a-Adrenoceptor-Mediated Prejunctional Effects of Chloroethylclonidine in the Canine Saphenous Vein

The present study was undertaken to look for the effect of chloroethylclonidine (CEC) on prejunctional alpha-2 autoreceptors of the canine saphenous vein. The effect was tested on tritium overflow evoked by electrical stimulation from tissues preloaded with 0.2 mM H-norepinephrine. Yohimbine (3–300 nM) and CEC (1–125 mM) increased and UK-14,304 reduced the overflow of tritium evoked by 300 pulses (1 Hz). The maximal increase of tritium overflow caused by yohimbine was much higher than that caused by CEC: 3.82 and 1.74 times, respectively. CEC (5 mM) abolished both the inhibition caused by UK-14,304 and the enhancement of tritium overflow caused by yohimbine. However, when CEC was added after yohimbine, it reduced the electrically evoked overflow of tritium, the maximal effect being a reduction of tritium overflow by 35%. Prazosin (1–100 nM) did not change either the inhibitory effect of UK-14,304 or the facilitatory effect of CEC. These results suggest that CEC acts on two different subtypes of prejunctional alpha-2 autoreceptors; on one of them it acts as an antagonist and increases the electrically evoked overflow of tritium (and inhibits both the effect of UK-14,304 and yohimbine); on the other it acts as an agonist and reduces the electrically evoked overflow of tritium. Alternatively, one can admit that CEC is able to inhibit alpha-2 autoreceptors, which causes an increase of the transmitter release, and to activate a nonadrenergic inhibitory receptor thus causing a reduction of the transmitter release. Chloroethylclonidine was described by Leclerc et al. (1980) as the first example of an alpha adrenoceptor agonist with an irreversible effect. Later on, CEC was found to bind irreversibly to alpha-1B adrenoceptors and is now used both in binding and functional studies as an antagonist in the current definition of alpha-1 subtypes (Han et al., 1987; Minneman et al., 1988). More recently CEC was found to be an irreversible agonist at postjunctional alpha-2 adrenoceptors of the canine saphenous vein; this effect involved the activation of receptors that differ from those activated by UK14,304 (Nunes and Guimarães, 1992, 1993; Low et al., 1994). Apart from the postjunctional effects of CEC, there is evidence for its action on prejunctional adrenoceptors. In the rat vas deferens, CEC reduces the release of norepinephrine and of a purinergic co-transmitter by irreversible stimulation of prejunctional alpha-2 adrenoceptors, and this effect is prevented by pretreatment with rauwolscine (Bültmann and Starke, 1993). Also in vivo (in the pithed rat), CEC (25 mg/kg) significantly reduced the pressor response to electrical stimulation of spinal sympathetic nerves and this inhibitory effect was antagonized by idazoxan, which indicates that CEC activates prejunctional alpha-2 autoreceptors (Vargas et al., 1994) The present investigation was undertaken to study the effect of CEC on prejunctional alpha adrenoceptors of the canine saphenous vein. A preliminary report of these results was presented at the 8th Meeting on Adrenergic Mechanisms (Guimarães and Paiva, 1994) Materials and Methods Tissue preparations. In the municipal dog pound, mongrel dogs, 10 to 16 kg in weight, of either sex, were anesthetized with pentobarbitone sodium (30 mg/kg). Immediately after removal, the saphenous veins were placed in small vials containing aerated (95% O2 and 5% CO2) and cold Krebs-Henseleit solution of the following composition (mM): NaCl, 118.6; KCl, 4.70; CaCl2, 2.52; KH2PO4, 1.18; MgSO4, 1.23; NaHCO3, 2.50; glucose, 10; ascorbic acid, 0.57; disodium EDTA, 0.027 (Guimarães et al., 1987). The animals were sacrificed by an overdose of pentobarbitone sodium (100 mg/kg). The veins were then transported to the laboratory where they were helically cut into small strips (of about 2.5 3 25 mm) which were preincubated for 30 min in medium containing 1 mM pargyline (to inhibit monoamine oxidase), 40 mM hydrocortisone and 50 mM Received for publication December 31, 1996. 1 This work was supported by Junta Nacional de Investigação Cientı́fica e Tecnológica (JNICT)-Project number PECS/P/SAU/80/95, by PRAXIS/2/2.1/ SAU/1293/95 and by Biomed 2 (EureCa project). 2 Present address: Departamento de Farmacologia. I.B. Botucatu-UNESP, 18600 Botucatu, SP, Brasil. ABBREVIATIONS: CEC, chloroethylclonidine; U-0521, 3,4-dihydroxy-2-methylpropiophenone. 0022-3565/97/2823-1326$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 282, No. 3 Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 282:1326–1330, 1997 1326 at A PE T Jornals on O cber 5, 2017 jpet.asjournals.org D ow nladed from U-0521 (3,4-dihydroxy-2-methylpropiophenone) (to inhibit extraneuronal removal of norepinephrine) (Guimarães et al., 1978). Hydrocortisone and U-0521 were also kept in the medium for the remainder of the experiment. After preincubation, the vessel segments were exposed for 60 min to H-norepinephrine (0.2 mM). Perifusion experiments. The vessel segments were mounted in 1-ml glass chambers between two platinum electrodes and perifused with amine-free medium (aerated and at 37°C) moving from bottom to top at a flow rate of 0.8 ml/min. From t 5 100 min (t 5 0 being the onset of the perifusion) the perifusion fluid was collected continuously as 5-min samples. Transmural electrical stimulation (1 Hz, 2 ms, 100 V, for 5 min; Stimulator II X, Hugo Sachs Elektronik, March-Hugstetten, Germany) was applied at min 120 (S1), 170 (S2), 220 (S3), 270 (S4) and 320 (S5).In addition to hydrocortisone and U-0521, cocaine (12 mM) was also present in the perifusion fluid from