Gα11: IMMUNOLOCALIZATION IN THE OLFACTORY EPITHELIUM OF THE RAT (RATTUS RATTUS) AND THE CHANNEL CATFISH (ICTALURUS PUNCTATUS)

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NeuroscienceVol. 74,No, 1, pp. 261-273,1996 CopyrightG 1996IBRO. Publishedby ElsevierScienceLtd Printed in Great Britain S0306-4522(96)00115-7 0306-4522/96$15M3+ 0.00

Gctq/Gctl, : IMMUNOLOCALIZATION IN THE OLFACTORY EPITHELIUMS OF THE RAT (RAZ’TUS R42TUS) AND THE CHANNEL CATFISH (IC7’ALURUS PUNCTATUS) C. DELLACORTE,*[[ D. RESTREPO,*T B. PH. M. MENCO,$ I. ANDREINI$ and D. L. KALINOSKI* *Monell Chemical Senses Center, Philadelphia, PA 19104,U.S.A. ~Department of Physiology,School of Medicine, University of Pennsylvania,Philadelphia, PA 19104, U.S.A. $Department of Neurobiology and Physiology, Northwestern University Evanston, IL 60208-3520, U.S.A. $Schoolof Veterinary of Pisa, Pisa, Italy 56124 Abstract-The immunohistochemicallocalizationof Gaq/Ga[l was studiedin the olfactoryand respiratory epitheliumsof two representativevertebrates, the rat and the channel catfish. Localizationin the rat was found at the apical surface of cells in the epitheliumsand within nerve tracts in the lamina propria. Immunostaining of neuronal cilia and supporting cell microvilliwas confirmedby electron microscopy. Immunoreactivity on the ipsilateral neuorepithelium was abolished five weeks following unilateral bulbectomy.An emergenceof patchy immunoreactivitywas found,however,after fifteenweeks.In catfish, Gaq/Gall antigenicity was found at the apical surface of cells within the olfactory epitheliums,at supranuclear regions within some cell bodies and in basal nerve tracts of the olfactory rosette. Immunroeactivitywas removedwith unilateral bulbectomy.Specificlabellingin both rat and catfish was eliminatedby preincubation of the Gaq/Ga,l antibodieswith the cognate peptide. Proteins were extracted from olfactory tissues of both species and solubilized.Using western blotting, bands corresponding in aPParent mokcular weight to a 38,000mol. wt protein were found. These data demonstrate the presence of Gaq/Ga,, in the olfactory tissues of these vertebrates and suggest a role in olfaction for this class of G-protein. Copyright G 1996IBRO. Published by Elsevier Science Ltd. Key words: G-proteins, olfaction, Gaq, Gal,,

immunocytochemistry.

G-proteins, heterotrimers composed of a, /?, and y involvedin a PTX-insensitivecoupling to PLC actisubunits, are important components in hormone and vation3’20catalyzing formation of inositol 1,4,5neurotransrnitter signal transduction in eukaryotic trisphosphate (IP~) and diacylglycerol. Ligandcells. These GTP-binding proteins relay signals with mediated stimulation of PLC by angiotensin, high fidelityfrom transmembrane receptors to intra- bradykinin, vasopressin and thromboxane A2recepcellular effecters such as phospholipase C (PLC), tors via subunits of the G~ class have been adenylyl cyclase and ion channels.7’29’s9 Recently, a described,*3.s3Activation of PLC by rhodopsin in class of G-proteins, G~, which includes Ga~, Gall, squid and stimulation of prolactin secretion in GU14and Gu15/Ga16,has been characterized.49’53’s8 anterior pituitary cells by thyrotropin releasing The u-subunit in members of this class lacks a hormone (TRH) are reportedly coupled by PTXcysteine residue at the extreme C-terminus, a region insensitiveG~ subunits.3’51 A wealth of evidence suggests G-proteins are reportedly involved in both receptor coupling and covalent modification by pertussis toxin (PTX).C involvedin olfactory stimulus-responsecoupling.2’14’w While u-subunits of the G12and G, classes also lack These proteins, as well as others involved in signal only the G~classis reportedly transduction in olfactory receptor neurons (ORNS), the cysteineresidue,49’ss are believedto be localized in cilia that extend from the olfactory knob at the apical end of the ORN. IITowhom correspondence should be addressed. Indeed, putative olfactory receptors,30’31>37 several of Abbreviations: EGTA, ethylene glycol-bis-(aminoethyl the enzymes involved in the second messenger ether)-N,N,N’,N’-tetra-acetate; HEPES, N-2-hydroxan IPJ receptor (IP3R)17’2S and a yethylpiperazine-N’-2-ethane sulphonic acid; IpJ, cascade,4’2e’u’45 inositol-l,4,5-triphosphate; IP3R, IP3 receptor; ORN, cAMP-gated channelUis have been localized to the olfactory receptor neuron; PBS, phosphate-buffered olfactory cilia. Olfactory receptor occupation by saline; PIP2, phosphatidylinositol (4,5)biphosphate; odorants stimulatesG-protein-mediatedformation of PLC, phospholipase C; PTX, pertussis toxin; TME, adenosine 3’5’-monophosphate (cAMP) or IP3 in tris(hydroxymethyl)-aminomethane(Tris) HC1, 3 mM lobster,]ofrog,52’cs MgC12, 2 mM EGTA; TRH, thyrotropin releasing many speciesincluding catfish,24’59 and rat.13’M’s5 hormone. 261

