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GABA在摄食和味觉机制中的作用
雷琦;闫剑群;施京红;杨雪娟
【期刊名称】《世界华人消化杂志》
【年(卷),期】2006(14)19
【摘要】γ-氨基丁酸(γ-aminobutyric acid,GABA)是哺乳动物中广泛分布的一种抑制性神经递质,GABA及其受体在下丘脑、杏仁核、孤束核等摄食和味觉中枢均有分布．GABA具有A、B和C三种受体,其中A和B受体参与对摄食行为和味觉感知．在不同脑区应用GABA选择性受体拮抗剂可不同程度的促进或抑制摄食,并对味觉的喜好和厌恶发生改变．另外,GABA与调节摄食和味觉的有关物质具有相互作用,他们共同参与摄食和味觉的调控．本文就GABA在摄食和味觉感受与调制中的研究进展进行了回顾．
【总页数】6页(P1906-1911)
【关键词】γ-氨基丁酸(GABA);传导通路;摄食;味觉
【作者】雷琦;闫剑群;施京红;杨雪娟
【作者单位】西安交通大学医学院生理与病理生理学系
【正文语种】中文
【中图分类】R333;R338
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γ-氨基丁酸A受体——抑制性神经递质GABA受体的A亚型一、受体的含义：GABAA受体，又称作γ-氨基丁酸A型受体，是一种离子型受体，而且是一类配体门控型离子通道，此通道的内源性配体是一种被称为GABA的神经递质。
它可使神经元膜超极化，并抑制神经元的兴奋性。
GABAA受体是一种递质调控的Cl-通道，由α、β、γ-和δ等多种亚单位以不同的组合组成；但是天然存在的GABAA受体则可能是由α、β和γ亚单位组成的杂合五聚体。
GABAA受体可被GABA快速地活化﹐从而直接激活内禀的阴离子通道﹐引起Cl-内流；此种作用可被比枯枯灵（bicuculline）所阻断。
二、亚单位的组成：受体的亚单位,它迄今已由cDNA文库中克隆到19个有关哺乳动物GABAA们都是由不同的基因编码的。
这19个亚单位是6α，4β，3γ，1δ，1ε，1π，和3ρ；并据此分为7个序列组(sequence groups)，即：α1-α6，β1-β4，γ1-γ3，δ，ε，π，ρ1-ρ3。
其中α1-亚单位是其中的主要组分，此已由用['H]-flunitrazepatm(氟硝西泮)所做的亲和标记所证实﹐其中最主要的氨基酸残基是His101;而γ-亚单位则是BZ对通道的功能调制所必需的。
三、受体的药理学：GABA受体可被GABA及其类似物所活化,后者包括菌类的天然产物蝇蕈醇A(musci-mol〉和合成的类似物如THIP (4，5，6，7-受体与GABA等激动tetrahydrydroisoxazolopyridin-3-ol）)等。
当GABAA剂相互作用后，即可调节其内禀离子通道的开启和闭合，由此介导相应的生受体还具有BZ、巴比妥和印防己毒物效应。
除GABA及其类似物外， GABAA素(picrotoxin)等的结合部位，并因此对它的功能产生调节作用。
GABA和受体的激动剂，但两者的作用部位和性质却不相同。
BZ均可视为GABAA早期进行的实验表明，GABA浓度反应曲线呈“S”形，其Hill系数约为2，提示至少要有两分子的GABA与受体结合，方能将天然的受体通道活化。
敌敌畏的化学原理
敌敌畏（DDT）是一种有机化合物，其化学结构为氯苯基二氯乙烷。
敌敌畏的化学原理基于其独特的结构和性质，使其在农业和公共卫生领域具有杀虫和杀菌作用。
敌敌畏的杀虫作用主要通过影响昆虫的神经系统产生。
敌敌畏能够干扰昆虫神经细胞的正常传递信号，导致昆虫神经毒性和麻痹，最终引起昆虫的死亡。
敌敌畏主要通过影响昆虫神经细胞上的钠通道和钙通道来发挥杀虫作用。
具体来说，敌敌畏能够阻断钠通道上的钠离子进出，导致神经细胞无法正常产生和传递信号。
这会导致昆虫的肌肉无法收缩和放松，最终导致瘫痪和死亡。
此外，敌敌畏还可以抑制昆虫神经细胞上的γ-氨基丁酸（GABA）受体通道的正常功能，GABA是一种神经递质，调节昆虫神经细胞的兴奋性。
通过抑制GABA受体通道，敌敌畏增加了昆虫神经细胞的兴奋性，导致异常兴奋和神经毒性。
此外，敌敌畏还具有杀菌作用。
它可以通过与水中的无机磷结合，形成稳定的结合物，抑制磷酸酶活性，从而阻断细菌和真菌的细胞壁合成和代谢，进而杀死它们。
需要注意的是，敌敌畏虽然具有很高的杀虫效果，但也具有较高的毒性和持久性。
长期和大量使用敌敌畏可能会对环境和生态系统造成负面影响，因此它已经被禁
用或严格限制使用。
γ-氨基丁酸GABAγ-氨基丁酸的发现已有近100 年的历史，在控制神经兴奋性与信息加工，神经可塑性与网络同步化等方面起到相当重要的作用。
在神经系统的发育过程中，GABA可能是最主要的兴奋性递质。
γ-氨基丁酸在中枢神经系统CNS中分布较广，几乎在所有区域充当抑制性递质。
一. γ-氨基丁酸受体及其调节机制GABA通过其受体发挥作用，GABA受体可分为三类：GABA A、GABA B、GABA C。
(一) GABA A受体1. 受体的结构GABA A受体是CNS 中分布最为广泛的GABA 受体。
近年来应用分子生物学方法，已克隆到15 种以上GABA A受体的亚单位(如α1-6、β1-3、γ1-3、δ、ε、π、θ等)。
2. 受体的作用GABA A受体是一种配体门控离子通道受体，与Cl通道偶联，受体激活时打开Cl通道，Cl-流动方向取决于细胞内外Cl-的浓度。
GABA受体在爪蟾卵细胞中已经成功表达。
果蝇GABA受体的定点突变产生的抗药性。
GABA受体并测得抗性相关的突变点（丙氨酸到丝氨酸）的第二跨膜域的功能性，第二跨膜域这个区域被认为是氯离子通道的附着点。
这种突变在所有的果蝇种群中都有发现存在。
在定义产生抗药性的变异，我们对整个开放的有抗性RNA等位基因上的Rdl R-MD阅读框进行扩增和测序。
（图1）在序列中，两个氨基酸的序列己检测的被替换一个是丙氨酸变成丝氨酸的302号位（Ala302到Ser）和另一个361号位上的甲硫氨酸变成异亮氨酸。
染色体组DNA上的等位基因上的适当的外显子进行测序，只有丝氨酸代替丙氨酸被发现与PTX或环戊二烯杀虫剂的抗性密切相关。
这种突变时位于第2个跨膜区域M2附近。
研究发现，PKA对GABA A受体β亚单位附近的磷酸化调节PKA的激活能够提高或减低神经元GABA A受体的功能，快速突触抑制的主要位点。
PKA 诱导的磷酸化在β亚单位受体的临近的保守位点引起不同的调节。
包含β3亚基的受体在408丝氨酸和409丝氨酸磷酸化增强了GABA活性反应，然而选择性突变408丝氨酸到丙氨酸是的强化变成抑制，和β1亚基对比，磷酸化只在409丝氨酸上。
药理学（药）形考任务二试题正文现在临床上最常用的镇静催眠药是（）。
A.巴比妥类B.苯二氮䓬类C.吩噻嗪类D.水合氯醛E.丁酰苯正确答案是：苯二氮䓬类试题正文苯二氮䓬类药发挥药理作用主要通过（）。
A.糖皮质激素B.前列腺素C.γ－氨基丁酸D.乙酰胆碱E.白三烯正确答案是：γ－氨基丁酸试题正文GABA与GABAA受体结合后使下列离子中（）的内流增加。
A.Mg2＋B.K＋C.Ca2＋D.Na＋E.Cl－正确答案是：Cl－试题正文下列苯二氮䓬类药口服后代谢最快、作用最强的是（）。
A.地西泮B.奥沙西泮C.三唑仑D.劳拉西泮E.氟西泮正确答案是：三唑仑试题正文苯二氮䓬类受体的分布应与中枢神经递质（）的分布一致。
A.去甲肾上腺素B.γ－氨基丁酸C.乙酰胆碱D.脑啡肽E.多巴胺正确答案是：γ－氨基丁酸试题正文麻醉前给予地西泮的理由错误的是（）。
A.减少呼吸道分泌物B.减少麻醉药用量C.镇静作用D.术后短时遗忘E.肌肉松弛正确答案是：减少呼吸道分泌物试题正文地西泮的药理作用不包括（）。
A.抗焦虑B.抗癫痫C.镇静催眠D.抗震颤麻痹E.抗惊厥正确答案是：抗震颤麻痹试题正文丁螺环酮具有的药理作用是（）。
A.中枢肌松作用B.抗惊厥作用C.抗焦虑作用D.催眠作用E.抗癫痫作用正确答案是：抗焦虑作用试题正文唑吡坦明显优于地西泮之处是（）。
A.催眠时间长B.延长深睡眠C.抗焦虑作用强D.同时抗抑郁E.可长期应用正确答案是：延长深睡眠试题正文扎来普隆最适用于（）类失眠患者。
A.入睡困难B.早醒C.伴有抑郁症D.中间易醒E.伴有恐惧症正确答案是：入睡困难试题正文对氯丙嗪的论述错误的是（）。
A.可抑制糖皮质激素的分泌B.可加强苯二氮卓类药物的催眠作用C.可阻断脑内多巴胺受体D.可使正常人体温下降E.对刺激前庭引起的呕吐有效正确答案是：对刺激前庭引起的呕吐有效试题正文氯丙嗪抗精神分裂症的作用机制是（）。
