Interactions between imidazoline compounds and sulphonylureas in the regulation of insulin secretion

Transcription

1 British Journl of Phrmcology (1997) 121, 799 ± Stockton Press All rights reserved 0007 ± 1188/97 $10 Interctions etween imidzoline compounds nd sulphonylures in the regultion of insulin secretion Mirn Mourtd, Colin A. Brown, Stephen A. Smith, Vlerie Piercy, Susn L.F. Chn & 1 Noel G. Morgn Cellulr Phrmcology Group, Deprtment of Biologicl Sciences, Keele University, St s ST5 5BG nd Deprtment of Vsculr Biology, SmithKline Beechm Phrmceuticls, Welwyn, Herts AL6 9AR 1 Imidzoline 2 -ntgonist drugs such s efroxn hve een shown to increse the insulin secretory response to sulphonylures from rt pncretic B-cells. We hve investigted whether this re ects inding to n islet imidzoline receptor or whether 2 -drenoceptor ntgonism is involved. 2 Administrtion of -efroxn or glienclmide to Wistr rts ws ssocited with trnsient increse in plsm insulin. When oth drugs were dministered together, the resultnt increse in insulin levels ws much greter thn tht otined with either drug lone. 3 Use of the resolved enntiomers of efroxn reveled tht the ility of the compound to enhnce the insulin secretory response to glienclmide resided only in the 2 -selective--enntiomer; the imidzoline receptor-selective-(7)-enntiomer ws ine ective. 4 In vitro, -efroxn incresed the insulin secretory response to glienclmide in rt freshly isolted nd cultured islets of Lngerhns, wheres (7)-efroxn ws inctive. By contrst, -efroxn did not potentite glucose-induced insulin secretion ut (7)-efroxn induced mrked increse in insulin secretion from islets incuted in the presence of 6 mm glucose. 5 Incution of rt islets under conditions designed to minimize the extent of 2 -drenoceptor signlling (y receptor lockde with phenoxyenzmine; receptor down-regultion or tretment with pertussis toxin) olished the cpcity of -nd -efroxn to enhnce the insulin secretory response to glienclmide. However, these mnoeuvres did not lter the ility of -efroxn to potentite glucose-induced insulin secretion. 6 The results indicte tht the enntiomers of efroxn exert di erentil e ects on insulin secretion which my result from inding to e ector sites hving opposite stereoselectivity. Binding of (7)-efroxn (presumly to imidzoline receptors) results in potentition of glucose-induced insulin secretion, wheres interction of -efroxn with second site leds to selective enhncement of sulphonylure-induced insulin relese. Keywords: Endocrine pncres; efroxn; glienclmide; dietes mellitus; nti-hyperglycemic drugs; pncretic -cell Introduction It is well estlished tht mny compounds which inhiit the outwrd ow of potssium ions through ATP-sensitive potssium (K-ATP) chnnels in pncretic B-cells re le to potentite nutrient-induced insulin secretion. Among these gents, sulphonylures re the est studied group nd currently represent the drugs of choice for tretment of the insulin secretory defect frequently ssocited with non-insulin-dependent dietes mellitus (NIDDIM) (Henquin, 1992; Stin, 1996). However, recent studies hve shown tht second group of compounds, chrcterized y the possession of n imidzoline ring (or closely relted moiety) cn lso elicit n increse in insulin secretion y inducing closure of K-ATP chnnels (Schulz & Hselltt, 1988; Chn & Morgn, 1990; Dunne, 1991; Jons et l., 1992; Brown et l., 1993, ; Chn et l., 1993; Chn, 1993; Berdeu et l., 1995). Such molecules my, therefore, represent useful new clss of therpeutic drugs in NIDDM nd severl imidzoline compounds hve lredy shown promise s nti-hyperglycemic gents in mn (Brodstone et l., 1987; Kwzu et l., 1987; Berlin et l., 1994). While there is good evidence tht imidzoline nd sulphonylure compounds re e ective insulin secretgogues in their own