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SeveralG-proteins are known to be associatedwith as confirmed by visual inspection with a dissecting the olfactory neuroepithelium.25,2b,39,a GO~ti, suggested microscope. to mediate olfactory transduction for those odors OIJactory bulbectomy in channel ca@sh that stimulate cAMP production, has been localized For unilateral bulbectomy,channel catfish were immobiThe presence of adenylate lized with gallamine triethiodate (Flaxedyl;Davis & Geck, to olfactory cilia.ZLVS cyclase-coupledG, has also been demonstrated.38,4,45 Pearl River, NY; 0.3mg/kg, intramuscular), and the fish The molecular identity and localization of the G- wereheld in place on a waxblock.The gills,skin and barbels were continuously perfused with aerated “artificial pond protein(s) linked to the PLC pathway in olfaction, water”12and the fish kept anesthetizedduring the course of however, has remained elusive. In other systems, the experiment with 3-aminobenzoic acid ethyl ester, PLC-/3 isozymes are known to be activated with methanesulfonate salt (1:10,000:Sigma Chemical Co., St varying effectivenessby Gu~.67,70 A recent study doc- Louis, MO). The surgicalsite was bathed with sterileteleost Ringer solution (100mM NaCl, 3.OmM KC1, 2.0mM umenting inhibition of odor-inducedinward currents MgC12,1.0mM CaC12,10mM HEPES).57An incision was in cultured lobster olfactory neurons by Ga~/Gall made in the skin adjacent to the posterior naris and the antibodies suggests that members of this family of frontal bone was exposed. Using a high speed micro-drill GTP-binding proteins may be involved in signal held at a 45°angle, the bone was opened and one olfactory bulb ablated. The cavity was filledwith gelfoam,the wound transduction in olfaction.21 laced with Furanace (Aquaria Products, Glen Burnie, In light of the possible functional importance of MD), and the skin sutured in place. The surgical site was the G~ class of proteins in signal transduction covered with Panolog (E. R. Squibb and Sons) to avoid or other functions in the olfactory epitheliums,we infection. The fish were returned to the aquarium and kept sought to use immunocytochemistry and Western at 11°C in 0.25mg/1 solution of Furanace for 48h and to fresh water tanks. The water temperature blotting to determine the presence and location of transferred was raised to 15°C, 15 days post-surgery. Fish were killed GU~/GCqlproteins in the peripheral olfactory neu- with a sharp blow to the head 21 to 26 days after bulbecroepithelium of the rat and the channel catfish. tomy. Visual inspection of the rosettes upon sacrifice An antiserum, selectivefor the C-terminal decapep- showedthe bulbectomizedrosettes were slightlydiminished tide sequencecommon to two membersof this class,c3 in size. Ga~ and Gcql was used to investigate the cellular Protein isolation, electrophoresis and western blotting distribution. Olfactory cilia were isolated in the presence of protease EXPERIMENTAL PROCEDURES Animals