A.阻断中枢α肾上腺素受体B.阻断中枢5-HT受体C.阻断中枢DA受体D.阻断中枢M胆碱受体E.阻断中枢GABA受体正确答案是：阻断中枢DA受体试题正文氯丙嗪临床不用于（）。
Cell Tissue Res (1998)294:161±168 Springer-Verlag 1998REGULAR ARTICLEMark J.Snyder ´Rik Van AntwerpenEvidence for a diazepam-binding inhibitor (DBI)benzodiazepine receptor-like mechanism in ecdysteroidogenesis by the insect prothoracic glandReceived:6March 1998/Accepted:6May 1998This work was supported by USDA grant 93±37302±9595and by a NIH Postdoctoral Fellowship through the University of Arizona,Center for Insect Science (to M.J.S.)and by NIH grant GM50551(to R.V.A.).M.J.Snyder ())Bodega Marine Laboratory,P.O.Box 247,Bodega Bay,CA 94923,USAFax:+1±707±875±2089;e-mail:mjsnyder@ M.J.Snyder ´R.Van AntwerpenCenter for Insect Science,University of Arizona,Tucson,Ariz.,USAR.Van AntwerpenDepartment of Biochemistry,University of Arizona,Tucson,Ariz.,USAAbstract The diazepam-binding inhibitor (DBI)is a 10-kDa highly evolutionarily conserved multifunctional pro-tein.In mammals,one of DBI's functions is in the activa-tion of steroid hormone biosynthesis via binding to a spe-cific outer mitochondrial membrane receptor (benzodiaz-epine receptor,BZD)and promoting cholesterol transport to the inner membrane.In this work,a multitiered ap-proach was utilized to study the role of this receptor-like activity in ecdysteroidogenesis by larval insect prothorac-ic glands (PGs).First,both DBI protein and messenger RNA (mRNA)levels were correlated with peak PG ecdy-steroid production.In vitro ecdysteroid production was stimulated by the diazepam analogue FGIN 1-27and in-hibited anti-DBI antibodies.The DBI protein was found distributed throughout PG cells,including regions of dense mitochondria,supposed subcellular sites of ecdy-steroid synthesis.Finally,a potential mitochondrial BZD receptor in PG cells was demonstrated by photoaf-finity labeling.These results suggest an important role for the insect DBI in the stimulation of steroidogenesis by prothoracic glands and indicate that a pathway for cho-lesterol mobilization leading to the production of steroid hormones appears to be conserved between arthropods and mammals.Key words Diazepam-binding inhibitor (DBI)´Benzodiazepine receptor (BZD)´Prothoracic gland (PG)´Manduca sexta (Insecta)IntroductionIn mammalian brain,an endogenous protein ligand was initially discovered with the ability to displace benzodiaz-epine analogs from the GABA A receptor (Guidotti et al.1983).The protein was termed the diazepam-binding in-hibitor (DBI)and has since been found in non-vertebrates such as yeast,insects,and plants (Rose et al.1992;Snyder and Feyereisen 1993;Kolmer et al.1994;Genbank T04081).All known DBI proteins are 9±10kDa in mole-cular weight,consist of 86±104residues,and share se-quence identities of 50%or more.The highly conserved sequence between phylogenetic groups is likely to indicate common functions for DBI.In addition to the previously mentioned nervous system function as a regulator of GABA receptor activity,DBI proteins have several other demonstrated functions ger-mane to this work.The first relates to the exact identity of DBI with another protein,the acyl-CoA-binding protein (ACBP),so named for its ability to specifically bind medi-um to long-chain acyl CoA fatty acid esters (Mikkelsen et al.1987;Rosendal et al.1993).The second function con-cerns its role in promoting steroidogenesis via a specific outer mitochondrial membrane receptor (BZD or PBR,pe-ripheral benzodiazepine receptor).Once DBI,its process-ing products,or benzodiazepine analogs bind to BZD,an increase in cholesterol movement to the inner membrane leads to pregnenolone formation by the action of the cyto-chrome P450side-chain cleavage enzyme (Papadopoulos et al.1991;Whitehouse 1992;Costa et