Regulation of the common carotid arterial blood flow by nicotinic receptors in the medulla of cats

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1 British Journl of Phrmology (6) 149, & 6 Nture Pulishing Group All rights reserved /6 $3. RSARCH PAPR Regultion of the ommon rotid rteril lood flow y niotini reeptors in the medull of ts C-L Gong 1, Y-T Chiu 2, N-N Lin 2,3, C-C Cheng 2,3, S-Z Lin 4, TJ-F Lee 4,5,6 nd J-S Kuo 4,5 1 Deprtment of Physiology, Shool of Mediine, Chin Medil University, Tihung, Tiwn; 2 Deprtment of dution nd Reserh, Tihung Veterns Generl Hospitl, Tihung, Tiwn; 3 Deprtment of Veterinry Mediine, Ntionl Chung Hsing University, Tihung, Tiwn; 4 Neuro-Medil Sientifi Center nd Center for Vsulr Mediine, Buddhist Tzu Chi Generl Hospitl nd Tzu Chi University, Hulien, Tiwn; 5 Institute of Phrmology nd Toxiology, Tzu Chi University, Hulien, Tiwn nd 6 Deprtment of Phrmology, Southern Illinois University, Shool of Mediine, Springfield, IL, USA Bkground nd purpose: Ations of glutmte nd serotonin on their respetive reeptors in the dorsl fil re (DFA) of the medull re known to regulte ommon rotid rteril (CCA) lood flow in ts. Less is known out etylholine tion on its niotini reeptor (nachr) sutypes in the DFA for regultion of CCA lood flow nd this spet ws investigted. xperimentl pproh: otini nd musrini gonists nd ntgonists were miroinjeted into the DFA through threerrel tuing in nesthetized ts. Results: CCA lood flow ws dose-dependently inresed y niotine ( non-seletive nachr gonist) nd holine ( seletive 7-nAChR gonist). These effets of niotine were ttenuted y -ungrotoxin (n 7-nAChR ntgonist), methyllyonitine (n 7-nAChR ntgonist), memylmine ( reltively seletive 34-nAChR ntgonist) nd dihydro--erythroidine ( reltively seletive 42-nAChR ntgonist). The holine-indued flow inrese ws ttenuted y -ungrotoxin nd memylmine, ut not y dihydro--erythroidine. Musrini gonists (musrine nd methholine) nd ntgonist (tropine) ffeted neither the sl nor the niotine-indued inrese in the CCA lood flow. Conlusions nd implitions: Funtionl 7, 42, nd 34 suunits of the nachr pper to e present on the DFA neurons. Ativtions of these reeptors inrese the CCA lood flow. The present findings do not prelude the presene of other nachrs suunits. Musrini reeptors, if ny, on the DFA re not involved in regultion of the CCA lood flow. Vrious sutypes of nachrs in the DFA my medite regultion of the CCA nd ererl lood flows. British Journl of Phrmology (6) 149, doi:1.138/sj.jp.76844; pulished online 7 August 6 Keywords: holinergi reeptor; rotid rtery; medull; nachr; prsymptheti; vsulr regultion Arevitions: A-BT, -ungrotoxin; CCA, ommon rotid rtery; CVLM, udl ventrolterl medull; DB, dihydro-erythroidine; DFA, dorsl fil re; DMNV, dorsl motor nuleus of the vgus;, glutmte;, hert rte; Me, memylmine; Met, methyllyonitine; M, men systemi rteril pressure; nachrs, niotini etylholine reeptors; nts, nuleus trtus solitrius; RVLM, rostrl ventrolterl medull;, systemi rteril pressure Introdution Kuo et l. (1987) first identified the dorsl fil re (DFA), retiulr re just dorsl to the fil nuleus in the t. Stimultion of the DFA with glutmte evoked minly n ipsilterl inrese in lood flow of the ommon rotid rtery (CCA) without signifint hnges in systemi rteril lood pressure (Kuo et l., 1987; Chyi et l., 1995). tmte nd serotonin, tonilly relesed in the DFA, indue n inrese nd derese, respetively, in the CCA lood flow Correspondene: Professor J-S Kuo, Institute of Phrmology nd Toxiology, Tzu Chi University, 71, Se 3, Chung-Yng Rod, Hulien 97, Tiwn. -mil: jskuo@mil.tu.edu.tw Reeived 3 My 6; revised 2 June 6; epted 28 June 6; pulished online 7 August 6 (Li et l., 1996; Kuo et l., 1999; Gong et l., 2). The DFA is prsymptheti nuleus (Kuo et l., 1987, 1992, 1995; Chyi et l., 1995, 5). It my e funtionlly nd ntomilly equivlent to the rt prsymptheti ererovsodiltor enter (Nki et l., 1993) tht is loted dorsolterlly to the fil nuleus. Therefore, oth res re likely to e the rostrl extension of the dorsl motor nuleus of the vgus (DMNV). linergi nerves re widely distriuted in the ortex (Sto et l., 1; Hott et l., 2), hippompus (Sto nd Sto, 1995), stritum (Kiser nd Wonnott, ; Zhou et l., 2), hypothlmus (Htton nd Yng, 2) s well s medull olongt suh s the rostrl ventrolterl medull (RVLM) (Kuo et l.,, 2), DMNV