right, it hs lso een demonstrted tht comintions of these drugs cn represent prticulrly e ccious formultions. For exmple in rt, dministrtion of high doses of sulphonylure in comintion with the imidzoline efroxn leds to 1 Author for correspondence. much greter rise in circulting insulin thn cn e otined with either drug lone (Berridge et l., 1992). These results imply tht imidzoline nd sulphonylure drugs cn interct functionlly t the level of the pncretic B-cell, concept strengthened y the recent oservtion tht molecule hving imidzoline ntgonist ctivity in islets (RX801080) lso inhiits the insulin secretory response to glienclmide (Brown et l., 1993). Mny of the imidzoline compounds which promote n increse in insulin secretion re lso 2 -ntgonists nd their e ects in vivo were initilly thought to e medited y the lockde of endogenous 2 -drenoceptor-medited tone which cts to restrin the rte of insulin secretion (Brodstone et l., 1987; Berlin et l., 1994). While this mechnism my ccount for prt of their e ects, the more recent discovery of second mode of ction for imidzoline compounds (which involves inding to n `imidzoline receptor' ssocited with K-ATP chnnels) hs prompted the suggestion tht their insulin secretgogue ctivity my result primrily from gonist ctivity t n islet imidzoline receptor rther thn ntgonism t 2 - drenoceptors (Brown et l., 1993; Chn 1993; Berdeu et l., 1995; Morgn et l., 1995; Wng et l., 1996). However, it is not known which of these two possile mechnisms (i.e. imidzoline receptor gonism or 2 -drenoceptor ntgonism) ccounts for the ility of imidzoline compounds to enhnce the insulin secretory e ects of sulphonylures. In the present study we hve ddressed this question y tking dvntge of the nding tht one of the most e ective imidzoline 2 -ntgonist insulin secretgogues, efroxn, exists in two enntiomeric forms which disply opposite

2 800 M. Mourtd et l selectivity for imidzoline receptors nd 2 -drenoceptors (Chn et l., 1993). -Efroxn is potent 2 -ntgonist ut hs miniml imidzoline receptor-medited insulin-secretgogue ctivity in vitro, wheres (7)-efroxn is wekly potent 2 -ntgonist ut stimultes insulin secretion strongly (Chn et l., 1993; Morgn et l., 1995). We hve now investigted the e ects of these two enntiomeric forms of efroxn on insulin secretion induced y the sulphonylure glienclmide, to estlish whether 2 -ntgonism or imidzoline receptor ctivtion is the more importnt determinnt for the enhncing e ect of efroxn. Methods in vivo studies For ssessment of the e ects of imidzoline drugs nd sulphonylures on plsm insulin levels in rt, lood smples were removed from conscious mle Wistr rts (200 ± 300g ody weight) vi indwelling ctheters permitting rteril smpling. Animls were treted with ntiiotic (Synulox; 0.1 ml 100 g 71 ) 1 h efore the induction of nesthesi y intrmusculr injection of Dormitor (30 ml 100 g 71 ) nd intrperitonel dministrtion of Sulimse (0.6 ml 100 g 71 ). Ctheters were introduced into the femorl rtery nd fed to the dominl portion of the ort. The ctheters were exteriorized t the ck of the neck. The nesthesi ws reversed with Antisedn & Nuin (20 ml 100 g 71 ) nd the nimls housed individully for period of 7 dys efore experimenttion. For ech experiment, nimls were fsted overnight nd plced in restrint cges. A zero time lood smple ws removed nd the nimls were then dosed orlly with test regents or sline vehicle. Further lood smples were removed t indicted time points, for mesurement of insulin levels y rdioimmunossy y use of commercil guinepig nti-insulin serum (Sigm, U.K.) nd humn insulin stndrds. in vitro studies E ects of test regents on insulin secretion in vitro were mesured in islets of Lngerhns isolted from mle Wistr rts y collgense digestion. Islets were selected y hnd under dissecting microscope