Adult female Spragu&Dawley rats (200-250g) were obtained from Charles River Laboratories (Wilmington, MA) and maintained in a temperature- and humiditycontrolled vivarium. Freshwater channel catfish, Zctalurus punctatus (200 to 1000g) were purchased from local suppliers and maintained at 17°C in 250 gallon aquaria unless otherwise stated. Oljaclory bulbec~omy in rats

Olfactory bulbectomieswere performed in rat according to the procedure described by Schwob et aLc2Briefly,after induction of anesthesia with 0.1rnl/100g body weight of a solution containing 100mg/ml ketamineHC1and 0.7mg/ml acepromazine maleate, an olfactory bulb was exposed and ablated by suction. The cavity was filledwith gelfoam,and the skin was sutured. Care was taken to avoiddamageto the contralateral bulb. After recovery, the animals were returned to the vivariumto be sacrificedby C02 inhalation at five and 15weeksafter surgery. The lesion was complete

inhibitors (10#g/ml leupeptin, 76.8nM aprotinin, 0.7 M pepstatin, 0.83mM benzarnide, 0.23mM phenylmethanesulfonyl fluoride and 1mM iodoacetamide) using a Ca2+ shock procedure as described previously.27,s8The final suspension of cilia was in TME medium [10mM tris(hydroxymethyl)-aminomethane (Tris) HC1, 3mM MgC12,2 mM EGTA],pH 8.2, with protease inhibitors at a final concentration of 200–500mg/ml. Deciliatedtissue preparations were homogenizedwith the aid of a Brinkmann Polytron tissue homogenizerand centrifuged at 30,000x g for 90min at O“C.The supernatant (S1)was decanted and reserved.The pellet was resuspended in TME medium containing protease inhibitors and 1°/0 Triton X-100,homogenizedand centrifugedat 30,000g for 90min at O“C.The solubilizedfraction (S2) was decanted and reserved. Brain tissue removed from rat and channel catfish was placed in TME medium containing protease inhibitors, homogenized,and centrifugedat 800g for 10min at O°Cto removeinsolubledebris.The supernatant was transferred to a new tube and centrifuged at 30,000g for 90min at O°C. The supematant (S1) was reserved. The pellet was resuspended in TME containing protease inhibitors and 1% Triton X-1OO,homogenized, and centrifuged at 30,000g

Fig. 1. Upper left. Immunolocalization of anti-Gcs~/Gal,in a coronal section through the olfactory epitheliumsof the rat. Antigenicityis seen on the apical surface of the olfactory epitheliumsof the nasal septum, and within nerve tracts in the lamina propria. Immunolocalizationis not seen within the cell bodies. Scale bar =20 pm. Fig. 2. Upper right. Immunolocalizationof anti-Ga~/Gall at endoturbinate IIa (Liebich36)in the olfactory epitheliumsof the rat. Apical surfacesof the olfactory epitheliumsand nerve tracts within the lamina propna exhibit immunoreactivity. In addition, labelling is found within supranuclear regionsof somecells.Scalebar = 30pm. Fig. 3. Lowerleft. Anti-Ga~/Gall reaction product on a coronal section through the ipsilateral olfactory epitheliumsof a bulbectomized rat, five weeks post-bulbectomy.Immunolocalizationis eliminated at the apical surface. Diminishedimmunoreactivity is found with nerve tracts of the lamina propria. Scale bar = 20pm. Fig. 4. Lower right. Anti-Gu~/Gall reaction product on a coronal section of the ipsilateral olfactory epitheliumsof a bulbectomized rat, 15 weeks post-bulbectomy. Patchy re-emergence of antigenicity is seen on the apical surface of the olfactory epitheliums.Scale bar = 30~m.