al.1995).Although vertebrate-type steroids are found in arthro-pods,the major steroid hormones involved in molting and reproduction are the polyhydroxylated ecdysteroids.Some of the possible steroid intermediates in the pathway leading from cholesterol to ecdysteroids have been iden-tified,but many of the biosynthetic steps are not yet162known(Grieneisen1994).Likewise,proteins involved in the biosynthetic pathway are less known in arthropod ste-roidogenic glands such as the prothoracic glands(PGs)of insects as compared to such pathways in mammalian tis-sues(Jefcoate et al.1992;Smith1995).We have previously demonstrated DBI mRNA expres-sion in insect(Manduca sexta)midgut,fat body,adult ovary and testis,and larval prothoracic glands tissues (Snyder and Feyereisen1993).This study extends these early findings and presents information supporting a role for a DBI/BZD-like pathway involved in ecdysteroid-ogensis by larval M.sexta PGs.Materials and methodsTissue expressionM.sexta larvae were reared as previously described(Snyder et al. 1993)and staged according to established methods(Goodman et al.1985).Total RNA,RNAid Kit(BIO101),was prepared from prothoracic glands dissected from pools of10±15larvae collected on days1±9of the fifth instar.For the Northern analyses,3m g of RNA was loaded per lane and blotted onto Gene Screen nylon mem-brane(Du Pont NEN).The blots were hybridized at42 C to the ran-dom-primed,32P-labeled M.sexta DBI cDNA in50%formamide, 5 SSPE,pH7.4,5 Denhardt's solution,and1%SDS.Final wash-ing was at65 C for15min in2 SSPE and2%SDS.The blots were stripped of bound DBI probe and reprobed with random-primed, 32P-labeled Drosophila actin cDNA to insure equal RNA loading. The scanned band intensities of exposed films from each blot were determined on an LKB Ultroscan Laser Densitometer and the result-ing values were corrected for equal loading by comparison with ac-tin levels.Western blotting and enzyme-linked immunosorbant assayAnti-DBI antiserum was produced in rabbits by immunization with a M.sexta DBI fusion protein expressed in E.coli according to estab-lished protocols(New England Biolabs).Reactivity of the affinity-purified polyclonal anti-DBI antibody to a single10-kDa protein was confirmed by western blotting of larval PG cytosol(Fig.1,in-sert).PG cytosols Were prepared by differential centrifugation methods following homogenization of individual larval PG pools (10±15PGs per sample)in20mM TRIS-HCl(pH7.2),1mM ED-TA,0.1mM DTT,and fresh1mM PMSF.An enzyme-linked im-munosorbant assay(ELISA)was developed to measure cytosol DBI levels according to the procedure of Kingan(1989)using a pu-rified DBI-fusion protein as standard(Snyder and Van Antwerpen 1997)and purified anti-DBI antibody conjugated to alkaline phos-phatase(anti-DBI-AP)as antibody.Effects of diazepam agonists and anti-DBI antiseraon ecdysteroid productionGrace's medium(GibcoBRL)was adjusted to pH7.2and sterile-fil-tered(0.22m m)before use.PGs were dissected from4-day(gate II larvae),fifth-instar M.sexta.This stage of PG is known to be the most responsive to prothoracicotropic hormone(PTTH)in resulting ecdysteroid production(Ciancio et al.1986;Smith and Pasquarello 1989).Brain extracts were prepared according to Smith and Pas-quarello(1989).Briefly,pupal brain extracts were boiled and then filtered through a Centricon10-spin column,thereby removing the small PTTH.Dose responses of initial brain equivalents(BR)were used until an optimum concentration of0.5brain equivalents was found that gave the highest stimulation of ecdysteroid production (Fig.5).Fidia-Georgetown Institute for the Neurosciences indoleacet-amide derivative obtained from RBI(FGIN1-27)was dissolved in 