2 Medullry niotini reeptors regulte rotid flow C-L Gong et l 7 nd nuleus trtus solitrius (nts) (Reynolds et l., 1994). Stimultion of niotini reeptors promotes glutmte relese tht modultes dopmine releses in rt stritl slie (Kiser nd Wonnott, ). linergi inputs to the RVLM ply vsopressor effet through musrini tion (Kuo et l.,, 2). The holinergi fiers to the rt ortex relese etylholine to inrese the ererl lood flow vi oth musrini nd niotini etylholine reeptors (nachrs) in the prenhym of the ortex (Sto et l., 1). In slie preprtions of the rt medull olongt, pplition of etylholine to the pregnglioni neurons of the DMNV results in mrked depolriztion through niotini tion (Ito et l., 1989). otini reeptors in speifi medullry regions, suh s the nts (Dhr et l., ; Ferguson et l., ; Ferreir et l., 1), the DMNV (Ferreir et l., 1), the RVLM nd udl ventrolterl medull (Hungfu et l., 1997; Aerger et l., 1), ply importnt roles in rdiovsulr regultion. The DFA s prsymptheti nuleus or the rostrl extension of the DMNV (Kuo et l., 1987, 1992, 1995; Chyi et l., 1995, 5), therefore, my quite possily shre some nture of the ove-mentioned nulei tht regulte rdiovsulr funtion. Whether niotini nd/or musrini tions nd their reeptors in the DFA were involved in regultion of the CCA lood flow ws not known. otini reeptors re undnt nd ply diverse roles in the entrl nervous system (Deker et l., 1995; Colquhoun nd Ptrik, 1997). The 7-nAChRs re present in the DMNV (Ferreir et l.,, 1), the hik symptheti gngli (Du nd Role, 1) on the stritl glutmtergi terminls (Kiser nd Wonnott, ) nd in the hypothlmi supropti nuleus (Htton nd Yng, 2). Both the 7- nd 34-nAChRs re present in the nts (Dhr et l., ). The 34-nAChRs re present in the hippompus (Alkondon nd Aluquerque, 2; Gioomo nd Hsselmo, 5; Co et l., 5). The 42-nAChRs re present in the sustnti geltinos (Kiyosw et l., 1). Both the 32- nd 42-nAChRs hve een found in the stritum (Kiser nd Wonnott, ). Nevertheless, nachrs ontining 7, 34 nd 42 suunits re most ommonly present in the entrl nervous system. Hene, we foused on these three suunits on the neurons of the DFA, whih hve not yet een investigted so fr s we know. Our novel findings demonstrte tht t lest three different sutypes of nachrs, 7, 34 nd 42 suunits, re present on the DFA neurons, nd tht their tivtions inrese the CCA lood flow. Musrini reeptors, if ny, on the DFA neurons re not involved in regultion of the CCA lood flow. Mterils nd methods Generl proedures The experiments were rried out in ordne with the guidelines of the Tzu-Chi University Institutionl Animl M 5 nmol M 3 mm 5 3 se nmol Figure 1 Typil trings show the reproduile inrese in the CCA lood flow indued y repeting miroinjetions ( nl s 1 for 5 s) of niotine nd holine into the DFA t n intervl of 3 min. The DFA ws first identified y eletril stimultion () nd then onfirmed y glutmte stimultion. Note the ipsilterl inrese in the CCA lood flow without hnges in, nd M. The dots on the drwing of medullry setions indite the injeted loi. Arevitions for this nd the following figures: B min 1, ets per min;, holine; DFA, dorsl fil re;, eletril stimultion ( Hz,.5 ms, ma, 1 s);, fil nuleus;, glutmte;, hert rte; M, men systemi rteril pressure;, niotine;, pyrmidl trt; or, right or left ommon rotid rteril lood flow;, systemi rteril pressure; 5ST, spinl trigeminl nuleus. British Journl of Phrmology (6)

