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Electrochemical and electrophysiological analysis of the effects of SK&F 104078 on prejunctlonal α2-adrenoceptors
European Journal of Phurmacology, 203 ( 199 1) 159- I63 0 1991 Elsevier Science Publishers B.V. All rights reserved 0014-2999/91/$03.50 ADONIS 001429999100655H
159
EJP 520~5
Jian-Xin Bao, Fraqois
Gonon ’ and Lennart Stjkne
Department of Physiology I, Karolinska Instit;rtet, S-104 01 Stockholm, Sweden and ’ rNSERM UI 71 -CNl?S UA 119.5, Centre Hospitalier Lyon-Sud-Pac. 4H, 69310 Pierre B&ire, France Received 4 April 1991, revised MS received 4 July 1991, accepted 23 July 19~1
SK&F lC4078 is proposed to be a selective postjunctional a*-adrenoceptor antagonist. We examined its effects on the electrically evoked release of endogenous noradrenaline (NA) from the rat tail artery and of ATP from the mouse vas deferens, as determined electrochemically or by extracellular recording, respectively. The cu,-adrenoceptor antagonist yohimbine (0.1 and 1 PM) increased NA release at both 2 and 20 Hz; the effect of yohimbine was further enhanced by the NA uptake blocker cocaine. SK&F 104078 (0.01-l FM) did not change NA release at 2 or 20 Hz in the absence or presence of cocaine. NA release at 2 Hz was dose dependently depressed by the a,-adrenoceptor agonist xylazine, an effect reversed by ycyhimbine (1 PM) but unaffected by SK&F 104078 (0.1 and 1 PM). Similarly, the inhibitory effect of xylazine on ATP release at 0.1 Hz was not influenced by SK&F 104078 (1 PM) but partially reversed by yohimbine (1 PM). The results show (i) that prejunctional n,-adrenoceptors mediate autoinhibition of the release of endogenous NA and ATP from sympathetic nerves in the rat tail artery and in the mouse vas deferens, respectively, and (ii) that SK&F 104078 does not block these receptors in the concentrarion range 0.01-l CLM. ATP; Electrochemistry;
Noradrenaline
(endogcnous);
Extracellu!ar recording; cY,-Adrenoceptors (prcjunctional);
1. Introduction SK & F 104078 (6-chloro-9-[(3-methyl-2-butenyl)oxy!]-3-methyl-lH-2,3,4,5,tetrohydro-3-benzazepine) has been proposed to be a selective antagonist of postjlmctional cu,-adrenoceptors (Hieble et al., 1986). The results of some in vitro and in vivo studies support this proposal (Ruffolo et al., 1987; Hieble et al., 1988; Daly et al., 1988; Kelly et al., 1989), but the results of others show that this compound also blocks the prejunctional effects of certain a,-adrenoceptor agonists, at least in some tissues (Connaughton and Docherty, 1988, 1990; Akers et al., 1989). The results are thus conflicting. One reason may be inadequacy of the parameters used to assess the activity of SK&F 104078 to block prejunctional a,-adrenoceptors, namely its effects on the nerve stimulation-induced overflow of exogenous (“H-labelled) noradrenaline (NA) or on smooth muscle contraction.
Correspondence lo: J.-X. Baa, Dcp;lrtment of Physiology I, Karolinska lnstitulet, S-104 01 Stockholm, Swcdcn. Tel. 46.46X.728 7225, fex 46.468.311 101.
SK&F 104078
We have confirmed that SK&F 104078 blocks postjunctional cY,-adrenoceptors in rat tail artery (manuscript in preparation). We now examine its effects cn the nerve stimulation-induced release of erzdogenous NA (in rat tail artery) and ATP (in lnouse vas deferens).