100%ethanol and preincubated with PGs for2h before the medium was changed and individual glands incubated an additional1h be-fore collection of the medium for ELISA measurement of ecdyste-roid production(Kingan1989).This assay measures ecdysone and 20-hydroxyecdysone with equal affinity,but fails to detect3-dehy-droecdysone.Similar patterns of in vitro total ecdysteroid produc-tion can be measured whether the detected product(s)are ecdy-sone-only or total excreted products including the major ecdyste-roid,3-dehydroecdysone(Keightly et al.1990;Smith1995).All me-dia samples were extracted in100%ethanol,centrifuged to remove precipates,and the resulting supernatant was dried and redissolved in PBS prior to ELISA measurements.Streptolysin O(SPO,from Streptococcus pyogenes)was ob-tained from Sigma.This bacterial toxin forms membrane pores of approximately1.5nm when incubated with cells in vitro(Bhakdi et al.1993).Glands were incubated in0.01±10lysis units per 100m l.For the SPO experiments,glands were preincubated for3h with the indicated SPO concentrations with or without dilutions of anti-DBI-AP conjugated-or AP-conjugated preimmune antiserum (as control for the possible effects of the added AP activity on ecdy-steroid production)in Grace's medium.The formation of pores in PG cells was confirmed after the3-h incubation by the presence of fluorescent Ab only in those glands also exposed to SPO.Contra-lateral glands from each insect served as a paired control for each measurement(analyzed by paired t-tests)as previous work demon-strated equivalent ecdysteroid production by both PGs of individual M.sexta larvae(see Smith1995).After3h,the media were replaced with fresh Grace's,including the same SPO and Ab concentrations for an additional1h when the media were collected for ecdysteroid mesurement by ELISA.ImmunohistochemistryAP was conjugated directly to the purified anti-DBI antibodies ob-tained as described previously(Snyder and Van Antwerpen1997). The resulting anti-DBI-AP was used at a dilution of1:10000in whole-mount immunocytochemistry of last-instar M.sexta larval PGs.Glands were dissected in PBS,fixed for1h in4%paraformal-dehyde in PBS,and washed six times(last wash overnight at4 C)in PBS containing0.25%Triton X-100and0.05%sodium azide (PBST).Glands were blocked for2h at room temperature in PBST containing5%normal goat serum and incubated overnight at4 C in the same solution containing the anti-DBI-AP.Control glands were incubated in the appropriate amount of flow through from anti-DBI-AP absorbed onto the DBI-affinity column prepared as described earlier(Snyder and Van Antwerpen1997).Tissues were stained us-ing nitroblue tetrazolium(NBT)and X-phosphate as substrates in a buffer of100mM TRIS-HCl,pH9.5,100mM NaCl,and50mM MgCl2for10min at room temperature,and stopped in10mM TRIS-HCl,pH8.0,1mM EDTA.Glands were mounted in80% glycerol before photography.Electron microscopyGlands from days2and7of the fifth instar were fixed for1h in2% paraformadehyde in PBS at room temperature,rinsed three times in PBS,and then45min each in0.5M,1M,and2M sucrose in PBS. Glands in2M sucrose were plunge-frozen onto metal studs and stored in liquid nitrogen.Prior to sectioning in a model MT6000XL ultramicrotome with model CR21cryo-attachment(RMC),the fro-zen tissue was transferred to the cryochamber,kept at±90 C,and 100-nm sections cut with a glass knife.Sections were picked up on Formvar/carbon-coated nickel grids in sucrose,placed on a drop of PBS,and then taken through a series as follows:PBS plus0.02M glycine(15min),PBS plus0.5%BSA and0.2%gelatin(PBSG,1h), primary antibody in PBSG(1h),washes in PBSG,secondary goat anti-rabbit antibody-gold(10nm particle