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for 90min at O“C. The solubilized fraction (S2) was decanted and reserved. Proteins were electrophoresedin the presence of sodium dodecyl sulphate on 12.5% gradient gels by the method ofLaemmli33and transferred to nitrocelltdosesheets. Western blotting was performedby the method of Towbinet al.71 In brief, blots were processed through incubations with blocking solution (5% skim milk in phosphate-buffered saline (PBS) with 0.1°4 Tween-20) for 12h at 4“C, immunostained with polyclonal antibodies raised against Gaq/Ga,, (NEN, Wilmington,DE) for 1h at 4°C (1:330), biotinylated anti-rabbit immunoglobulins,and an avidinhorseradish peroxidase complex (Vector Laboratories, Burlingame, CA) according to manufacturer’s directions. Conjugates were visualized using a 4-chloro-l-naphthol/peroxidase substrate system (Kirkegaard & Perry, Gaithersburg, MD). For preabsorption experiments,monospecificpolyclonal Gaq/Gal, antibodies were absorbed with a C-terminal decapeptide (QLNLKEYNLV; QCB Hopkinton, MA) common to the two members of this classb3at. a concentration of 500pg/rnl. The preparation was allowed to incubate at 4°C for 24h with gentle stirring. The supernatant was decanted after centrifugation at 14,000g for 15min at 4°C and was used for Western blotting. Other control procedures includeddeletion of secondary antibodies and substitution of the primary antiserum with non-immune serum.

peptide(QLNLKEYNLv; QCB,Hopkinton, MA) common to the two membersof this class63at concentrationsranging from 50rig/ml to 500pg/ml. The preparation was allowed to incubate at 4°C for 24h with gentle stirring. The supernatant was decanted after centrifugation at 14,000g for 15rnin at 4°C and was used for immunoblotting. Other control procedures included deletion of secondary antibodies and substitution of the primary antiserum with non-immuneserum. Electron microscopic immunocytochemi.rtry

For ukrastructural post-embedding electron immunocytochemistry,specimensof rat tissue were fixed with 4°/0 paraforrnaldehydeby transcardial perfusion.Free aldehydes were quenchedwith O.lOAsodium borohydride/50mM glycine/PBS overnight. Catfish rosettes were removed and placed in 4% paraformaldehyde/O.l?40glutaraldehyde.Tissues were washed and cryoprotected in an ascending series of l@30°/0 glycerol/PBSfor 2 h each.72,73 Samples were plunge-frozen in liquid propane using a Gentleman Jimquick-freeze apparatus in the plunge modess and freezesubstituted in methanol/O.l uranyl acetate.43,5b Specimens were embeddedat —60°Cin LowicrylK1IM,+,75sectioned (150 to 200nm), and incubated with primary antibody at 1:100,1:250and 1:1000dilutionsovernightat 4“C. Binding of monospecificpolyclonal anti-Ga~/Gall anti-body was visualized with goat-anti-rabbit IgG conjugated to 10nm colloidal gold particles (Aunon, Wageningen, The Netherlands) as a secondary probe. Tris-buffered saline Light microscopic immunocytochemistry (0.01M, 500mM NaCl, pH 8.0) supplementedwith 0.1% Tissues, consisting of whole rosette from catfish and the acetylated bovine serum albumin3swas used for blocking, septum with attached turbinates from rat (not decalcified), incubations, dilutions and most washes. For washing after were placed in 40/o paraforrnaldehydein pH 7.4, for 8h at gold incubation, this buffer was supplemented with O.lYO 4“C. Sampleswere rinsed in PBS, cryoprotected by immer- Tween-20 which was used first, followed by washing in sion for 12h each in 10°/0,20°/0and 300/o sucrosein PBSat water. Sections were stained with 0.50/0uranyl acetate in 4°C, and placed in Tissue-Tek(Miles Inc., Elkhart, IN) to 50°76methanol and observed in a JEOL 100CXelectron be frozen in dry ice. Twenty pm (rat) and 8pm (catfish) microscopeat 120kV. sectionsweremade on a Microm 500M Cryostat (ZeissInc., Thornwood, NY) at –20°C and thaw-mounted on subbed Statistics Quantitative evaluations were performed to substantiate slides. Slides were placed in PBS for 20rein, endogenous peroxidases inactivated with ().s~o hydrogen peroxide in the immunolabelledpatterns observed in electron microPBS for 20rein, and rinsed in three successive 15min scopic studies. Counts were made on photographs at magchanges of PBS. Specimenswere incubated for 1h in PBS nifications of 35,000x to 50,000x, using a grid graded in containing 0.3°/0 Triton X-1OOand 10°/0normal serum cmzand the number of particles within a rectangle of 3 to (Vector Laboratories, Burlingame,CA), and placed in pri- 10 squared cm were counted. The number of particles per mary antisera to Gaq/Gall (NEN, Wilmington, DE) at a pm2was then calculated by taking into account the magnifi1:3000dilution for 24h at 4“C.PBSrinseswere followedby cation factor, and the means and standard deviations were incubation in a biotinylated secondary antibody, and a estimated for each region. As noted in the Results, not all preformed avidin/biotinylatedhorseradish peroxidasecom- cilia were labelled. For the counts, all areas with cilia were plex(Vector Laboratories, Burlingame,CA) followingman- included regardless of whether they contained particles or ufacturer’s instructions. Tissues were incubated in a not. In most cases, a minimum of four sections at two chromagen solution composed of equal volumes of O.lO/O differentdilutions, on about 30 micrographs,were counted. hydrogen peroxide and 0.5?40 3,3’-diaminobenzidine A later repeat count, not includedin thesecalculations,gave tetrahydrochloride (DAB, Sigma Chemical Co., St Louis, the samepattern of labelling.Differencesin particle densities among regionswere determinedusing a one-wayanalysisof MO) in PBS. For preabsorption experiments,monospecificpolyclonal variance (ANOVA)and Fisher’s least significantdifference Gaq/Gatl antibodieswere absorbed with a C-terminaldeca- (LSD) tests.”