to minimize the contminting exocrine tissue. Islets were either used immeditely (`fresh islets') or were cultured in RPMI-1640 for 18 h with regents, s descried in the Results section, efore the ssessment of their secretory function. These methods hve een descried in detil previously (Brown et l., 1993, ; Chn et l., 1993). Insulin secretion experiments involved incution of groups of 3 isolted islets of Lngerhns in ml of icronteu ered solution (Gey & Gey, 1936) gssed with O 2 :CO 2 (95 : 5) nd supplemented with vrying concentrtions of glucose, 1 mm clcium chloride nd 1 mg ml 71 ovine serum lumin (frction V). Incution ws performed t 378C for 1 h, fter which time smples of medium were removed for ssy of insulin secretion y rdioimmunossy. Sttisticl nlysis Results were processed y nlysis of vrince nd di erences etween experimentl groups were considered signi cnt when P Mterils -Efroxn, its resolved enntiomers nd glienclmide were kindly provided y SmithKline Beechm Phrmceuticls (Welwyn, Herts, U.K.). Idzoxn, phenoxyenzmine, ntzoline, nordrenline nd pertussis toxin were purchsed from Sigm Chemicls (Poole, Dorset, U.K.). UK14304 (5-romo- 6-[2-imidzolin-2-ylnino]-quinoxlene) ws gift from P zer Imidzolines nd sulphonylures in islets Phrmceuticls, Sndwich, Kent, UK. All other regents were of nlyticl regent grde. Results E ects of efroxn nd glienclmide on circulting insulin levels in vivo Initilly, experiments were crried out in vivo in rts, to con rm tht -efroxn nd glienclmide cn interct functionlly to elicit potentited rise in plsm insulin levels nd to estlish the time course of this response. Orl dministrtion of single dose of -efroxn (10 mg kg 71 ody weight) to fsted rts, resulted in signi cnt rise in plsm insulin with 30 min, compred to sline treted nimls. This ws mintined for up to 1 h, fter which time the insulin levels declined towrds the sl vlue (Figure 1). Tretment of rts with glienclmide lso resulted in rise in plsm insulin levels, nd the extent nd time course of this increse ws identicl to tht seen fter dministrtion of -efroxn (Figure 1). When oth drugs were dministered together, the resultnt rise in insulin ws much lrger thn tht oserved with either gent lone (Figure 1) nd circulting insulin ws mintined t high level for the entire durtion of the experiment. The e ects of the enntiomers of efroxn on plsm insulin levels were then explored, ut lower doses were used (3.5 mg kg 71 ) ecuse of the limited vilility of these regents (Figure 2). In common with the rcemte (3.5 mg kg 71 ) ech enntiomer of efroxn induced smll increse in insulin levels within 45 min of dministrtion to fsted rts (Figure 2) nd this e ect ws lost y 90 min (Figure 2). However, mrked di erence etween the enntiomers ws oserved when the drugs were dministered in comintion with glienclmide. As expected, rcemic -efroxn cused signi cnt potentition of the glienclmide response (Figure 2 nd ) nd similr e ect ws lso seen when nimls were treted with -efroxn in the presence of glienclmide. However, the comintion of (7)-efroxn nd glienclmide did not elicit ny increse in insulin levels eyond tht induced y ech drug lone (Figure 2 nd ). Plsm insulin (ng ml 1 ) Time (min) Figure 1 E ects of efroxn nd glienclmide on plsm insulin levels in fsted rts. Groups of experimentl nimls were fsted overnight then treted orlly with sline (control, no symol) rcemic efroxn (10 mg kg 71, &), glienclmide ( mg kg 71, ) or efroxn plus glienclmide (~). Blood smples were removed t the times indicted nd ssyed for insulin y rdioimmunossy. Dt re presented s men vlues nd verticl lines show s.e.men (n=8 nimls per group). Comintions of efroxn nd glienclmide induced sttisticlly signi cnt increse in insulin levels eyond tht with ech gent lone (P50.001) t the 30, 60 nd 90 minute time points.