2. Materials and methods 2.1. Preparations The proximal part of the central caudal artery from male Sprague-Dawley rats (200-300 g) and the vas deferens from mice (30-50 g) were dissected out and prepared as described previously (Stjlrne and Stjiirne, 1989; Mermet et al., 1990). Segments of the artery (about 2 cm) or the vas deferens were pinned to the Sylgard layer covering the bottom of a 3-ml Perspex chamber, and perfused at 37’ C at a rate of 2 or 1 ml #ith phosphate-buffered solution (PBS) or min-’ Tris-Tyrode solution, respectively. Both solutions were continuously bubble I Ah oxygen (pH 7.2-7.4). PBS had the following composition (mM): NaCl 136.9, KCI
2.7. CaCl, 1.3. MgCl, 0.5, Na, HPO, 4.06, KH ,PO, 0.73, D-glucose 5.6 and ascorbic acid 0.1. Prazosin (0.1 FM) was routinely added to PBS to inhibit neurogenic contractions. The Tris-Tyrode solution had the following composition (mhl): NaCl 136.9, KC1 2.7, CaCl, 1.3, MgClz 0.5. Tris 20, D-glucose 5.6. The proximal end of the artery or the prostatic end of the vas deferens was drawn into a suction electrode and sympathetic uostgcngiionic nerves were excited by nerve stimulation at 0.1-20 Hz (0.3 ms, 0.1-0.5 mA). 2.2. Analysis of the release of endogenous NA in rat tail artery The release of endogenous NA was studied by means of an electrochemical technique which has been described elsewhere (see Mermst et al., 1990). Briefly, differential normal pulse voltammetry (DNPV) and differential pulse amperometry (DPA) were used to measure the local concentration of endogenous NA at the surface of a pretreated carbon fibre electrode (length: 500 pm, tip diameter: 8 or 12 pm). By means of a micromanipulator, the carbon fibre electrode was lowered vertically towards the rat tail artery until its tip made a slight impression in the tissue surface. This electrode detects the presence of NA, which causes an oxidation current in DNPV with a maximum at + lcJ0 mV. The amplitude of this NA oxidation current was measured every 1 s by DPA. Brief electrical stimulation, consisting of 100 pulses either at 2 or at 20 Hz, induced an increase in this oxidation current which lasted as long as the stimulation did (see fig. 1). The maximal amplitude of this increase was used as a measure of the ‘ncreasr: in the local concentration of NA at the surface of the carbon fibre (A[NA]CF). It reflects the evoked release of NA minus its clearance (by reuptake and/or diffusion). The perivascular sympathetic nerves of the rat tail artery were stimulated with 100 pulses at 2 or 20 Hz, usually at 5-min intervals. Agents to be tested for effects on A[NAlCF were added to the reservoir proximal to the pump. Their effects were examined after they had been present in the bath for 15-20 min at full concentration. Results are expressed as % change in A[NA]CF in relation to the mean of two to three controls. 2.3. Analysis of the release of endogenous ATP in mouse L’asdeferens The release of cndogenous ATP was detected by means of an electrophysiological technique as described previously (see Stjtirne and Stjlrne, 1989). Briefly, a window was cut in the sheath of the mouse vas deferens and the tip (inner diameter. 100-230 pm) of a glass micro-electrode filled with Tris-Tyrode solu-
tion coniaining 1 PM tetrodotoxin, applied to the bare surface of the smooth muscle, was used to monitor the positive-going excitatory junction current (EJC), which reflects the release of ATP from sites outside the patch. Electrical activity was recorded with an a.~. amplifier, stored on magnetic tape and digitized on an IBM AT microcomputer. The sympathetic nerves of the vas deferens were continuously stimulated at 0.1 Hz with recording of EJC for 5 min (30 pulses) at lo- to 15-min intervals, during the control period and after each addition of a pharmacological agent. The EJC responses were stable for l- 1.5 h, under control conditions (not shown). Agents to be tested were added to the reservoir proximal to the pump. Their effects were expressed as % change in EJC peak amplitude in relation to the mean of the last 10 control EJCs in 5-min control recording periods. 2.4. Drugs The following drugs were used: cocaine hydrochloride (gift from Karolinska Apoteket, Sweden), guanethidine sulphate (Ciba-Geigy, Switzerland), prazosin hydrochloride (Pfizer, U.K.), SK&F 104078 maleate (gift from Smith Kline & French Laboratories, PA, U.S.A.) and tetrodotoxin, xylazine hydrochloride, yohimbine hydrochloride (Sigma). 2.5. Statistics All data in the text and figures are given as means f S.E.; n is the number of preparations. Means were compared by using Student’s paired or unpaired t-test or a one-way analysis of variance (ANOVA) followed by Fisher test, as appropriate. P Q 0.05 was regarded as significant.