Fig. 5. Upper left. Horizontal section through the olfactory rosette of the channel catfish (Zctalurus punctatus) immunostainedwith antibody generatedto Ga~/Gall. Antigenicityis seen at the apical surface of cells(arrows)withinsensoryregionsof the rosette, withinsupranuclearregionsof cellbodiesand within basal nerve tracts adjacent to the midline raphe (asterisk). Scale bar= 20#m. Fig. 6. Upper right. Ga~/Gall immunolabellingwithin the sensory regions of the ipsilateral rosette of the channel catfish is lost at the apical surface of cells(arrows)within the sensoryregionsof the rosette and withinsupranuclear regions of cell bodies 21 days after unilateral bulbectomy.Light immunolocalizationis still seen within basal nerve tracts adjacent to the midline raphe (asterisk). Scale bar= 20pm. Fig. 7. Lower left. Preincubation of the Gaq/Gall antibody with peptide. In the rat, the specificbindingof the antibody was abolished at the apical surfaces of cells within the olfactory neuroepithelium using 50pg of cognate peptide. Scale bar= 30pm. Fig. 8. Lower right. Preincubation of the Ga /Gall antibody with peptide. Specificbinding of the Ga,/Ga,, antibody to channel catfish protein was,e!iminatedby preincubation of the antibodies with 50pg of cognate peptide prior to incubation with tissue sections. Scalebar= 30pm.

