A RAPID AND SENSITIVE METHOD TO ASSAY ENDOGENOUS ACETYLCHOLINE OVERFLOW FROM MYENTERIC NEURONS BY MEANS OF HPLC AND ELECTROCHEMICAL DETECTION C. Giaroni, F. Madno, M. "Cosentino, O. LeoN, L. Somaini, S. Lecchini and G.M. Frigo Department of Internal Medicine and Therapeutics , II Faculty of Medicine University of Pavia, Via O.Rossi n.9, 1-21100 Varese, Italy.
A N A B A S I N E IS A N I C O T I N I C ANALGESIC C.Ghelardini, *P.C. Lazareno, *D.Wilkison, *B.Collins Dept. of Pharmacol., Florence, Italy and *hast. of Neuropharmac. Med. Res. Center, Bradford, U.K. Anabasine [2-(3-pyridyl)piperidinel is a Solanaceae alkaloid structurally similar to epibatidine. Epibatidine is very potent non-sedating and non-opioid analgesic endowed with nicotinic agonist properties (Quian et al. 1993). In our studies anabasine produced a decrease in responsiveness to radiant heat in the tail-flick test. The onset of antinociceptive action was rapid with a maximum antinociception at 10 min and duration was 30 min for anabasine. Ten rain after anabasine administration in mice the antinociceptive ED50 was determined to be 64.7 I.tg/kg LP. Rats were more sensitive than mice to anabasine in tail-flick test. In rats the ED50 was 22 ~tg/kg i.p. The centrally active nicotinic receptor blocker mecamylamine (0.5 mg/kg i.p.) shifted the ED50 value of anabasine induced antinociception b y 4.4fold in rats. The quaternary nicotinic receptor blocker hexametonium (3 mg/kg i.p.) which hardly passes into the central nervous system did not show antagonism to anabasine. Naloxone (2 mg/kg i.p.) and atropine (5 mg/kg i.p.) were inactive in antagonizing anabasine antinociception. Similar to the results obtained in mice the same antagonists did not block the anabasineinduced antinociception in rats. Anabasine doses which are able to increase pain threshold did not show any behavioural impairment in mice and rats. Nicotine receptor has been found to be involved in the mediation of several diseases such as Parkinson disease, Alzheimer disease and tobacco dependence. Anabasine as a potent central nicotinic agonist may be a useful tool to investigate the role of nicotinic receptor in the above mentioned disease.
The study of acetylcholine (ACh) overflow from the myenteric neurons provides useful information on the physiopharmacology of the autonomic nervous system. However, while several different methods have been used to identify and quantitate ACh in biological specimens, including bioassay, gas chromatography-mass spectrometry and radiochemical assay, their use is often limited by many drawbacks. We have developed a simple and sensitive method to assay ACh released from myenteric neurons by means of HPLC technique and electrochemical detection. ACh separation w a s accomplished by means of a cation exchanger column. A post column immobilized enzyme reactor loaded with chemically bonded acetylcholinesterase and choline oxidase converted ACh to betaine and H202 which was in turn electrochemically detected by means of an amperometric cell. Sample analysis required approximately 7 minutes. The detection limit was 200 fmole. The relative standard deviation for 10 injections of ACh was 1.5% for 2 pmole injected. The detector response was proportional to the amount of ACh for at least three orders of magnitude (500 fmole-500 pmole). The method was tested by determining endogenous ACh released from guinea pig isolated colonic segments. Specimens were suspended isotonically (weight: 1 g) in organ baths containing Tyrode solution with added physostigmine sulphate (15 gM), maintained at 35,5°C, and continuosly bubbled with 95%O2-5%CO2.. Samples of the medium were collected at fixed intervals and directly analyzed after filtration through 0.45 Fm filters. Spontaneus endogenous ACh was 18,7+I,8 (mean:l:SEM, n=13). Electrically evoked overflow (bipolar square w a v e pulses, 1Hz, 1ms, 10 min duration, silver wire electrodes) was 85.45+9.8 (n=19). In conclusion, the method has proved rapid, sensitive and reproducible, thus representing a useful tool for the measurement of endogenous ACh released from myenteric neurons.
Tobacco Res. Council provided financial support fi~r these studies.
MUSCARINIC RECEPTOR SUBTYPES IN HUMAN
NICOTINIC ACETYLCHOLINE RECEPTORS IN HUMAN NEUROBLASTOMA CELLS C. Gotti, B. Balestra, C. Verderio, M. Oortgiesen*,L; Briscini, M. Moretti, and F. ClementL CNR Center of Cytopharmacology, Department of Medical Pharmacology, University of Milan, Milan, Italy, and *Research Institute of Toxicology, University of Utrecht, Utrecht, The Netherlands.
LYMPHOCYTES : FROM PERIPHERIAL BLOOD AND
LEUKAEMIC CELL LINES H . Laskowska Bo~ek , T. Burakowski, U. Bany, J. Ry~.ewski. Institute of Rheumathology, Dept. ofPathophysiology, Spartafiska str. 1 02-637 Warsaw, Poland.
Neuronal nicotinic acetylcholine receptors (nAChRs) belong to the ligand-gated ion channel family and are found throughout the central and peripheral nervous system. Recently it has been demonstrated a large variety of nAChRs receptors in the mammalian nervous system. In order to study the structure and function of the native nAChRs we used the human neuroblastoma cell lines MR32 and SKNBE. These cells are derived from neural crest and display several features of ortosymphathetic neurons, such as neuron specific enzymes, receptors and ion channels. IMR32 and SKNBE cells, although with different ratio, express two classes of surface nicotinic receptors: those labeled with high affinity by 1251Neuronal toxin, and those labeled by 1251_c~Bungarotoxin. Whole-cell patch clamp recordings indicate that both classes of receptor are able to elicit inward currants, totally blocked by d.Tubocurarine, but only partially blocked by ~zBungarotoxin. In IMR32 cells, nicotine induces an increase in the intracellular level of free Ca2+. This increase, which is also completely blocked by d-Tubocurarine and only partially by ~Bungarotoxin and Cd 2+, is due to extracellular calcium influx through both the nicotinic receptors and the voltageactivated Ca2+ channels. By using subunit-spacific polyclonal antibodies, we have demonstrated that the aBungarotoxin receptors contain the ¢7 subunit, but none of the other subunits whose transcripts are present in IMR32 cells. The pharmacological profile of these human c¢7-containing ~Bungarotoxin receptors is similar to that observed in the native chick ~7 receptor, but there are also some species-specific differences.
Muscafinic receptor activation involves several effector mechanisms: activation of the phosphoinositide turnover, inhibition of adenylate cyclase, activation ofguanylate cyclaze, and Ca~+ channels. Binding studies with new specyfic muscafinic antagonists provided evidence for the existence of a heterogenous muscarinic receptor system .In this study we have compared the binding characteristics of 3H QNB to human lymphocytes from peripheral blood and lymphocytes from the leukaemic cell lines : Jurkat and Rail. The results indicate the presence of muscarinic cholinergic receptors : 1) with a predominance of M2 subtypes on human peripheral blood lymphocytes 2) with a predominance of M3 subtypes on Jurkat cells 3) M1 ,M2 and M3 subtypes on Raji cells.These results are important for understanding the various biochemical responses mediated by the putative subtypes of muscarinic receptors.