3 (±)- ( )- (±)- ( )- M. Mourtd et l E ects of the enntiomeric forms of efroxn on insulin secretion in vitro The ctions of the enntiomers of efroxn on insulin secretion were explored further in in vitro experiments employing isolted islets of Lngerhns. As expected, stimultion of insulin secretion (in the presence of 6 mm glucose) y the two enntiomers reveled di erence in potency (Figures 3 nd 4) with the (7)-enntiomer eing fr more e ective thn - efroxn over the dose rnge 10 ± 50 mm. Exmintion of the interction etween the rcemte nd ech isomer of efroxn (25 mm) with glienclmide in vitro reveled surprising result (Tle 1). Rcemic efroxn incresed the insulin secretory response to glienclmide signi cntly nd similr response ws oserved with the -enntiomer, even though this drug did not induce ny signi cnt increse in insulin secretion, lone. By contrst, despite cusing n increse in insulin secretion itself, (7)-efroxn did not potentite the response to glienclmide (Tle 1). Imidzolines nd sulphonylures in islets 801 The interctions of -efroxn with glienclmide were then investigted over wider rnge of concentrtions (Figure 4). -Efroxn did not directly promote insulin secretion from rt isolted islets t concentrtions up to 50 mm (Figure 4). Glienclmide (1 mm) induced signi cnt rise in insulin secretion from rt isolted islets nd ddition of incresing concentrtions of -efroxn resulted in further enhncement of the secretory response to glienclmide, in dosedependent mnner (Figure 4). (7)-Efroxn did not produce signi cnt e ect over this concentrtion rnge (not shown). E ects of other imidzoline nd 2 -ntgonist drugs on insulin secretion from rt islets The study ws extended y investigtion of the e ects of two further imidzoline drugs on the insulin secretory response to glienclmide in rt isolted islets. Idzoxn is potent 2-80 Tle 1 Effects of rcemic efroxn nd its resolved enntiomers on glienclmide-induced insulin secretion from rt isolted islets of Lngerhns Plsm insulin (ng ml 1 ) Control (±)- Ef Gli ( )- Ef - Ef - Efroxn Glienclmide Insulin secretion (25 mm) (1 mm) (ng/islet h 71 ) None None (7) (7) Asent Asent Asent Asent ,NS Groups of 3 isolted islets were incuted in the presence of 6mM glucose in the presence or sence of test regents, s shown, for 1 h, fter which time the medium ws smpled nd ssyed for insulin secretion. Efroxn ws used s the rcemte () or s the resolved enntiomers, shown s (7) or. Results re presented s men vlues+s.e.men for 12 ± 16 oservtions in ech cse. P50.01 reltive to sence of efroxn or glienclmide. P50.01, reltive to efroxn or glienclmide lone. NS Not signi cntly di erent from (7)-efroxn in the sence of glienclmide. Plsm insulin (ng ml 1 ) Control (±)- Ef Gli ( )- Ef - Ef - Figure 2 E ects of rcemic efroxn nd the resolved enntiomers on plsm insulin levels in fsted rts. Groups of experimentl nimls were fsted overnight then dosed orlly with rcemic efroxn (3.5 mg kg 71, -Ef), (7)-efroxn (3.5 mg kg 71 ), - efroxn (3.5 mg kg 71 ) or glienclmide ( mg kg 71, Gli) s shown. Blood smples were removed fter 45 min () or 90 min () nd ssyed for insulin y rdioimmunossy. Results re presented s men vlues +s.e.men for 8 nimls per group. P50.01 reltive to sline control. P reltive to either efroxn or glienclmide lone Lignd concentrtion (µm) Figure 3 Stimultion of insulin secretion y (7)-efroxn. Groups of rt isolted islets were treted with (7)-efroxn t incresing concentrtions in the presence of 6 mm glucose. Following incution for 1 h, smples of the incution medium were removed nd ssyed for insulin secretion. Dt represent men rtes of insulin secretion (n=12); verticl lines show s.e.men.