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normal rate within the ipsilateral margin. The average life-span of these neurons is reduced, however, Immunolabelling in the rat and the majority of mature neurons disappear within The cellular distribution of the a-subunit of two weeks of ablation, Loss of mature ORNS in our Ga~/Gall was determined immunohistochemicallyin experimentswas confirmed by the loss of immunosthe olfactory ,neuroepitheliurn of two phylogeneti- taining with olfactory marker proteina in the ipsilat,cally distant species using an antiserum directed eral epitheliums at the end of five weeks. The hgainst the predicted C-terminal decapeptide com- contralateral epitheliumsretained normal patterns of mon to these two G-proteins.b3Strong reactivity in staining (not shown). Five weeks after unilateral the rat was seen at the apical surfaces of cells along bulbectomy, Gaq/Gal, apical immunoreactivity the septum, the dorsal recess,and the endoturbinates appeared to be abolished on the ipsilateral olfactory within the olfactoryepitheliums.Immunolabellingwas epitheliums(Fig. 3). Unilateral bulbectomy did not also found in nerve tracts within the lamina propria affect the pattern or extent of immunolocalizationat (Fig. 1). The apical surface of cells of the respiratory the apical surface of the respiratory tissue (not epitheliumswas also found to be reactive(not shown). shown). However,patterns ‘ofpatchy antigenicity on Immunolocalization was not observed within the the apical surface of cells within the olfactory epibodies of cells along the olfactory neuroepithelium, theliumswere seen on the ipsilateral side fifteenweeks or within the cellsof the respiratory epitheliumsin the after unilateral bulbectomy (Fig. 4). This pattern rat. Labellingof supranuclear areas of cellswithin the suggests initial loss and subsequent resurgence of endoturbinates was noted (Fig. 2). Control prep- Gaq/Gall antigenicitypossiblyon ORNSand on cells arations in which the Guq/Gall antibodies were other than ORNS. replaced with non-immune serum exhibited no Antigenicity in the channel catjsh staining. To confirm the presence of Gaq/Gctllat the apical We also investigated the presence of Gaq/Gall in surface of mature rat ORNS, we carried out the olfactory neuroepithelium of the channel catfish. immunostaining on tissues from bulbectomizedrats. ORNS in this species are confined to discrete epiAcute and chronic effects of bulbectomy on ORNS thelial regions distributed on each lamella within the Our results showed antihave been described.W’4]@ Following unilateral bul- olfactory rosette.W*SO bectomy, which allows direct comparison of the Gaq/Gal, reaction product was predominantly localnormal and denervated sides of the epitheliums,neu- ized to sensory regions of the lamellae (Fig. 5). rogenesis takes place at a two-fold greater than Antigenicity was also seen at supranuclear regions RESULTS

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. Fig. 9. Electron rnicrograph(left) and schematicrepresentation(right) of anti-Ga~/Gall immunoreactivity within the olfactory epitheliurn of the rat. Immunolocalizationof 10nm goldparticles is seen on the cilium projecting from the olfactory knob (0) of a sensory neuron. Scale bar= 0.70pm. A schematic representation of the photograph shows gold particle localization. Antibody dilution 1:250.

Gaq/Gall in the olfactory epitheliums

within some cell bodies as well as at apical cell surfaces and basally, within the nerve tracts. Unilateral bulbectomy in the catfish resulted in a loss of Gaq/GIX1limrnunostaining within the sensory regions of the rosette on the ipsilateral side (Fig. 6). In catfish, the sensory region of the rosette contains two morphologically distinct, spatially distributed receptor cells: ciliated ORNS and microvillous cells.ls’79ORNS are preferentially distributed in the ventrolateral area

of the sensoryregion of the lamella, whilethe density of the microvillouscellsis greatest in the dorsomedial portion of the sensory zone.15J9Close examination did not revealdifferencesin the spatial distribution of immunolocalization. In a manner similar to rat preparations, when Ga~/GLY1l primary antibodies were omitted no staining was found. Specz&ity of the Gaq/GuIL antibody The specificbinding of the Guq/Gall antibody to rat and channel catfish protein was demonstrated by

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preincubation of the antibodies with peptide prior to incubation with tissue sections from these species. Using 50 pg of cognate peptide, immunoreactivity was abolished at the apical surfaces of the olfactory neuroepithelium and the respiratory tissue in the rat (Fig. 7), and in the sensory lamellae of the catfish (Fig. 8). Electron microscopy

Fine-structural localization was provided by electron microscopicstudies. Examination of the rat and catfish olfactory epitheliums confirmed the pattern and location ofimmunoreactivity. Antibodies to Ga~/Grql displayed the same relative pattern of Iabellingin all cellularcompartments considered.The cilia of ORNS (Fig. 9) and the microvilliof sutmorting cells in the rat (Fig. 10), and the cilia of O-RNS in the catfish (Fig. 11), were labelled with immunogold. Antigenicity was also found in nerve tracts within the lamina propria (not shown).