3. Results 3.1. Effects on A[NA]CF 3.1.1. Control actit&y
The only peak detected by DNPV under control conditions, namely at - 80 mV, represents the ascorbic acid in the perfusion medium. Nerve stimulation with 100 pulses at 0.5-20 Hz caused the appearance of a second DNPV peak at + 100 mV, the oxidation potential of NA (not shown, but see Mermet et al., 19901, and a distinct increase in the oxidation current of NA measured by DPA (see figs. 1, 4). This stimulus-induced signal was abolished by tetrodotoxin (1 PM),, guanethidine
Ob-
8
7
6
5
-log [xylazine] @I)
Fig. 3. Xyla~ine-induced concent~ti~~n-d~~ndent depression of d[NAKF at 2 (open circle) or 20 Hz (closed circle) in rat tail artery. Abscissa: minus log molar concentration of xylazine; ordinate: A[NA]CF response normalised 10 the average of two to three controls at 2 or 20 Hz before addition of xylazinr. n = 4. * P < 0.05 and ** * P < 0.001 when compared to controls.
non-intermittent, essentially non-fluctuating and very stable (not shown, but see StjZrne and StjBrne, 1989).
Fig. 5. Summary of results from three experiments in rat tail ;irtery showing the depression of A[NA]CF by xylazine (Xyl, 1 FM) at 2 Hz and the effects of cumulative addition of SK&F 104078 (SKF, 0.1-I PM) and yohimbine (Yoh, 1 &MI. Ordinate: A[NA]CF response normalised tu the average of two to three controls (100%) before addition of drugs. ** P < 0.01 and *** P < 0.001 when compared to controls.
3.2.2. Effect of SK&F 204078 At 0.01-l PM SK&F 104078 did not significantly affect the EJCs (four experiments), but at 10 FM consistentiy depressed the EJCs (by 51.5 + I 1.O%; fig. 6). 3.2.3. SK&F f04078 and the xylazitze-induced inhibition Xylazine (0.001-l ,uM) depressed the EJCs at 0.1 Hz in a concentration-dependent manner (not shown). The inhibition by 0.1 PM xylazine (80.4 of:3.6%, n = 4) was not affected by SK&F 104078 (1 PM) but partially reversed by yohimbine (1 PM) (fig. 7).
4. Oiscussion
Fig. 6. Concentration-dependent effects of SK&F 104078 (SKF) on the EJCs (which reflect ATP release outside the recording electrode) in mouse vas deferens caused by stimulation at 0.1 Hz. Abscissa: minus log molar concentration of SKF; ordinate: mean of the last 10 EJC responses during S-min recording periods normalised to that recorded during a control period (100%) before addition of drugs; n = 4. * * P c: 0.01 when compared to controls.
The aim of the present study was to find out whether SK&F 104078 blocks prejunctional cw,-adrenoceptors in sympathetic nerve terminals. The strategy was to study as directly as possible its effects on the nerve stimulation-induced release of endogenous syinpathetic transmitters in hvo model tissues. The effects of SK&F 104078 were compared with those of yohimbine, a known blocker of prejunctional a,-adrenoceptors, in the absence as well as in the presence of cocaine, a
blocker of NA reuptake. Of the two signals recorded, the EJCs reflect on an impulse by impulse basis the release of one sympathetic transmitter (i.e. ATP) (Stjgrne and StjZrne, 19891. In contrast, d[NA]CF reflects for a different sympathetic transmitter (i.e. NA) release minus clearance (by reuptake and diffusion), not release per se. However, the change in this signal caused by drugs which do not affect NA clearance
Fig.4. Representativeoriginal recording in rat tail artery showing A[NA]CF responses induced by stimulation with 100 pulses at 2 Hz (closed bars), and the effect of xylazine (Xyl, I PM), SK&F 10407X (SKF. 0.1 and 1 PM) and yohimbinc (Yoh, 1 pM). Time intervals between stimultis trains: 5 min.
