2 NEUROPEPTIDES

newborn but not to adult rats produced analgesia in the Effects of CGRF’ (32-37), Somatostatin-28 and tail immersion test and completely reversed the hyperal- gesia induced by sympathectomy, while did not affect SP-

~G*-Vasopressin on the Monkey Iris

LI content of tail skin. Sphincter; Interactions with CCK_A Receptors

As capsaicin desensitization of adult or newborn rats B. Almegird and A. Bill

has been shown to affect a different percentage of primary Department of Physiology and Medical Biophysics,

afferents (3), these findings may indicate that capsaicin Uppsala University Sweden

2

would usefully discriminate among subsets of primary afferents involved in the processing of thermal nocicep-

Cholecystokinin (CCK) - A receptors have been demon-

tive input. strated to be present in the monkey iris sphincter muscle; CCK is a potent non-muscarinic miotic and its effect is

1. Tyers, M. B. (1980). Br. J. Pharmac. 69,503. 2. Gamse, R. (1982) Naunyn-Schmiedeberg’s Arch. Pharmac.

320,205. 3. Maggi, C. A. (1990). Behav. Brain Sci. 13,312.

Extraction and Quantitative Analysis of Neuropeptides in Bone and Joint M. Ahmed, G. Srinivasan, A. Bjurholm, A. Kreicbergs and E. Theodorsson Departments of Orthopedics and Clinical Chemistry, Karolinska Hospital, 104 0 1 Stockholm, Sweden

Immunoreactive substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) were analysed by radioim- munoassay (RIA) in extracts of 50 knee joints and 30 femur bones in rat.

Knee joints were dissected, and extracted in five dif- ferent boiling (10 mm) media: I) 1M acetic acid, 2) 2M acetic acid, 3) neutral water and 4) 95% alcohol in 2M acetic acid, all containing 4% EDTA and 5) 2M acetic acid without EDTA. The samples were subsequently soni- cated, centrifuged and the supematants lyophilized. The lyophilisates were dissolved in RIA buffer before RIA. The highest yield of SP (1 .O + 0.4 pmol/g) and CGRP (1 .O + 0.3 pmolig) was obtained with 2M acetic acid without EDTA, whereas 2M acetic acid in 4% EDTA gave max- imum of NPY (3.8 + 0.2 pmol/g) and VIP (0.6 + 0.1 pmol/g). The optimum common extraction method for all neuropeptides proved to be 2M acetic acid in 4% EDTA, which consequently was used in the ensuing experiments.

The diaphyses of 30 femora were dissected and divided into three sample groups consisting of 1) cortical bone, 2) periosteum and 3) bone marrow. After homogenisation, extraction was performed with 2M acetic acid in 4% EDTA. For SP the concentration in cortical bone was 0.1 pmoVg compared to 0.8 pmol/g in periosteum and 2.3 pmol/g in marrow. The corresponding concentrations for CGRP were 0.2, 1.0, 1.0, for NPY 1.0, 2.4,4.2, and for VIP 0.1,0.9,0.3. Hence, cortical bone consistently exhib- ited the lowest levels. In marrow the level of SP and NPY was clearly higher than in periosteum.

We previously identified the described neuropeptides in bone and joints by immunohistochemical technique. The procedure now presented offers a means of quanti- tating these neuropeptides in normal as well as pathologic conditions.

antagonized by CCK-A receptor antagonists such as lor- glumide (1). It has been reported that C-terminal peptides of calcitonin gene-related peptide (CGRP) (2) as well as the NH2-terminally extended form of somatostatin, somatostatin-28 (S,,) (3) act as agonists at CCK-receptors in the pancreas.

In the present study we have examined if CGRP(32-37) and SZ8 exert any contractile effects via the CCK-recep- tors in the monkey iris. The contractile effect of arg*-vaso- pressin was also evaluated, since we have found that also this peptide contracts isolatedmonkey iris sphincters. The peptides were administered intracamerally to anesthe- tized cynomolgus monkeys pretreated with atropine. CGRP(32-37) induced a miosis with 50% of the maximal response at a concentration of37 pmole. Pretreatment with loxiglumide caused a rightward shift of the dose response relationship, indicating interaction with CCK-A recep- tors. No contractile effect was elicited by 70 pmole - 7 nmole Szs. A small pupillary contraction was observed after administration of 350 pmole arg*-vasopressin. A higher dose (3.5 nmole) produced no further constriction. The contraction was not abolished by loxiglumide pre- treatment.

1. Bill et al. (1990). Acta Physiol. Stand. 138: 479-485. 2. Maton et al. (1990). Peptides 11:1163-l 167. 3. Tahiri-Jouti et al. (1991). Neuropeptides 19: 65-71.

Effect of Neurokinin Antagonists on Noxious Stimulation-Induced Reflexes in the Rabbit R. Atnann and M. Eder Department of Experimental and Clinical Pharmacology, Graz University, Univ.-Platz 4, A-8010 Graz, Austria

In order to evaluate a possible analgesic action of NK antagonists, the isolated perfused rabbit ear with intact neuronal connection (1) was used. In rabbits, systemic administration of the NK- 1 selective neurokinin antago- nist CP-96, 345 (2) (60-600 nmovkg iv.) did not significantly attenuate the depressor reflex following injection of capsaicin into the central artery of the vascu- larly isolated ear. At 600 nmoVkg i.v., CP-96,345 on its own produced a hypotensive response, thus preventing the evaluation of a possible depressant effect on the nocicep- tive reflex.