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Fig. 10.Electron micrograph (left) and schematicrepresentation (right) of anti-Ga~/Gall immunoreactivity within the olfactory epitheliumsof the rat. Gold particle immunolocalizationto anti-Ga~/Gall on the microvilli of a supporting cell (S) within the olfactory epitheliums.Scale bar = 1#m. A schematic representation of the photograph demonstrates the gold particle localization. Antibody dilution 1:100. NSC 74/1—1

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Iabellingin the periphery and dendrites of neuronal cellbodies in the cerebellum,hippocamus,cortex and striatum.qsInterestingly, high molecular weight secondary bands 78,000mol. wt were seen in both the catfish cilia preparation and in the deciliated membrane preparation of catfish olfactory tissue. Experiments in which (i) the primary antibody was deleted, or (ii) the antibodies were preincubated with peptide Western blotting prior to immunoblotting, resulted in loss of both the To characterize the protein(s) recognized by the 38,000and 78,000mol. wt bands, suggestingthat the Gaq/Gall antibodies in the olfactoryepitheliums,reac- high molecular weight protein may share some comtive bands were visualizedby immunoblotting. Elec- mon amino acid sequenceswith the Ga~/Gall protein trophoretic protein separation and identification by in catfish. Western blotting using monospecific polyclonal DISCUSSION G~q/Gall antiserum resulted in the labellingof bands of 38,000mol. wt in preparations of cilia isolated An antiserum selectivefor the C-terminal decapepfrom both the rat and the catfish (Fig. 13). Specific tide sequence common to two members of the class labelling was also seen with membrane homogenates of G~proteins, Ga~ and GaL1,was used to study the prepared from deciliated olfactory tissue of the rat cellular distribution of this protein in the olfactory and channel catfish, and with rat brain where epitheliumsof the rat and the channel catfish. The immunocytochernical localization studies report intense immunocytochemical staining of apical

Although immunoreactivitywas present on cilia, not all cilia present on the same ORN were labelled. Olfactory dendritic knobs showed little labelling. Quantitative evaluations in the rat showedsignificant differencesin the amount of labelling between olfactory cilia and all other regions (Fig. 12; Table 1). VSB2

Fig. 11. Electron micrograph. (Left) Gold particle anti-Gaq/Gall irnmunoreactivityon cilia of ORNS withinthe olfactoryepitheliums of the catfish.Antibodydilution1:1000.Scale bar= 0.07pm. (Right) Immunoreactivityon cross-sectionsof catiish ORN cilia. Antibodydilution 1:1000.Scalebar = 0.03pm.

Gctq/Ga,, in the olfactory epitheliums

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immunolabelling,is suggestiveof an initial decrease of Gaq/Gull protein expression in both ORNS 3.on80brykn0b and supporting cells, followed by supporting cell 4.Mlcrwllli s. R~ciu~ stabilization and re-appearance of Gaq/Gctll 6. Rw@mtorymioIwIlli expression. ~ An2ib0dyfUiut10n l:250 EJ ~~~n~:$oo Results of control experiments support our conclusion that there is expressionof an authentic form of Ga~/Gal[ in the olfactory tissues in these vertebrates. Immunoreactivitywas suppressedwhen the antiserum was preabsorbed with the Gaq/Gallspecific peptide. Support was also found in our Western blot analysis of protein extracts. Antiserum recognizeda band of appropriate molecularweightin CelllRegion of Cell a tissue-specificmanner, corresponding to the distriFig. 12. Bar graph plot of grid counts (mean+ SD) of bution of immunoreactive staining in these prepanti-Ga~/Gall immunogoldlabellingin different regions of arations. The appearance of an additional band of the olfactory epithelimnof the rat at two differentantibody higher molecular mass in extracts of the catfish may dilutions. The counts in olfactory and respiratory cilia were significantlydifferent from the counts in the apex of the reflect cross-reactivityof the antiserum with another supportingcell(seeTable 1).The number of fields(n) scored protein, although all positive signals in the tissue for each region at 1:250 and 1:100dilutions, respectively: sections were blocked by preabsorption of this antiapex of sustentactdar cells: 9, 8; olfactory cilia: 9, 6; serum with G~~/Gall-specific peptide. Antibodies olfactory knob: 9, 7; microvilli: 10, 7; respiratory cilia: 3, directed against a peptide sequence-specific for respiratory microvilli: 1. Gaq/Gall have been used in vertebrates to discriminate between forms of G-proteins expressed by epithelial cell surfacesin these species,compartments specific cell types and to elucidate the function of which in olfactory neurons are known to be involved specific domains of the Ga~ molecule. While we in signal transduction processes, in addition to believe that the immunohistochernical procedures identification of 38,000mol. wt proteins upon used in this paper provide a sensitive means of immunoblotting, are consistent with the presence of detecting Ga~/Gall-related protein in these species, a functional Ga~/Gcql-like protein. Furthermore, the results will require confirmation with analysis of immunolocalization within axon bundles of both Gaq/Gall gene expression in these systems. Based on these observations and on research in species further supported a neuronal localization of Ga~/Gall, although additional glial staining cannot other systems,it would be tempting to suggesta role for Gaq/Gall in the hydrolysisof phosphatidylinosibe ruled out. The marked decrease and subsequent patchy tol (4,5)bisphosphate (PIPZ) by PLC, leading to increase in immunolabelling after olfactory bulbec- increases in IP~ concentrations and activation of tomy in the rat was notable. A wealth of information ciliaryIP3-gatedcation channels,whoseimmunocytodetails changes in the olfactory neuronal population chemical distribution, both at the apical surface and following bulbectomy. While information on the within nervetracts, is comparable to that of Gaq/Gall molecular alterations sustained by sustentacular cells in the rat and catfish olfactory neuroepitheliumk’ during these procedures is limited, some evidence (DellaCorte, unpublished observations). Selective suggests significant changes are also found in odorant stimulation in isolated rat olfactory cilia has the membrane properties of this cell population fol- been found to induce the formation of IP3.9The data, 42me changing reactivity however, indicate mediation by specific PTX-sensitive lowing nerve transection. pattern, loss and subsequent development of patchy G-proteins.g This apparent discrepancy could be ‘E a