163
a
t
t
t
b
present study are in agreement with the results of some studies but at variance with those of other studies (see Introduction). The discrepancies may be due to differences in methodoio~. In conclusion, our results show that 0.01-l FM SK&F 104078 does not block prejunctiona1 cY,-adrenoceptors in the rat tail artery or in the mouse vas deferens.
Acknowledgements
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3
-
G’f1 !y,o,rLL*
Fig. 7. Effects of SK&F 104078 (SKF) and yohimbine (Yoh) on the depression by xylazine (Xyl) of the EJCs caused by continuous stimulation of mouse vas deferens at 0.1 Hz. (a) Original recording from one representative experiment, averages of the last 10 EJCs during S-min recording periods; fbf summary of rest&s from four experiments. Ordinate: mean of the last 10 EJC responses normalised to the corresponding control EJCs ( = 100%) before addition of drugs. * * * P < 0.001 when compared to controls and *+ P < 0.01 when compared to the preceding column.
should give a good measure of their effects on NA release. The effects on A[NA]CF and on the EJCs observed in the present study strongly indicate (i) that the selective cw,-adrenoceptor agonist xylazine concentration dependently inhibited the nerve stimulation-induced release of endogenous NA as well as ATP, an effect which was inversely related to the stimulation frequency, (ii) that the inhibitory effect of xylazine could be effectively antagonised or prevented by the cy2adrenoceptor antagonist yohimbine, (iii) that yohimbine itself increased the release of endogenous NA at 2 and even more strongly at 20 Hz, and (iv) that the release-enhancing effect of yohimbine was more marked in the presence of the NA uptake blocker cocaine, and, in the presence of cocaine, was more marked at 2 than at 20 Hz. This effect may imply that high frequency stimuIation depresses per se the neuronal reuptake of NA (Webb et al., 1980). Unlike yohimbine, 0.01-l PM SK&F 104078 did not increase the release of NA or ATP, at 2 and 20 or at 0.1 Hz, respectively, neither in the absence nor in the presence of cocaine, and, furthermore~ did not antagonise the inhibitor effects of xylazine on the release of NA or ATP. The reason why 10 PM SK&F 104078 increased A[NA]CF but decreased EJCs remains to be established. The effects of SK&F 104078 on the release of endogenous sympathetic transmitters observed in the
This work was supported by the Swedish Medical Research Council (project B91-14X-03027-22B), Karolinska lnstitutets Fonder and a European Training Programme Twinning Grant
References /. ’ “1s. i.. J. Coates, G.M. Drew and A.T. Sullivan, 1989, Antagonist profile of SK&F 104078 at pre- and post-junctional uz-adrenoceptors, Br. J. Pharmacol. 98 (Proc. Suppl.), 655~. Connaughton, S. and J.R. Docherty, 1988, Evidence that SK&F 104078 does not differentiate between pre- and postjunctional ~~-adrenoceptors, Naunyn-Schmiedeb. Arch. Pharmacol. 338, 379. Connaughton, S. and J.R. Docherty, 1990, No evidence for differences between pre- and postjunctional cul-adrenoceptors in the periphery, Br. J, Pharmacol. 99, 97. Daly, R.N., A.C. Sulpizio, 8. Levitt, R.M. DeMarinis, J.W. Regan, R.R. Ruffolo, Jr. and J.P. Hieble, 1988, Evidence for heterogeneity between pre- and postjunctional alpha-2 adrenoceptors using rl-substituted 3-benzazepines, J. Pharmacol, Exp. Ther. 247, 122. Hieble, J.P., AC. Sulpizio, A.J. Nichols, R.M. DeMarinis, F.R. Pfeiffer, P.G. Lavanchy and R.R. Ruffolo, Jr., 1986, Pharmacological differentiation of pre- and post-junctional alpha,-adre:roceptors, J Hypert. 4 (Suppl. 