GR 64349

The suitability of various neurokinin-2 (NK2) receptor agonists and routes of administration to elicit on-demand voiding of the bladder and bowel, as future therapy for individuals with spinal cord injury, was examined using a rat model. The current study examined the feasibility of alternative routes of administration, which are more practical for clinical use than intravenous (IV) administration. Voiding and isovolumetric cystometry were recorded in anesthetized, acutely spinalized, female rats after IV, subcutaneous (SC), intramuscular (IM), intranasal (IN), or sublingual (SL) administration of [Lys;5;,MeLeu;9;,Nle;10;]-NKA;(4-10); (LMN-NKA). Administration of LMN-NKA (1-10μg/kg IV; 10-300μg/kg SC or IM; 15-1000μg/kg IN or 300-1500μg/kg SL) elicited rapid-onset, short-duration, dose-related increases in bladder pressure and voiding with the rank order for time of both onset and duration being IV < IN < SC = IM < SL. The incidence of voiding was dependent on the dose and route, with all routes resulting in a high voiding efficiency (~ 70%). Like LMN-NKA, neurokinin A (NKA 1-100μg/kg IV) and GR 64349 (0.1-30μg/kg IV or 1-300μg/kg SC) produced rapid-onset, short-duration increases in bladder pressure, as well as colorectal pressure.

Substance P (SP) nerve terminals innervate the intermediolateral cell column (IML) of the thoracic spinal cord, where SP coexists with serotonin (5-HT), neurokinin A (NKA) and thyrotropin-releasing hormone (TRH). Neither the depolarization-induced release of SP nor the presence of other neurochemicals in the regulation of SP release has been directly studied in this system. In the present study, basal and K(+)-stimulated release of SP from the microdissected intermediate area (including the IML, intercalated nucleus and central autonomic nucleus) of the rat thoracic spinal cord, and the regulation of SP release by presynaptic autoreceptors and by coexisting neurochemicals (5-HT, NKA and TRH) were studied using an in vitro superfusion system. Potassium evoked a concentration- and extracellular Ca(2+)-dependent release of SP. In rats pretreated with the serotoninergic neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT), both SP content and the absolute amount of SP released were decreased. However, the fraction of the remaining tissue content of SP released by K+ depolarization was not changed subsequent to 5,7-DHT treatment. Moreover, 5-HT, 5-HT1B agonists (CGS-12066B and RU 24969) and a 5-HT3 agonist (2-methyl-5-HT) did not alter the K(+)-evoked release of SP.

The tachykinin NK2 receptors present in membranes of rat small intestine were characterized by means of tachykinin receptor agonists and antagonists, by using the natural agonist, NKA, or the nonpeptide antagonist SR 48968, as radioligands. The affinity of the antagonists was independent of the radioligand used, whereas the NK2 receptor selective agonists showed a different binding profile according to the radioligand. In particular, when using [125I]NKA, NKA and NKA(4-10) bound to a high affinity site, whilst [beta-Ala8]NKA(4-10) and GR 64349 bound to a high and a low affinity site; the high affinity site was still detected in the presence of 100 microM GppNHp which produced a strong inhibition of the specific binding of [125I]NKA. On the other hand, when using [3H]SR 48968, NKA bound to a high affinity site, [beta-Ala8]NKA(4-10) bound to a low affinity site and NKA(4-10) and GR 64349 bound to a high and a low affinity site; in this case, the high affinity site was no longer detected in the presence of 1 microM GppNHp, which did not reduce the specific binding of [3H]SR 48968 to NK2 receptors. We interpreted these data as an indication that in the rat small intestine membranes [125I]NKA labels multiple conformations of G protein-coupled NK2 receptor which are distinguished by the use of selective receptor agonists, but not by peptide or nonpeptide antagonists.