3 8 Medullry niotini reeptors regulte rotid flow C-L Gong et l Cre nd Use Committee nd of the Chin Medil University thil Committee for Animl Reserh, nd were pproved y oth ommittees. Cts ( kg) of either sex were nesthetized intrperitonelly with -hlorlose ( mg kg 1 ) nd urethne ( mg kg 1 ). nd expirtory CO 2 onentrtion ws mintined t y rtifiil ventiltion. The retl temperture ws mesured nd kept t C y n eletril heting pd. Right femorl rtery nd vein were nnulted with P-9 polyethylene tuing for mesurement of the systemi rteril pressure () nd supplement of fluid, respetively. The ultrsound Doppler proes (dimeter mm) were pled round the right nd left CCA nd monitored with Diretionl Pulsed Doppler Flowmeter (University of Iow, Bioengineering, 545C-4, Iow, USA). The, hert rte () nd CCA lood flows were routinely reorded on Gould Reorder RS3 (Clevelnd, OH, USA) s desried in our previous ppers (Li et l., 1996; Gong et l., 2). Miroinjetion tehnique The hed of ts ws immoilized in Dvid-Kopf stereotxi instrument. The stereotxi oordintes of the DFA were out 6 mm rostrl to the oex, 3.5 mm lterl to the midline nd 3.5 mm ventrl to the floor of the fourth ererl ventrile. The three-rrel eletrode tuing ws onstruted with three stinless-steel tuings (.3 mm in dimeter) glued together nd insulted, exept the tip t one end. It ws inserted into the DFA t n ngle of 341 from the vertil xis of the stereotxi instrument. This filitted the tuing insertion to e perpendiulr to the floor of the fourth ererl ventrile. h rrel of this tuing ws filled with one of the following hemils: sodium glutmte, niotine ( non-seletive nachr gonist), holine (n 7-nAChR gonist), -ungrotoxin (n 7-nAChR ntgonist), methyllyonitine (n 7-nAChR ntgonist), memylmine (n 34-nAChR ntgonist), dihydro-erythroidine (n 42-nAChR ntgonist), musrine ( musrini reeptor gonist) nd methholine ( musrini reeptor gonist). All these drugs were dissolved in rtifiil ererospinl fluid (CSF) ontining the hemils (mm) NCl 119, KCl 2.5, MgCl 2 4, CCl 2 4, NHCO , NH 2 PO 4 1 nd gluose 11, nd gssed with 95 O 2 nd 5 CO 2 t ph 7.4. The CSF ws used s vehile ontrol. h hemil with volume of nl ws miroinjeted into the DFA in 5 s with the miroinjetion pump (CMA/, Crnegie Mediin, North Chelmsford, MA, USA). All hemils were purhsed from Sigm-Aldrih In. (St Louis, MO, USA). Blood Flow Inrese ( of sl level) otine (nmol) 16 M nmol d Blood Flow Inrese ( of sl level) e d line (nmol) 16 M se 16 nmol 3 mm Figure 2 The inrese of the CCA lood flow ws dose-dependently indued y miroinjetions into the DFA of niotine (n ¼ 4) (, ) or holine (n ¼ 4) (, d). (, ) Sttistil nlysis; (, d) originl trings nd injetion loi (indited y dots) in the DFA. Dt re expressed s mens7s.e.m. nd nlyzed y Student s t-test. Po.1 vs vehile; Po.1 vs 1 nmol; Po.1 vs nmol; Po.5 vs 1 nmol; d Po.5 vs nmol; e Po.1 vs nmol. British Journl of Phrmology (6)

4 Medullry niotini reeptors regulte rotid flow C-L Gong et l 9 xperimentl designs For loliztion of the DFA y eletril stimultion of it, ts were further prlyzed with trurium (GlxoSmithKline S.p.A., Prm, Itly), initilly.5 mg kg 1 nd.2 mg kg 1 intrvenously every min, to eliminte interferene in reording of lood flow owing to stimultion-indued musle ontrtion. The DFA ws identified y n inrese of the CCA lood flow first indued y the eletril stimultion ( Hz,.5 ms, ma, 1 s) nd then y glutmte (5 nmol) stimultion of the DFA through the eletrode tuing. The eletrode tuing ws then mintined there throughout the whole ourse of the experiment. The intervl for reproduile inreses in the CCA lood flow y repeted miroinjetions of niotine ( nmol) nd holine ( nmol) ws 3 min (Figure 1, n ¼ 3 for eh drug). For the susequent experiments, eh drug ws lso injeted t n intervl of 3 min. Dose responsive effet of niotine nd holine ws determined t doses of 1,,, nd 16 nmol (n ¼ 5 for eh drug). We determined nachr suunits in the DFA for regultion of the CCA lood flow. otine ( nmol) nd holine ( nmol) were miroinjeted into the DFA to inrese the CCA lood flow. This effet ws then sujeted to the effets of niotini ntgonists, inluding -ungrotoxin (2., 4. nd 8. pmol), methyllyonitine (.25,.5 nd.1 nmol), memylmine (1., 2. nd 4. nmol) nd dihydro--erythroidine (.25,.5 nd 1. nmol) (n ¼ 4 for eh ntgonist). In detil, niotine ( nmol) ws miroinjeted to indue n inrese in the CCA lood flow. After 3 min, -ungrotoxin (2 pmol) ws injeted. After 5 min, the sme dose of niotine ws repeted. After 3 min, the sme proess ws repeted for 4. nd 8. pmol -ungrotoxin. xmintion of other nachr ntgonists followed the sme