6), Sl89, Hieble. J.P., AC. Sulpizio, A.J. Nichols, R.N. Willette and R.R. Ruffolo, Jr., 1988, Pharmacologic characteri~?ion of SK&F 104078, a novel alpha-2 adrenoc-otor antagonist which discriminates between pre- and postjunctional alpha-2 adrenoceptors, J. Pharmacol. Exp. Ther. 247, 645. Kelly, J., S.P. Roberts and M.V. Sennitt, 1989, The effects of SK&F 104078 at pre- and postjunctional a,adrenoceptors in vitro, Br. J. Pharmacol. 98 (Proc. XuppU,727p. Mermet, C., F.G. Gonon and L. Stjarne, 1990, Online electrochemical monitoring of the local noradrenaline release evoked by electrical stimulation of the sympathetic nerves in isolated rat tail artery, Acta Physiol. Stand. 140, 323. Ruffolo, R.R., Jr., A.C. Sulpizio. A.J. Nichols, R.M. DeMarinis and J.P. Hieble, 1987, Pharmacologic differentiation between preand postjunctional ~z-adrenoceptors by SK&F 104078, NaunynSchmiedeb. Arch. Pharmacol. 336, 415. Stjiirne, L. and E. Stjiirne, 1989, Basic features of an extracellufar recording method to study secretion of a sympathetic cotransmitter, presumably ATP, Acta Physiol. Stand. 135, 217. Webb, R.C., P.M. Vanhoutte and D.F. Bohr, 1980, Inactivation of released norcpinephrine in rat tail artery by neuronal uptake, J. Cardiova~. Pharmacol. 2, 121.
159
EJP 520~5
Jian-Xin Bao, Fraqois
Gonon ’ and Lennart Stjkne
Department of Physiology I, Karolinska Instit;rtet, S-104 01 Stockholm, Sweden and ’ rNSERM UI 71 -CNl?S UA 119.5, Centre Hospitalier Lyon-Sud-Pac. 4H, 69310 Pierre B&ire, France Received 4 April 1991, revised MS received 4 July 1991, accepted 23 July 19~1
SK&F lC4078 is proposed to be a selective postjunctional a*-adrenoceptor antagonist. We examined its effects on the electrically evoked release of endogenous noradrenaline (NA) from the rat tail artery and of ATP from the mouse vas deferens, as determined electrochemically or by extracellular recording, respectively. The cu,-adrenoceptor antagonist yohimbine (0.1 and 1 PM) increased NA release at both 2 and 20 Hz; the effect of yohimbine was further enhanced by the NA uptake blocker cocaine. SK&F 104078 (0.01-l FM) did not change NA release at 2 or 20 Hz in the absence or presence of cocaine. NA release at 2 Hz was dose dependently depressed by the a,-adrenoceptor agonist xylazine, an effect reversed by ycyhimbine (1 PM) but unaffected by SK&F 104078 (0.1 and 1 PM). Similarly, the inhibitory effect of xylazine on ATP release at 0.1 Hz was not influenced by SK&F 104078 (1 PM) but partially reversed by yohimbine (1 PM). The results show (i) that prejunctional n,-adrenoceptors mediate autoinhibition of the release of endogenous NA and ATP from sympathetic nerves in the rat tail artery and in the mouse vas deferens, respectively, and (ii) that SK&F 104078 does not block these receptors in the concentrarion range 0.01-l CLM. ATP; Electrochemistry;
Noradrenaline
(endogcnous);
Extracellu!ar recording; cY,-Adrenoceptors (prcjunctional);
1. Introduction SK & F 104078 (6-chloro-9-[(3-methyl-2-butenyl)oxy!]-3-methyl-lH-2,3,4,5,tetrohydro-3-benzazepine) has been proposed to be a selective antagonist of postjlmctional cu,-adrenoceptors (Hieble et al., 1986). The results of some in vitro and in vivo studies support this proposal (Ruffolo et al., 1987; Hieble et al., 1988; Daly et al., 1988; Kelly et al., 1989), but the results of others show that this compound also blocks the prejunctional effects of certain a,-adrenoceptor agonists, at least in some tissues (Connaughton and Docherty, 1988, 1990; Akers et al., 1989). The results are thus conflicting. One reason may be inadequacy of the parameters used to assess the activity of SK&F 104078 to block prejunctional a,-adrenoceptors, namely its effects on the nerve stimulation-induced overflow of exogenous (“H-labelled) noradrenaline (NA) or on smooth muscle contraction.