size)conjugate(EM GAR G10;Amersham;1h),PBSG and PBS washes,PBS plus1633%glutaraldehyde(15min),distilled water washes,methyl cellu-lose-uranyl acetate on ice(10min),and blotted dry before viewing on a Philips EM420electron microscope.Photoaffinity labeling/fluorographyPGs(80pairs)were dissected and homogenized in20mM sodium phosphate(pH7.2),150mM NaCl,0.25M sucrose,1mM EDTA, and fresh1mM PMSF at4 C,and centrifuged for10min at3000g to pellet cellular debris.The supernatant was centrifuged for30min at14000g and the crude mitochondrial pellet was resuspended in buffer,an aliquot was removed for protein determination(BCA as-say),and the remainder was stored at±80 C.[3H]-PK14105(specific activity)was obtained from Dr.H.E. Laird II,University of beling was performed in24-well culture plates in0.5-ml volumes of the same buffer(minus sucrose and PMSF)containing0.5mg mitochondrial protein,5.0nM[3H]-PK14105with or without2.5m M nonlabeled benzodiazepine com-petitor PK11195.Samples were allowed to mix on ice in the dark for15min before the1-h irradiation on ice3cm below the UV light source(400nm maximum wavelength).At the end of1h,samples were centrifuged at14000g for30min,the pellets resuspended in buffer containing2.5m M nonlabeled PK11195,and reincubated for 30min before recentrifugation to obtain washed mitochondria.This incubation and recentrifugation was repeated a second time to wash out and prevent binding of nonphotolyzed[3H]-PK14105.The washed mitochondrial pellets were redissolved in a small volume of buffer and an aliquot was removed and added to SDS sample buffer.Samples were run on12%separating gels,stained with Coo-massie,destained,treated with En3Hance(Dupont),rinsed in water, dried,and placed on film to obtain the corresponding fluorograms.ResultsCorrelation of DBI levels with ecdysteroidogenesis Figure1(insert)demonstrates that the anti-M.sexta DBI antiserum recognizes a single10-kDa band in larval PGs as previously found for larval midgut(Snyder and Van Antwerpen1997)and fat body and ovary(data not shown).Measurements of DBI in pools of PGs dissected each day of the fifth larval instar show that protein levels peak on day7,the most active time of ecdysteroid pro-duction in vivo(Smith1995).These results were con-firmed by measurements of PG mRNA expression of66164DBI over the same period (Fig.2),indicating that peak DBI expression occurs on day 7of the fifth instar also and is significantly lower during times of low ecdysteroid synthesis.In last-instar larval midgut and mandibular glands,peak DBI protein and mRNA levels are found in days 1±4of active feeding,demonstrating tissue-spe-cific regulation of this gene (Snyder and Van Antwerpen 1997,and unpublished data).In vitro PG ecdysteroidogenesisAssays measuring the effects of added factors were per-formed on isolated PGs from day-4M.sexta larvae,as these are known as most sensitive to factors that stimulate ecdysteroid production in vitro (Ciancio et al.1986;Smith and Pasquarello 1989).Figure 3shows that PG ec-dysteroid production is stimulated 4.5-fold by brain ex-tracts containing the large subunit of PTTH.The addition of the peripheral benzodiazepine (valium)receptor ligand FGIN 1-27(0.1±10m M)provided 1.5-to 3.0-fold stimu-lation of ecdysteroidogenesis,similar to that found for steroidogenesis increases in mammalian tissues (Papa-dopoulos et al.1991).Isolated glands were permeabilized using the pore-forming toxin streptolysin O (SPO)from Staphylococcus aureus (Bhakdi et al.1993).They remained viable,con-tinued to produce ecdysteroids at levels slightly higher than contralateral control glands without SPO,and were permeable to rhodamine-conjugated anti-DBI antisera,as demonstrated