proedure s -ungrotoxin. A similr protool for holine ( nmol) ws followed. Whether musrini reeptors in the DFA might regulte the CCA lood flow ws exmined in nine nimls. Musrine (1 nmol), methholine ( nmol) nd tropine ( nmol) were miroinjeted into the DFA (n ¼ 3 for eh drug) to determine if these gonists or ntgonist ffeted the sl CCA lood flow. Histology The stimulted site of the DFA ws mrked with pontmine lue (.1, nl) miroinjetion or lesion produed y DC urrent of 2 ma for 1 s through the three-rrel eletrode tuing. At the end of the experiment, the t ws killed y sturted KCl dministered intrvenously. The rin ws removed nd frozen-setioned t mm thikness on Cr mirotome (2 Frigout). Proper plement of the proe ws onfirmed upon mirosopi exmintion. Only ts with orretly positioned eletrode tuing in the DFA were onsidered for dt nlysis. Dt nlysis Chnges in the, nd CCA lood flow responding to miroinjetions of hemils were lulted s (response vlue ontrol vlue)/(ontrol vlue). Dt were expressed s mens7s.e.m. nd nlyzed sttistilly y Student s t-test. The proility level of signifint differene ws Po.5. Results Dose-dependent responses of niotine nd holine The men systemi rteril pressure (M), nd CCA lood flow in the norml ontrol ts were 1728, 2387 ets min 1 nd 375 ml min 1, respetively (n ¼ 52). In ontrol experiments, miroinjetions into the DFA of ny drug vehile used did not ffet M, nd CCA lood flow. Repeted miroinjetions of either nmol niotine or nmol holine into the DFA t n intervl of 3 min (n ¼ 3 for eh drug) indued reproduile inreses in the CCA lood flow in eh niml (Figure 1). Miroinjetions of niotine (Figure 2 nd, n ¼ 5) or holine (Figure 2 nd d, n ¼ 5) into the DFA used dosedependent inreses of the CCA lood flow, ut did not ffet the M nd (Tle 1). otine t doses of 1 nmol eliited dose-dependent inreses in the CCA lood flow, rehing mximl inrese of 114 s ompred with the sl level (Figure 2). The inrese ws redued t higher Tle 1 ffet of intr-dfa miroinjetion of niotine or holine on hnges of M, nd oth sides of CCA lood flow nmol 1 16 otine M () () ICCA () , , CCCA () line M () () ICCA () , 54714,d 18711,e CCCA () CCA, ommon rotid rteril lood flow; CCCA, ontrlterl ommon rotid rtery lood flow; DFA, dorsl fil re;, hert rte; ICCA, ipsilterl ommon rotid rtery lood flow; M, men systemi rteril pressure. N ¼ 5 for either niotine or holine group. Vlues re men7s.e.m., Po.1 vs vehile; Po.1 vs niotine 1 nmol; Po.1 vs niotine nmol; Po.5 vs holine 1 nmol; d Po.5 vs holine nmol; e Po.1 vs holine nmol y Student s t-test. British Journl of Phrmology (6)

5 21 Medullry niotini reeptors regulte rotid flow C-L Gong et l A-BT, 8. pmol M 5 nmol Me, 4. nmol M 5 3 se nmol Figure 3 Typil trings demonstrte tht the inrese of CCA lood flow indued y miroinjetion of niotine into the DFA ws mrkedly inhiited y pretretment of -ungrotoxin (7 nachr ntgonist) or memylmine (34 nachr ntgonist) miroinjeted into the DFA. Arevitions: A-BT, -ungrotoxin; Me, memylmine. 3 mm doses of nd 16 nmol, proly owing to development of thyphylxis to niotine (Zhng et l., 1998). Miroinjetions of holine, t doses similr to niotine, lso dosedependently inresed the CCA lood flow (Figure 2). line t 1 nmol eliited dose-dependent inreses in the CCA lood flow, rehing mximl inrese of 18 s ompred with the sl level. The flow inrese ws not further inresed y greter dose of 16 nmol. Bsed on these results, nmol of niotine nd nmol of holine were seleted for susequent studies. ffets of nachr ntgonists on niotine-indued inrese in the CCA lood flow To determine whether niotini tion ws medited y 7-, 34- nd 42-nAChR suunits in the DFA, seletive ntgonists were used. The inresed CCA lood flow indued y miroinjetion of nmol niotine into the DFA ws mrkedly inhiited y pretretment with 8. pmol -ungrotoxin (A-BT, seletive 7-nAChR ntgonist) nd 4. nmol memylmine (Me, reltive seletive 34- nachr ntgonist) miroinjeted into the DFA (Figure 3). The niotine-indued inreses in the CCA lood flow were dose-dependently suppressed y pretretments of A-BT (Figure 4, n ¼ 4), methyllyonitine (Met, seletive 7- nachr