Correspondence lo: J.-X. Baa, Dcp;lrtment of Physiology I, Karolinska lnstitulet, S-104 01 Stockholm, Swcdcn. Tel. 46.46X.728 7225, fex 46.468.311 101.
SK&F 104078
We have confirmed that SK&F 104078 blocks postjunctional cY,-adrenoceptors in rat tail artery (manuscript in preparation). We now examine its effects cn the nerve stimulation-induced release of erzdogenous NA (in rat tail artery) and ATP (in lnouse vas deferens).
2. Materials and methods 2.1. Preparations The proximal part of the central caudal artery from male Sprague-Dawley rats (200-300 g) and the vas deferens from mice (30-50 g) were dissected out and prepared as described previously (Stjlrne and Stjiirne, 1989; Mermet et al., 1990). Segments of the artery (about 2 cm) or the vas deferens were pinned to the Sylgard layer covering the bottom of a 3-ml Perspex chamber, and perfused at 37’ C at a rate of 2 or 1 ml #ith phosphate-buffered solution (PBS) or min-’ Tris-Tyrode solution, respectively. Both solutions were continuously bubble I Ah oxygen (pH 7.2-7.4). PBS had the following composition (mM): NaCl 136.9, KCI
2.7. CaCl, 1.3. MgCl, 0.5, Na, HPO, 4.06, KH ,PO, 0.73, D-glucose 5.6 and ascorbic acid 0.1. Prazosin (0.1 FM) was routinely added to PBS to inhibit neurogenic contractions. The Tris-Tyrode solution had the following composition (mhl): NaCl 136.9, KC1 2.7, CaCl, 1.3, MgClz 0.5. Tris 20, D-glucose 5.6. The proximal end of the artery or the prostatic end of the vas deferens was drawn into a suction electrode and sympathetic uostgcngiionic nerves were excited by nerve stimulation at 0.1-20 Hz (0.3 ms, 0.1-0.5 mA). 2.2. Analysis of the release of endogenous NA in rat tail artery The release of endogenous NA was studied by means of an electrochemical technique which has been described elsewhere (see Mermst et al., 1990). Briefly, differential normal pulse voltammetry (DNPV) and differential pulse amperometry (DPA) were used to measure the local concentration of endogenous NA at the surface of a pretreated carbon fibre electrode (length: 500 pm, tip diameter: 8 or 12 pm). By means of a micromanipulator, the carbon fibre electrode was lowered vertically towards the rat tail artery until its tip made a slight impression in the tissue surface. This electrode detects the presence of NA, which causes an oxidation current in DNPV with a maximum at + lcJ0 mV. The amplitude of this NA oxidation current was measured every 1 s by DPA. Brief electrical stimulation, consisting of 100 pulses either at 2 or at 20 Hz, induced an increase in this oxidation current which lasted as long as the stimulation did (see fig. 1). The maximal amplitude of this increase was used as a measure of the ‘ncreasr: in the local concentration of NA at the surface of the carbon fibre (A[NA]CF). It reflects the evoked release of NA minus its clearance (by reuptake and/or diffusion). The perivascular sympathetic nerves of the rat tail artery were stimulated with 100 pulses at 2 or 20 Hz, usually at 5-min intervals. Agents to be tested for effects on A[NAlCF were added to the reservoir proximal to the pump. Their effects were examined after they had been present in the bath for 15-20 min at full concentration. Results are expressed as % change in A[NA]CF in relation to the mean of two to three controls. 2.3. Analysis of the release of endogenous ATP in mouse L’asdeferens The release of cndogenous ATP was detected by means of an electrophysiological technique as described previously (see Stjtirne and Stjlrne, 1989). Briefly, a window was cut in the sheath of the mouse vas deferens and the tip (inner diameter. 100-230 pm) of a glass micro-electrode filled with Tris-Tyrode solu-