4 802 M. Mourtd et l Imidzolines nd sulphonylures in islets Lignd concentrtion (µm) Figure 4 E ects of -efroxn on insulin secretion in the presence nd sence of glienclmide. Groups of rt isolted islets were treted with -efroxn t incresing concentrtions in the presence of 6 mm glucose () or 6 mm glucose plus 1 mm glienclmide (~). Following incution for 1 h smples of the incution medium were removed nd ssyed for insulin secretion. Dt represent men rtes of insulin secretion (n=12 ± 18); verticl lines show s.e.men. 0.0 Control Antz (±)-Ef Control Antz (±)-Ef Figure 6 E ects of ntzoline nd rcemic efroxn on insulin secretion in the presence nd sence of glienclmide. Groups of rt isolted islets were treted with ntzoline (50 mm; Antz) or - efroxn (50 mm; Ef) in the presence of 6 mm glucose lone (htched columns) or 6 mm glucose plus 1 mm glienclmide (stippled columns). Following incution for 1 h, smples of the incution medium were removed nd ssyed for insulin secretion. Dt represent men rtes of insulin secretion +s.e.men (n=12). P50.01 reltive to glienclmide lone Lignd concentrtion (µm) Figure 5 E ects of idzoxn on insulin secretion in the presence nd sence of glienclmide. Groups of rt isolted islets were treted with idzoxn t incresing concentrtions in the presence of 6 mm glucose () or6mmglucose plus 1 mm glienclmide (~). Following incution for 1 h, smples of the incution medium were removed nd ssyed for insulin secretion. Dt represent men rtes of insulin secretion (n=12 ± 18); verticl lines show s.e.men. ntgonist which, in rt islets, lcks the ility to stimulte insulin secretion vi imidzoline receptor-medited lockde of K-ATP chnnels (Figure 5; Chn & Morgn, 1990; Berdeu et l., 1995). Despite its potency s n 2 -ntgonist (which is similr to -efroxn; Stillings et l., 1985) idzoxn filed to enhnce the secretory response to glienclmide in rt isolted islets (Figure 5). Similrly, ntzoline (n imidzoline insulin secretgogue which hs very low nity for 2 -drenoceptors; Berdeu et l., 1995) did not enhnce the secretory response to glienclmide under conditions where it cused direct stimultion of insulin secretion in the sence of the sulphonylure (Figure 6). E ect of mnoeuvres designed to inhiit 2 -drenoceptor responses, on the ility of efroxn to potentite the insulin secretory response to glienclmide In the nl section of the work, numer of di erent experimentl prdigms were devised to minimize the extent of 2 - drenoceptor signlling in rt isolted islets of Lngerhns. The consequences of these mnoeuvres on the ility of efroxn to potentite the insulin secretory response to glienclmide were studied. Initilly, the covlent 2 -ntgonist phenoxyenzmine (Pz) ws used to inctive irreversily islet 2 -drenoceptors. Rt isolted islets were treted with either sline (control) or Pz (0.1 mm) during 30 min preincution period, then exposed to test regents (Figure 7). In control islets, glienclmide lone incresed insulin secretion nd the ddition of -efroxn, resulted in n dditionl enhncement of insulin secretion (Figure 7). In islets treted with Pz, glienclmide still elicited n increse in insulin secretion when dded lone, ut the comintion of -efroxn nd glienclmide filed to enhnce further insulin secretion under these conditions (Figure 7). Control experiments with the 2 -gonist nordrenline con rmed tht Pz tretment resulted in complete 2 -lockde. Nordrenline 1 mm filed to inhiit signi cntly glucose-induced insulin secretion fter Pz tretment, wheres it olished insulin secretion from untreted islets (Figure 7). In the next series of experiments, we exploited the results of previous studies showing tht culture of rt islets in the presence of the selective 2 -gonist UK14304 leds to loss of 2 - responsiveness (Chn et l., 1993). In support of this, prior exposure of islets to 10 mm UK14304 for 18 h, resulted in complete loss of the ility of 1 mm nordrenline to inhiit glucose-induced insulin secretion in susequent incution experiments (Figure 8). The response to nordrenline ws mintined in control islets not exposed to UK14304 during the culture period. The ility of either -efroxn or glienclmide to promote insulin secretion ws un ected y culture with UK14304 (Figure 8), wheres the enhncement of