ntgonist) (Figure 4, n ¼ 4), Me (Figure 5, n ¼ 4) nd dihydro--erythroidine (DB, reltive seletive 42- nachr ntgonist) (Figure 5, n ¼ 4). In ontrol experiments, ll ntgonists used did not ffet the sl CCA lood flow. ffets of nachr ntgonists on holine-indued inrese in the CCA lood flow To further test the involvement of 7-, 34- nd 42- nachrs in niotini tion in the DFA, holine, seletive 7-nAChR gonist, ws used to eliit inreses in the CCA lood flow. The inresed CCA lood flow ws dosedependently suppressed y prior miroinjetions in the DFA of A-BT (Figure 6, n ¼ 4) nd Me (Figure 6, n ¼ 4). A-BT nd Me lone did not ffet the sl CCA lood flow. DB ffeted neither the sl nor the holine- British Journl of Phrmology (6)

6 Medullry niotini reeptors regulte rotid flow C-L Gong et l 211 Blood Flow Inrese ( of sl level) Blood Flow Inrese ( of sl level) A-BT Met nmol pmol nmol nmol Figure 4 Intertions of niotine with seletive 7-nAChR ntgonists (-ungrotoxin) (n ¼ 4) () nd methyllyonitine (n ¼ 4) (), in the DFA. () Miroinjetion of niotine in the DFA used n inrese of CCA lood flow. The flow inrese ws dose-dependently redued y -ungrotoxin. () Miroinjetion of the sme dose of niotine in other group produed n inrese of CCA lood flow. This flow inrese ws dose-dependently redued y methyllyonitine. Arevitions: Met, methyllyonitine. Dt re expressed s mens7s.e.m. nd nlyzed y Student s t-test. Po.1 vs nmol; Po.1 vs A-BT 2. pmol; Po.5 vs Met.25 nmol. Blood Flow Inrese ( of sl level) Blood Flow Inrese ( of sl level) Me DB nmol nmol nmol nmol Figure 5 Intertions of niotine with memylmine, 34 nachr ntgonist (n ¼ 4) (), nd dihydro--erythroidine, 42 nachr ntgonist (n ¼ 4) (), in the DFA. () Miroinjetion of niotine indued n inrese of the CCA lood flow. The flow inrese ws dose-dependently ttenuted y pretretment with memylmine. () Miroinjetion of the sme dose of niotine in nother group produed n essentilly similr inrese of the CCA lood flow. This flow inrese ws dose-dependently redued y pretretment with dihydro--erythroidine. Dt re expressed s mens7s.e.m. nd re nlyzed y Student s t-test. Po.1 vs nmol; Arevitions: DB, dihydro--erythroidine. Po.5 vs Me 1. nmol; Po.1 vs DB.25 nmol; Po.5 vs DB.5 nmol. indued inrese in the CCA lood flow (n ¼ 3, dt not shown). ffets of musrine nd methholine Whether musrini reeptors were involved in the DFA ws ssessed y miroinjetions in the DFA of musrini gonists, musrine (5 nmol in one niml nd 1 nmol in two nimls) nd methholine (1 nmol in one niml nd nmol in two nimls) s well s musrini ntgonist, tropine sulfte (1 nmol in one niml nd nmol in two nimls). Neither gonists nor ntgonist ffeted the sl CCA lood flow or the M. Verifition of injetion site Figure 7 shows photogrph of the oronl setion of the medull inditing the injeted site in the DFA. This site is loted 6 mm rostrl to the oex, 3.5 mm lterl to the midline nd 3.5 mm ventrl to the floor of the fourth ererl ventrile. British Journl of Phrmology (6)

7 212 Medullry niotini reeptors regulte rotid flow C-L Gong et l Blood Flow Inrese ( of sl level) A-BT nmol pmol M A-BT, 8. pmol 5 nmol Blood Flow Inrese ( of sl level) Me d nmol nmol d M Me, 4. nmol 3 se 5 nmol Figure 6 Intertions of holine with -ungrotoxin, 7 nachr ntgonist (n ¼ 4) (, ), nd memylmine, 34 nachr ntgonist (n ¼ 4) (, d), in the DFA. () Miroinjetion of holine resulted in n inrese of the CCA lood flow. The flow inrese ws dose-dependently redued y -ungrotoxin. () Miroinjetions of the sme dose of holine in other group () produed n essentilly similr inrese of the CCA lood flow. This flow inrese ws redued y memylmine. (, ) Sttistil nlysis; (, d) originl experimentl trings nd miroinjetion sites. The dots on the drwing medull setions indite the injeted loi. Dt re expressed s mens7s.e.m. nd nlyzed y Student s t-test. Po.1 vs nmol; Po.1 vs A-BT 2. pmol; Po.1 vs A-BT 4. pmol; Po.5 vs Me 1. nmol; d Po.5 vs Me 2. nmol. 3 mm Disussion In the present experiment, we ssessed the effets of vrious niotini nd musrini gonists nd ntgonists on the DFA in regultion of the CCA lood flow. The results suggest for the first time