tion coniaining 1 PM tetrodotoxin, applied to the bare surface of the smooth muscle, was used to monitor the positive-going excitatory junction current (EJC), which reflects the release of ATP from sites outside the patch. Electrical activity was recorded with an a.~. amplifier, stored on magnetic tape and digitized on an IBM AT microcomputer. The sympathetic nerves of the vas deferens were continuously stimulated at 0.1 Hz with recording of EJC for 5 min (30 pulses) at lo- to 15-min intervals, during the control period and after each addition of a pharmacological agent. The EJC responses were stable for l- 1.5 h, under control conditions (not shown). Agents to be tested were added to the reservoir proximal to the pump. Their effects were expressed as % change in EJC peak amplitude in relation to the mean of the last 10 control EJCs in 5-min control recording periods. 2.4. Drugs The following drugs were used: cocaine hydrochloride (gift from Karolinska Apoteket, Sweden), guanethidine sulphate (Ciba-Geigy, Switzerland), prazosin hydrochloride (Pfizer, U.K.), SK&F 104078 maleate (gift from Smith Kline & French Laboratories, PA, U.S.A.) and tetrodotoxin, xylazine hydrochloride, yohimbine hydrochloride (Sigma). 2.5. Statistics All data in the text and figures are given as means f S.E.; n is the number of preparations. Means were compared by using Student’s paired or unpaired t-test or a one-way analysis of variance (ANOVA) followed by Fisher test, as appropriate. P Q 0.05 was regarded as significant.
3. Results 3.1. Effects on A[NA]CF 3.1.1. Control actit&y
The only peak detected by DNPV under control conditions, namely at - 80 mV, represents the ascorbic acid in the perfusion medium. Nerve stimulation with 100 pulses at 0.5-20 Hz caused the appearance of a second DNPV peak at + 100 mV, the oxidation potential of NA (not shown, but see Mermet et al., 19901, and a distinct increase in the oxidation current of NA measured by DPA (see figs. 1, 4). This stimulus-induced signal was abolished by tetrodotoxin (1 PM),, guanethidine
Ob-
8
7
6
5
-log [xylazine] @I)
Fig. 3. Xyla~ine-induced concent~ti~~n-d~~ndent depression of d[NAKF at 2 (open circle) or 20 Hz (closed circle) in rat tail artery. Abscissa: minus log molar concentration of xylazine; ordinate: A[NA]CF response normalised 10 the average of two to three controls at 2 or 20 Hz before addition of xylazinr. n = 4. * P < 0.05 and ** * P < 0.001 when compared to controls.
non-intermittent, essentially non-fluctuating and very stable (not shown, but see StjZrne and StjBrne, 1989).
Fig. 5. Summary of results from three experiments in rat tail ;irtery showing the depression of A[NA]CF by xylazine (Xyl, 1 FM) at 2 Hz and the effects of cumulative addition of SK&F 104078 (SKF, 0.1-I PM) and yohimbine (Yoh, 1 &MI. Ordinate: A[NA]CF response normalised tu the average of two to three controls (100%) before addition of drugs. ** P < 0.01 and *** P < 0.001 when compared to controls.
3.2.2. Effect of SK&F 204078 At 0.01-l PM SK&F 104078 did not significantly affect the EJCs (four experiments), but at 10 FM consistentiy depressed the EJCs (by 51.5 + I 1.O%; fig. 6). 3.2.3. SK&F f04078 and the xylazitze-induced inhibition Xylazine (0.001-l ,uM) depressed the EJCs at 0.1 Hz in a concentration-dependent manner (not shown). The inhibition by 0.1 PM xylazine (80.4 of:3.6%, n = 4) was not affected by SK&F 104078 (1 PM) but partially reversed by yohimbine (1 PM) (fig. 7).