5 M. Mourtd et l Imidzolines nd sulphonylures in islets insulin secretion induced y the comintion of -efroxn nd glienclmide in control islets, ws lost fter exposure to UK14304 (Figure 8). Finlly, groups of rt isolted islets were cultured for 18 h under either control conditions or in the presence of 200 ng ml 71 pertussis toxin. In the ltter circumstnces, susequent exposure of the islets to nordrenline (1 mm) did not led to inhiition of glucose-induced insulin secretion (Figure 9), con rming tht the 2 -drenoceptors hd ecome uncoupled from the control of insulin secretion y the pertussis toxin tretment. In these islets, the comintion of -efroxn nd glienclmide did not enhnce insulin secretion eyond tht induced y either drug lone. As expected, the comintion of -efroxn nd glienclmide did further enhnce insulin secretion from the sme tches of islets cultured in the sence of pertussis toxin (Figure 9). Discussion A B C D E F A B C D E F [Glucose] (mm) Figure 7 E ect of 2 -drenoceptor lockde on the ility of efroxn to increse glienclmide-induced insulin secretion. Groups of isolted islets of Lngerhns were preincuted for 30 min in the sence () or presence () of 0.1 mm phenoxyenzmine. They were then wshed nd exposed to test regents: (B) 20 mm glucose; (C) 20 mm glucose +1 mm nordrenline; (D) 1 mm glienclmide; (E) 25 mm -efroxn; (F) -efroxn + glienclmide, during further incution period of 60 min (A, control response to glucose). After this time the medium ws smpled nd ssyed for insulin content. Dt re presented s men vlues+s.e.men (n=12). P reltive to glienclmide or efroxn lone. The results presented here demonstrte tht, oth in vivo nd in vitro, comintions of -efroxn nd glienclmide cuse greter relese of insulin from rt islets thn when either drug is given lone. These dt con rm the oservtions of Berridge et l. (1992) in fed rts, ut extend the work to fsted nimls nd lso revel the isomeric speci city of the response. The results indicte tht there my e crucil mechnistic difference etween the ility of imidzoline 2 -ntgonists to potentite glucose-induced insulin secretion nd to enhnce the secretory response to glienclmide. In the former cse, the response proly results from inding of drugs to imidzoline receptors ssocited with K-ATP chnnels, s suggested in previous work (Schulz & Hselltt, 1988; Chn & Morgn, 1990; Dunne, 1991; Jons et l., 1992; Brown et l., 1993, ; Chn, 1993; Chn et l., 1993; Berdeu et l., 1995). By contrst, in the presence of glienclmide, the enntiomer of efroxn (which is only very wekly potent s n imidzoline receptor lignd ut is potent 2 -ntgonist; Chn et l., 1993) Insulin secretion (n/islet h 1 ) 0.0 A B C D E F A B C D E F [Glucose] (mm) Figure 8 E ect of 2 -drenoceptor downregultion on the ility of efroxn to increse glienclmide-induced insulin secretion. Groups of isolted islets of Lngerhns were cultured for 18 h in the sence () or presence () of 1 mm UK They were then wshed nd exposed to test regents: (B) 20 mm glucose; (C) 20 mm glucose +1 mm nordrenline; (D) 1 mm glienclmide; (E) 50 mm - efroxn; (F) -efroxn + glienclmide; during n incution period of 60 min (A, control response to glucose). After this time the medium ws smpled nd ssyed for insulin content. Dt re presented s men vlues+s.e.men (n=24). P reltive to glienclmide or efroxn lone A B C D E F A B C D E F [Glucose] (mm) Figure 9 E ect of culture with pertussis toxin on the ility of efroxn to increse glienclmide-induced insulin secretion. Groups of isolted islets of Lngerhns were cultured for 18 h in the sence () or presence () of 200 ng ml 71 pertussis toxin. They were then wshed nd exposed to test regents: (B) 20 mm glucose; (C) 20 mm glucose +1 mm nordrenline; (D) 1 mm glienclmide; (E) 50 mm -efroxn; (F) efroxn + glienclmide, during n incution period of 60 min (A, control response to glucose). After this time the medium ws smpled nd ssyed for insulin content. Dt re presented s men vlues +s.e.men (n=24). P reltive to efroxn or glienclmide lone. promoted further rise in insulin secretion, despite the fct tht it ws ine ective in the sence of the sulphonylure. Tken together, therefore, these oservtions suggest tht the potentition of glienclmide-induced insulin secretion my involve the inding of imidzoline drugs to 2 -drenoceptors rther thn to imidzoline receptors. In support of this conclusion, we hve found tht ny one of series of procedures designed to prevent or to minimize 2 -drenoceptor signlling in islet cells lso prevented the potentition of glienclmide-induced insulin secretion y efroxn. This ws true when the 2 -receptor ws locked