tht three funtionl suunits of nachrs, nmely, 7, 42 nd 34 suunits, on the neurons in the DFA prtiipte in regultion of the CCA lood flow. On the other hnd, musrini reeptors, if ny, on the DFA neurons do not pper to e involved in regultion of the CCA lood flow. The presene of nachrs on the DFA neurons (or synpti terminls) ws suggested from the present finding tht miroinjetions into the DFA of vrious doses of niotine, non-seletive nachr gonist tht stimultes vrious suunits of nachrs (Chvez-Norieg et l., 1997; Si nd Lee, 2; Amtge et l., 4), resulted in dose-dependent inrese in the CCA lood flow (Figure 2 nd d). This inrese ws dose-dependently inhiited y different niotini reeptor ntgonists, A-BT, Met, Me nd DB (Figures 4 nd 5). The presene of 7-nAChR on the DFA neuron ws suggested sed on the following findings. First, holine, preursor of etylholine nd metoli produt of etylholine, ts s reltively seletive gonist for 7- ontining nachr (Alkondon et l., 1997; Si nd Lee, 2). Miroinjetions of vrious doses of holine in the DFA resulted in dose-dependent inrese of CCA lood flow (Figure 2 nd ). Seond, A-BT (CChelin nd Rust, 1995; Lopez et l., 1998; Ferreir et l.,, 1; Si nd Lee, 2) nd Met (Alkondon et l., 1997; Si nd Lee, 2), seletive ntgonists for nachrs ontining 7 suunit, dose-dependently suppressed the inrese in the CCA lood flow indued y niotine (Figure 4) nd holine (Figure 6 nd ). The presene of 34- nd 42-nAChRs on the neurons of the DFA ws evident from our findings tht pretretments in the DFA of vrious doses of either Me (Figure 5) or DB (Figure 5) ttenuted the niotine-indued inrese in the CCA lood flow in dose-dependent mnner. Me nd DB re prtil ntgonists for nachrs ontining 34 nd 42 suunits, respetively (Alkondon et l., 1997; Wester et l., British Journl of Phrmology (6)

8 Medullry niotini reeptors regulte rotid flow C-L Gong et l 213 Figure 7 A photogrph of oronl medull setion, 6 mm rostrl to the oex, 3.5 mm lterl to the midline nd 3.5 mm ventrl to the dorsl surfe of the medull, indites miroinjetion site (indited y n rrow) in the DFA. 1999). They, however, hve een onsidered s unseletive nachr ntgonists tht my t t nachrs suunits other thn 34 nd 42 in humn (Chvez-Norieg et l., 1997; Amtge et l., 4; Si nd Lee, 2). Therefore, the present findings do not prelude the presene of nachrs suunits other thn 34 nd 42. line, seletive 7 nachr gonist (Alkondon et l., 1997; Si nd Lee, 2), ppers to t s prtil gonist t 34-nAChRs on PC12 ells (Alkondon et l., 1997). In onert with the finding, the present experiment demonstrted tht Me, prtil 34-nAChR ntgonist (Alkondon et l., 1997; Wester et l., 1999), ttenuted the holine-indued inrese in the CCA lood flow (Figure 6), inditing tht Me lso prtilly loked 7-nAChR. The order of preferentil trgets for Me in humn ppers to e 44B24422B42B7 (Chvez-Norieg et l., 1997; Amtge et l., 4). line, however, did not t s n gonist on 42-nAChR, s DB did not ffet the holineindued inrese in the CCA lood flow (dt not shown), supporting the finding tht holine did not tivte 42- nachrs on hippompl neurons (Alkondon et l., 1997). The DFA gives rise to xons tht ontriute to the prsymptheti pregnglioni fiers of the seventh nd ninth rnil nerves (Chyi et l., 1995, 5). Through these nerves, glutmte stimultion of the DFA inreses the CCA lood flow (Kuo et l., 1987, 1992, 1995; Chyi et l., 1995, 5) vi AMPA nd NMDA reeptors on the neurons in the DFA (Gong et l., 2). The relese of serotonin (5-HT) in the DFA, ting through 5-HT2 reeptor, suppresses the relese of glutmte in the DFA (Li et l., 1996). The present experiment for the first time demonstrted tht tivtion of 7-, 42- nd 34-nAChRs in the DFA inresed the CCA lood flow. It is not yet known whether these nachrs re modulted y glutmtergi nd serotonergi reeptors in the DFA. Nevertheless, niotini tivtion to the DFA my likely medite through the seventh nd ninth prsymptheti nerves to inrese the CCA lood flow s glutmtergi nd serotonergi tivtions. letril or glutmte stimultion of the DFA indues not only the inrese in the CCA lood flow ut lso the inrese in the ererl