antagonists/covalent blockers is necessary in order to use the same blocking conditions in a model tissue assay of receptor activation. The development of the binding assay will involve the determination of the affinity of several mu-,delta-and kappa1- selective as well as nonselective antagonists at the kappa2-site, currently assayed with the other opiate sites blocked with selective agonists. The effect of preincubation with various concentrations of covalent blockers on kappa2-binding sites will also be determined by calculating the number of kappa2-sites that can be detected before and after each treatment. From these assays, the appropriate blocking agents and concentrations will be determined and verified by comparison of this assay system to that in which the other receptors are masked with agonists. Several agonists will be assayed using this newly developed assay system in order to determine which bind to and therefore could produce action through the kappa2-sites. Those compounds with significant affinity for the kappa2-receptors will be used, in the second step, to screen for kappa2-mediated activity in the available model tissue systems made to single kappa2-receptor-containing tissues by treatment with the antagonists/covalent blocking agents under the conditions determined by the receptor binding studies. The model tissues to be investigated include: guinea pig ileum, known to contain mu-and kappa-receptors; mouse vas deferens, known to contain mu-,delta-and kappa-receptors; and rat vas deferens, proposed to contain mu-as well as epsilon-sites, which have been proposed by some investigators to be kappa2-receptors. The development of such in vitro assay systems will greatly facilitate the exploration of this seemingly important opiate receptor subtype and may lead to the development of new drugs for the control of pain.