by heavy fluorescence seen only in per-meabilized PGs (not shown).It is possible that perme-abilization of glands may promote the secretion of ecdy-steroids that occur through a granule-like exocytosis mechanism (Hanton et al.1993).Coincubation of permea-bilized PGs with anti-DBI antiserum significantly inhibit-ed ecdysteroid production by 40%(P `0.01,paired t -test)versus contralateral permeabilized glands incubated with an equivalent quantity of similarly purified preimmune serum (data not included in Fig.3).Thus antibodies against the M.sexta DBI are able to block at least a por-tion of the total ecdysteroid biosynthetic pathway strongly indicating a role of DBI in ecdysteroidogenesis.Cellular DBI distributionTo determine whether the subcellular localization of DBI is consistent with a role in ecdysteroidogenesis,light-and electron-microscopy studies were conducted.Whole mounts of PGs from days 2and 7of the fifth instar were incubated with anti-M.sexta DBI antiserum to assess any differences in cellular distribution patterns (Fig.4).Stain-ing was always minimal in controls with preabsorbed an-tibody (Fig.4A,C)indicating specificity of the antibody for the DBI protein as shown also by the western blot re-sults above.The whole-mount immunocytochemistry re-sults show a more concentrated pattern of tissue localiza-tion amongst mitochondrial organizing centers in day 7(Fig.4D)but not day 2PGs.Staining in day-2glands was more evenly distributed (Fig.4B).As found in Dro-sophila melanogaster (Kolmer et al.1994),DBI is present in many different M.sexta tissues (Snyder and Van Antwerpen 1997,and unpublished data on ovary,fat body,and mandibular glands).The developmental chang-es in tissue levels have not yet been characterized in other arthropods.The distribution of gold particles in sections of day-7glands shows the DBI protein throughout the PG cell and associated with regions of numerous mitochondria (Fig.5A).The association with mitochondria did not oc-cur in larval midgut (Snyder and Van Antwerpen1997).Fig.4Whole-mount immuno-cytochemistry of 5th-instar PGs collected on days 2(A,B )and 7(C,D ).Glands were fixed for 1h in 2%paraformaldehyde,washed in PBS,incubated with anti-DBI Ab (B,D )or preab-sorbed antibody (A,C ),stained for alkaline phosphatase,and mounted in 60%glycerol for photography.Bars 50m m165Gold particles were negligible in control sections (Fig.5B)and reduced in tissues thought to have lower DBI levels such as muscle from the oral region of the head (Fig.5D).In PG cells,DBI protein was also found in the nucleus (Fig.5C).This correlates with findings in lar-val M.sexta midgut columnar cells (Snyder and Van Ant-werpen 1997)and is explained by the fact that DBI is small enough to pass through nuclear pores (Silver 1991).Photoaffinity labelingThe availability of a BZD photoaffinty label ([3H]-PK 14105;Doble et al.1987)for mammalian receptors al-lowed the search for a similar receptor protein in the in-sect PG.Photoaffinty labeling of last-instar M.sexta lar-val PG mitochondria resulted in the detection of a specif-ically labeled protein of 17kDa (Fig.6).This was the on-ly protein band whose labeling by [3H]-PK 14105was competed for by 500-fold excess of the PBR agonist PK 11195.This result agrees with the expected size of report-ed mammalian BZDs of 17±18kDa (Papadopoulos et al.1997)and represents the first report of a BZD-like protein in aninvertebrate.Fig.5A±D Electron-microscope immunocytochemistry of 5th-in-star PGs collected on day 7.Glands and head cavity muscle tissues were fixed in 2%paraformaldehyde 1h,washed in PBS,infiltrated in 0.5M and 2M sucrose in PBS,frozen in liquid nitrogen in fresh 2M sucrose in PBS,cryosectioned at 150nm at ±80 C,and gold-labeled following incubation with anti-DBI Ab (A,C,D )or preab-sorbed antibody (B ).Arrowheads indicate mitochondria. 