lood flow (Kuo et l., 1995). tmte stimultion of the prsymptheti ererovsodiltor enter in rts (Nki et l., 1993), n re likely equivlent to the DFA in ts (Kuo et l., 1987, 1992, 1995, 1999; Chyi et l., 1995, 5; Li et l., 1996; Gong et l., 2), inreses ortil lood flow. Whether the ererl or ortil lood flow is lso regulted y niotini tion in the DFA deserves further investigtion. Musrini reeptors my not e present on the DFA neurons to regulte CCA lood flow. This ws evident from the present finding tht miroinjetions of musrini reeptor gonists (musrine nd methholine) nd ntgonist (tropine) did not ffet the sl or the niotineinresed CCA lood flow. Musrini reeptors, if ny, on the DFA neurons re not likely involved in regultion of the CCA lood flow. In summry, we demonstrte for the first time tht the neurons in the DFA ontin t lest three funtionl suunits of nachrs, nmely, 7, 42 nd 34-nAChRs. Ativtion of these reeptors results in inresed CCA lood flow. Nevertheless, the present findings do not prelude the presene of nachrs suunits other thn 7, 34 nd 42. On the other hnd, musrini reeptors, if ny, in the DFA re not likely involved in regultion of the CCA lood flow. Bsed on our previous (Li et l., 1996; Kuo et l., 1999; Gong et l., 2) nd the present findings, glutmte, serotonin nd nachrs re present in the DFA nd my ply roles in regultion of the CCA nd possily the ererl lood flows (Kuo et l., 1995). Aknowledgements This work ws supported y grnts from the Ntionl Siene Counil (NSC92-23-B-3-17 to JSK nd NSC92WFD27 42 to TJFL) nd Tihung Veterns Generl Hospitl (TCVGH D to JSK), Tiwn. Conflit of interest The uthors stte no onflit of interest. Referenes Aerger K, Chitrvnshi VC, Spru HN (1). Crdiovsulr responses to miroinjetion into the udl ventrolterl medull of the rt. Brin Res 892: Alkondon M, Aluquerque X (2). A non-lph7 niotini etylholine reeptor modultes exittory input to hippompl CA1 interneurons. J Neurosi 87: Alkondon M, Pereir F, Bros CT, Aluquerque X (1997). Neuronl niotini etylholine reeptor tivtion modultes gmm-minoutyri id relese from CA1 neurons of rt hippompl slies. J Phrmol xp Ther 283: British Journl of Phrmology (6)

9 214 Medullry niotini reeptors regulte rotid flow C-L Gong et l Amtge F, Neugheuer B, MIntosh JM, Freimn T, Zentner J, Feuerstein TJ et l. (4). Chrteriztion of niotine reeptors induing nordrenline relese nd sene of niotini utoreeptors in humn neoortex. Brin Res Bull 62: Chelin AB, Rust G (1995). Bet suunits o-determine the sensitivity of rt neuronl niotine reeptors to ntgonists. Pflugers Arh ur J Physiol 429: Co YJ, Surowy CS, Puttfrken PS (5). otini etylholine reeptor-medited [ 3 H] dopmine relese from hippompus. J Phrmol xp Ther 312: Chvez-Norieg L, Cron JH, Wshurn MS, Urruti A, lliott KJ, Johnson C (1997). Phrmologil hrteriztion of reominnt humn neurl niotini etylholine reeptors h lph 2 et 2, h lph 2 et 4, h lph 3 et 2, h lph 3 et 4, h lph 4 et 2, h lph 4 et 4, h lph 7 expressed in xenopus ooytes. J Phrmol xp Ther 2: Chyi T, Cheng V, Chi CY, Kuo JS (1995). Vsodilttion produed y stimultion of pvoellulr retiulr formtion in the medull of nesthetised-deererted ts. J Auton Nerv Syst 56: Chyi T, Wng SD, Gong CL, Lin SZ, Cheng V, Kuo JS (5). Pregnglioni neurons of the sphenopltine gngli reside in the dorsl fil re of the medull in ts. Chin J Physiol 48: 31. Colquhoun LM, Ptrik JW (1997). Phrmology of neuronl niotini etylholine reeptor sutypes. Adv Phrmol 39: Deker MV, Brioni JD, Bnnon AW, Arneri SP (1995). Diversity of neuronl niotini etylholine reeptors: lessons from ehvior nd implitions for CNS therpeutis. Life Si 56: Dhr S, Ngy F, MIntosh JM, Spru HN (). Reeptor sutypes mediting depressor responses to miroinjetions of niotine into the medil nts of the rt. Am J Physiol 279: R132 R1. Du C, Role LW (1). Differentil modultion of niotini etylholine reeptor sutypes nd synpti trnsmission in hik symptheti gngli y PG (2). J Neurophysiol 85: Ferguson DG, Hxhiu MA, To AJ, rokwu B, Dreshj IA (). The lph3 sutype of the niotini etylholine reeptor is expressed in irwy-relted neurons of the nuleus trtus