6 804 M. Mourtd et l Imidzolines nd sulphonylures in islets covlently with low dose of phenoxyenzmine (Figure 7), when the receptor ws down-regulted y exposure to the gonist UK14304 for 18 h (Figure 8) nd when receptore ector coupling ws interrupted y culture in the presence of pertussin toxin (Figure 9). In ech cse the loss of 2 -responsiveness ws con rmed y performing control experiments with nordrenline. It is importnt to note tht these experimentl mnipultions did not prevent the direct stimultion of insulin secretion y -efroxn in the presence of 6 mm glucose, suggesting tht the islet imidzoline receptor is insensitive to UK14304 nd tht it is not coupled to K-ATP chnnels vi pertussis toxin-sensitive G-protein. Overll, therefore, the dt imply tht there my e speci c interction etween 2 -drenoceptors nd sulphonylure responses in pncretic B-cells, which cn result in potentition of sulphonylure-induced insulin secretion. This conclusion is t vrince with the ndings of Grrino nd Henquin (1990) who rgued tht interctions etween drenoceptor lignds nd sulphonylures result from unde ned `non-speci c' events. However, evidence for n interction etween sulphonylure receptors nd islet 2 - drenoceptors hs een provided in other work. For exmple, Sirek et l. (1974) showed tht the 2 -ntgonist dihydroergotmine could mplify the insulin secretory response to sulphonylures in conscious dogs. In ddition, Cherksey & Altszuler (1984) hve suggested tht sulphonylures my in- uence the inding of lignds to 2 -drenoceptors on pncretic islet cells nd Rusteneck et l. (1995) hve shown tht tretment of islets with the 2 -ntgonist phentolmine, in the presence of tolutmide, results in n dditive increse in cytosolic clcium concentrtion. Despite the considertions ove, there lso remin numer of nomlies which do not t redily with the concept tht 2 -drenoceptor lockde is importnt for the enhncement of glienclmide-induced insulin secretion. For exmple, it is cler tht lockde of the 2 -receptor with the drugs idzoxn or phenoxyenzmine did not directly promote ny increse in the response to glienclmide (Figure 7), wheres occuption of the receptor y phenoxyenzmine did prevent the enhncing e ect of efroxn. Furthermore, the concentrtions of -efroxn required to enhnce the secretory response to glienclmide were in the rnge 10 ± 50 mm (Figures 4 nd 5). These levels seem unusully high given the potency of this drug s n 2 -drenoceptor lignd. Nevertheless, when tken s whole, the results indicte tht 2 -drenoceptors exert level of control over sulphonylure-induced insulin secretion such tht occuption of the receptors y certin ntgonists results in enhncement of the response to sulphonylures. It is conceivle tht this might re ect the ility of selected ntgonists to stilize 2 -drenoceptors in n inctive conformtion nd therey limit their sl level of signlling ctivity. Such `negtive' gonism hs een shown for some ntgonists of G-protein coupled receptors (Schutz & Freissmuth, 1992) nd could ccount for the present results. However, if this is the cse, it remins to e explined why the response exhiits selectivity for sulphonylures reltive to glucose. An lterntive explntion for the present results cnnot e excluded; nmely tht -efroxn cts s selective gonist t previously unde ned `imidzoline' receptor which cn in uence the functionl ctivity of the sulphonylure receptor. This site would need to e sensitive to phenoxyenzmine nd UK14304 nd coupled to pertussis toxin-sensitive G-protein. No such receptor hs yet een de ned in islets, lthough it is noteworthy tht the new insulin secretgogue S21663 displys properties which hve led its developers to propose tht it my interct with novel `imidzoline' receptor in the pncretic B- cell (Wng et l., 1996). In ddition, Zitsev et l. (1996) hve postulted tht n unde ned imidzoline receptor my control distl step in B-cell exocytosis which lies eyond the regultion of potssium permeility. In spite of the mechnistic miguities, the present dt suggest tht further considertion should e given to the development of therpeutic formultions in which selected 2 - ntgonists re comined with sulphonylures for the tretment of NIDDM. In this context, one dditionl point lso merits comment. During the in vivo experiments with efroxn, it ws oserved tht oth enntiomers cused smll, ut sttisticlly signi cnt, rise in circulting insulin levels in the sence of glienclmide. This implies tht, in vivo, it my e possile to promote n increse in insulin relese y either of two mechnisms ± dministrtion of n imidzoline receptor gonist (such s (7)-efroxn) or tretment with selective 2 - ntgonist (e.g. -efroxn). Only in the ltter cse does codministrtion of glienclmide then led to still greter increse in secretion rte. 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