43000.Bars 0.5m m (Cy cytoplasm,Nu nucleus,MF muscle fibers)166DiscussionBoth biochemical and ultrastructural evidence reported here indicate that the insect DBI appears to have a func-tion related to steroidogenesis in PGs.The localization of DBI protein in subcellular mitochondrial clusters(Hanton et al.1993)associates the protein with cellular compart-ments thought to be involved in ecdysteroidogenesis. Likewise,levels of both DBI mRNA and DBI protein peak at the time of the highest level of ecdysteroid pro-duction associated with the prepupal molt(Smith1995). The most active benzodiazepine analog in mammalian steroidogenesis(Romeo et al.1992;Costa et al.1995) also stimulates PG ecdysteroid production(Fig.3).Ecdy-steroid synthesis in PGs can also be diminished by the ad-dition of anti-M.sexta DBI antibodies to gland cultures. Finally,a specific BZD-like receptor protein is present in the insect PG(Fig.6)and may have a similar role to the mammalian counterpart in promoting steroidogenesis (Papadopoulos et al.1997).These results strongly indi-cate an evolutionary conservation of proteins involved in steroidogenesis between mammals and arthropods and perhaps the conservation of a BZD-like activation mechanism as well.Increases in size,shape changes,and the development of extensive mitochondrial latices(see also Fig.5),and intercellular bridges and gap junctions define the matura-tion of insect PG steroidogenic function(Hanton et al. 1993;Dai et al.1994).Glands become competent to pro-duce ecdysteroids on day4of the fifth instar,coinciding with an increase in total protein(Warren et al.1988; Smith and Pasquarello1989).Peak ecdysteroid produc-tion occurs on day7of the final instar leading to the prep-ual ecdysis on day9(recently reviewed by Smith1995). Glands are stimulated by PTTH via second messenger pathways,leading to increased cAMP and phosphoryla-tion of a number of proteins,including one of34kDa re-cently identified as ribosomal protein S6(Smith et al. 1986;Song and Gilbert1995).Both RNA and protein synthesis are absolutely required for gland activation (Keightly et al.1990),leading to an increase in the ap-pearence of a number of proteins(Rybczynski and Gilbert 1994).Inhibition of protein synthesis in mammalian ste-roidogenic tissues blocks cholesterol transfer to mito-chondria,possibly by disruption of DBI processing steps (Cavallaro et al.1992).Likewise,the involvement of cy-toskeletal proteins in the PTTH-stimulation of ecdysteroid production was suggested following exposure of PGs to mictotubule inhibitors(Watson et al.1996).Nothing is yet known about arthropod proteins other than DBI, known to have roles in promoting mammalian steroido-genesis,such as sterol-carrier protein,steroidogenesis ac-tivator peptide,and mitochondrial p30proteins(Jefcoate et al.1992).Currently it is not clear exactly how DBI and/or FGIN 1-27function in insect ecdysteroidogenic tissues,but re-cent work in progress(Ackerman-Morris and Smith 1994)reported that valium analogs stimulated PG mito-chondrial metabolism of cholesterol to7-dehydrocholes-terol in vitro,the first committed step of ecdysteroid bio-synthesis in arthropod glands(Grieneisen1994).Perhaps this first step promoting transport of cholesterol to the in-ner mitochondrial membrane by a DBI-receptor-based, BZD receptor-like mechanism is the conserved pathway. In mammals,steroid production in cultured steroidogenic cells can be inhibited by incubation with antisense oligo-nucleotides to DBI(Boujrad et al.1993).Such conclusive evidence for