solitrius, ut is not essentil for reflex ronhoonstrition in ferrets. Neurosi Lett 287: Ferreir M, ert SN, Perry DC, Ysud RP, Bker CM (1). videne of funtionl lph7-neuronl niotini reeptor sutype loted on motor neurons of the dorsl motor nuleus of the vgus. J Phrmol xp Ther 296: Ferreir M, Singh A, Drethen KL, Kellr KJ, Gill RA (). Brinstem niotini reeptor sutypes tht influene intrgstri nd rteril lood pressures. J Phrmol xp Ther 294: Gioomo LM, Hsselmo M (5). otini modultion of glutmtergi synpti trnsmission in region CA3 of the hippompus. ur J Neurosi 22: Gong CL, Lin NN, Kuo JS (2). tmtergi nd serotonergi mehnisms in the dorsl fil re for ommon rotid rtery lood flow ontrol in the t. Auton Neurosi 11: Htton GI, Yng QZ (2). Synpti potentils medited y lph 7 niotini etylholine reeptors in supropti nuleus. J Neurosi 22: Hott H, Uhid S, Kgitni F (2). ffets of stimulting the nuleus slis of Meynert on lood flow nd delyed neuronl deth following trnsient ishemi in the rt ererl ortex. Jpn J Physiol 52: Hungfu D, Shreihofer M, Guyenet PG (1997). ffet of holinergi gonists on ulospinl C1 neurons in rts. Am J Physiol 272: R249 R258. Ito C, Fukud A, Nekur J, Oomur Y (1989). Aetylholine uses niotini depolriztion in rt dorsl motor nuleus of the vgus, in vivo. Brin Res 53: Kiser S, Wonnott S (). Alph-ungrotoxin-sensitive niotini reeptors indiretly modulte [(3)H]dopmine relese in rt stritl slies vi glutmte relese. Mol Phrmol 58: Kiyosw A, Ktsuryshi S, Akike N, Png ZP, Akike N (1). otine filittes glyine relese in the rt spinl dorsl horn. J Physiol 536: Kuo T, Hgiwr Y, ndo S, Fukumori R (2). Ativtion of hypothlmi ngiotensin reeptors produes pressor responses vi holinergi inputs to the rostrl ventrolterl medull in normotensive nd hypertensive rts. Brin Res 953: Kuo T, Hgiwr Y, Sekiy D, Chi S, Fukumori R (). linergi inputs to rostrl ventrolterl medull pressor neurons from hypothlmus. Brin Res Bull 53: Kuo JS, Chyi T, Cheng V, Wng JY (1992). Immunoytohemil hrteristis of dorsl fil re of the medull in ts. So Neurosi Astr 22: Kuo JS, Chyi T, Yng MCM, Chi CY (1995). Chnges in intr- nd extr rnil tissue lood flow upon stimultion of retiulr re dorsl to the fil nuleus in ts. Clin xp Phrmol Physiol 22: Kuo JS, Li HT, Lin NN, Yng CS, Cheng FC (1999). Dorsl fil re of t medull: 5-HT2 tion on glutmte relese in regulting ommon rotid lood flow. Neurosi Lett 266: Kuo JS, Wng MR, Liu RH, Yu CY, Ching BN, Chi CY (1987). Redution of ommon rotid resistne upon stimultion of n re dorsl to the fil nuleus of ts. Brin Res 417: Li HT, Chen WY, Liu L, Yng CS, Cheng FC, Chi CY et l. (1996). The dorsl fil re of the medull in ts: inhiitory tion of serotonin on glutmte relese in regulting ommon rotid lood flow. Neurosi Lett 21: Lopez MG, Montiel C, Herrero CJ, Gri-Plomero, Myorgs I, Hernndez-Guijo JM et l. (1998). Unmsking the funtions of the hromffin ell lph7 niotini reeptor y using short pulses of etylholine nd seletive lokers. Pro Ntl Ad Si USA 95: Nki M, Tmki K, Ogt J, Mtsui Y, Med M (1993). Prsymptheti ererovsodiltor enter of the fil nerve. Cir Res 72: Reynolds DJ, Lowenstein PR, Moormn JM, Grhme-Smith DG, Leslie RA (1994). videne for holinergi vgl fferents nd vgl presynpti M1 reeptors in the ferret. Neurohem Int 25: Sto A, Sto Y (1995). linergi neurl regultion of regionl ererl lood flow. Alzheimer Dis Asso Disord 9: Sto A, Sto Y, Uhid S (1). Regultion of regionl ererl lood flow y holinergi fiers originting in the sl forerin. Int J Dev Neurosi 19: Si ML, Lee TJF (2). Alph7-nAChRs on ererl perivsulr symptheti nerves medite holine-indued nitrergi neurogeni vsodiltion. Cir Res 91: Wester JC, Frnis MM, Porter JK, Roinson G, Stokes C, Horenstein B et l. (1999). Antgonist tivities of memylmine nd niotine show reiprol dependene on et suunit sequene in the seond trnsmemrne domin. Br J Phrmol 127: Zhng W, dvinsson L, Lee TJF (1998). Mehnism of niotineindued relxtion in the porine silr rtery. J Phrmol xp Ther 284: Zhou FM, Wilson CJ, Dni JA (2). linergi interneuron hrteristis nd niotini properties in the stritum. J Neuroiol 53: British Journl of Phrmology (6)