4. Oiscussion
Fig. 6. Concentration-dependent effects of SK&F 104078 (SKF) on the EJCs (which reflect ATP release outside the recording electrode) in mouse vas deferens caused by stimulation at 0.1 Hz. Abscissa: minus log molar concentration of SKF; ordinate: mean of the last 10 EJC responses during S-min recording periods normalised to that recorded during a control period (100%) before addition of drugs; n = 4. * * P c: 0.01 when compared to controls.
The aim of the present study was to find out whether SK&F 104078 blocks prejunctional cw,-adrenoceptors in sympathetic nerve terminals. The strategy was to study as directly as possible its effects on the nerve stimulation-induced release of endogenous syinpathetic transmitters in hvo model tissues. The effects of SK&F 104078 were compared with those of yohimbine, a known blocker of prejunctional a,-adrenoceptors, in the absence as well as in the presence of cocaine, a
blocker of NA reuptake. Of the two signals recorded, the EJCs reflect on an impulse by impulse basis the release of one sympathetic transmitter (i.e. ATP) (Stjgrne and StjZrne, 19891. In contrast, d[NA]CF reflects for a different sympathetic transmitter (i.e. NA) release minus clearance (by reuptake and diffusion), not release per se. However, the change in this signal caused by drugs which do not affect NA clearance
Fig.4. Representativeoriginal recording in rat tail artery showing A[NA]CF responses induced by stimulation with 100 pulses at 2 Hz (closed bars), and the effect of xylazine (Xyl, I PM), SK&F 10407X (SKF. 0.1 and 1 PM) and yohimbinc (Yoh, 1 pM). Time intervals between stimultis trains: 5 min.
163
a
t
t
t
b
present study are in agreement with the results of some studies but at variance with those of other studies (see Introduction). The discrepancies may be due to differences in methodoio~. In conclusion, our results show that 0.01-l FM SK&F 104078 does not block prejunctiona1 cY,-adrenoceptors in the rat tail artery or in the mouse vas deferens.
Acknowledgements
e
L 0
3
-
G’f1 !y,o,rLL*
Fig. 7. Effects of SK&F 104078 (SKF) and yohimbine (Yoh) on the depression by xylazine (Xyl) of the EJCs caused by continuous stimulation of mouse vas deferens at 0.1 Hz. (a) Original recording from one representative experiment, averages of the last 10 EJCs during S-min recording periods; fbf summary of rest&s from four experiments. Ordinate: mean of the last 10 EJC responses normalised to the corresponding control EJCs ( = 100%) before addition of drugs. * * * P < 0.001 when compared to controls and *+ P < 0.01 when compared to the preceding column.
should give a good measure of their effects on NA release. The effects on A[NA]CF and on the EJCs observed in the present study strongly indicate (i) that the selective cw,-adrenoceptor agonist xylazine concentration dependently inhibited the nerve stimulation-induced release of endogenous NA as well as ATP, an effect which was inversely related to the stimulation frequency, (ii) that the inhibitory effect of xylazine could be effectively antagonised or prevented by the cy2adrenoceptor antagonist yohimbine, (iii) that yohimbine itself increased the release of endogenous NA at 2 and even more strongly at 20 Hz, and (iv) that the release-enhancing effect of yohimbine was more marked in the presence of the NA uptake blocker cocaine, and, in the presence of cocaine, was more marked at 2 than at 20 Hz. This effect may imply that high frequency stimuIation depresses per se the neuronal reuptake of NA (Webb et al., 1980). Unlike yohimbine, 0.01-l PM SK&F 104078 did not increase the release of NA or ATP, at 2 and 20 or at 0.1 Hz, respectively, neither in the absence nor in the presence of cocaine, and, furthermore~ did not antagonise the inhibitor effects of xylazine on the release of NA or ATP. The reason why 10 PM SK&F 104078 increased A[NA]CF but decreased EJCs remains to be established. The effects of SK&F 104078 on the release of endogenous sympathetic transmitters observed in the
This work was supported by the Swedish Medical Research Council (project B91-14X-03027-22B), Karolinska lnstitutets Fonder and a European Training Programme Twinning Grant
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