the involvement of DBI in arthropod steroi-dogenesis awaits further study.Our assay detects primarily ecdysone produced by iso-lated PGs(Kingan1989),though the major gland product is3-dehydroecdysone(Smith1995).Levels of PG ecdy-sone production are known to be effective indicators of glandular ecdysteroidogenesis(Smith and Pasquarello 1989).However,the stimulation of PGs by benzodiaze-pines such as FGIN1-27,may lead to the production of other unknown products not dectected by these methods. These potential problems may be solved once the entire biosynthetic pathway of ecdysteroid production has been identified(Grieneisen1994).Where DBI,and/or benzodi-azepines,may act if not to stimulate cholesterol move-ment into mitochondria is unclear.More information to answer these questions may come from future studies to identify the insect mitochondrial DBI receptor. Changes in both mRNA and DBI protein levels in M. sexta PGs over the course of the last larval stage(Figs.1, 2)indicate precise regulation of tissue expression.Simi-larly,M.sexta larval midgut exhibits high DBI expression correlated with days1±4of active feeding prior to wan-dering and pupation in the last instar(Snyder and Van An-twerpen1997).This high expression is greatly diminished in both larval midgut and mandibular glands(data not shown)upon cessation of feeding in the last instar.The mammalian DBI gene was initially thought to contain fea-tures labeling DBI as a typical housekeeping protein (Mandrup et al.1992).Later studies have found evidence for5©regulatory elements that may explain differences in DBI expression found in specialized tissues such as hepa-tocytes and adrenal gland(Kolmer et al.1993;Mandrup et al.1993).Likewise,the Drosophila DBI gene also con-Fig.6Fluorogram of[3H]-PK 14105photoaffinity-labeled lar-val M.sexta prothoracic gland mitochondrial proteins in the presence(+)or absence(±)of 500-fold excess PK11195.Af-ter labeling,proteins were sep-arated on a12%SDS gel along with molecular weight standards (indicated to the right of the gel) and visualized after autoradiog-raphy.A specificially photola-beled protein of17kDa was identified by this procedure167tains many features characteristic of housekeeping pro-teins with additional tissue-specific regulatory elements (Kolmer et al.1994).The finding of the10-kDa DBI pro-tein in the nucleus of both larval PGs and midgut(Snyder and Van Antwerpen1997)may reflect the fact that nucle-ar pore sizes allow free passage of molecules of less than 30kDa(Silver1991).The presence of a BZD receptor-like protein represents a novel finding outside of the vertebrates.The ability to photoaffinity label an insect BZD receptor will lead to fu-ture immunological studies to determine its tissue local-ization which can vary substantially.For instance the rat BZD receptor has distinct cellular localization patterns within such tissues as the adrenal gland,kidney,testis, and brain,signifying perhaps differential role(s)for this protein in specific tissues(Moynagh et al.1991).In addi-tion BZD mRNA levels are manyfold higher in steroido-genic tissues(Sprengel et al.1989;Parola et al.1991). These studies will form a groundwork to characterize the physiological role of this protein and perhaps give more support to the idea of a DBI/BZD pathway in inver-tebrate steroidogenic processes.In summary,the data reported here demonstrate that the DBI protein,and perhaps a BZD receptor mechanism are involved in the production of insect steroid hormones. These results suggest that particular components of the biochemistry of steroidogenesis are evolutionarily con-served between arthropods and mammals. 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