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Table 1. Levels of significancefor differencesin immunogoldlabeling for Fig. 11 Microvilli Apex Olfactory Olfactory supporting supporting Respiratory cell cilia knob cell cilia Olfactory knob Apex supporting cell Microvilli supporting cell Respiratory cilia Respiratory microvilli

0.0001” (0.0001)” 0.0001 (0.0001)” O.0001 (0.0001)* 0.0015” 0.0024”

0.8923 (0.7336) 0.2290 (0.1974) 0.0383* 0.7930

0.1812 (0.0764) 0.0309 0.7468

0.1936 0.7883

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*Significancelevel: 50/o;0.0000: 1:250 dilution of primary antibody; (0.0000): 1:100 dilution of primary antibody. P-values for the differencebetweengold particle counts in a number of subcellularregions of cells within the olfactory epitheliumsof the rat. (See Fig. 12 for n values.)

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12

Fig. 13. Western Blot. Lane 1:Molecularweightmarker; Lane 2:catish ciliapreparation, 11pg/lane; Lane 3: deciliatedcatfish cytosolicfraction (S1), 10#g/lane; Lane 4: deciliated catfish membrane fraction (S2), 13pg/lane; Lane 5:catfishbrain cytosolicfraction (S1),11.6pg/larre;Lane 6:membranefraction of catfish brain (S2), 10pg/lane; Lane 7: rat cilia preparation, 12pg/lane; Lane 8: deciliated rat cytosolicfraction (S1), 15pg/lane; Lane 9: deciliated rat membrane fraction (S2), 15pg/lane; Lane 10:rat brain cytosolic fraction (S1), 12pg/lane; Lane 11:membrane fraction of rat brain (S2), 11yg/lane; Lane 12:Molecular weight marker.

explained if other odorants, as yet undefined,couple re%ptor proteins in the olfactory epithelium30’31 through a PTX-insensitivepathway to PLC, or if the and to the detection of odorant receptor mRNAs antigen detected by the Gaq/Gull antibody in the rat within the axon terminals of sensory neurons,74the and catfish olfactory epitheliumsis a PTX-sensitive presenceof this protein(s)in basal nerve bundlesmay variant of Gaq/Gall. In addition, use of a monospe- suggest coupling of Ga~/Gal, to another effecter cific polyclonal antibody selective for a C-terminal system. The presence Gaq/Gall immunoreactivity in susdecapeptide sequence common to two members of this class, may mean results show the presence of tentacular cellsalso raises the question of function in either Ga~ or Gall in the tissues, or the presence this cell population. Sustentacular cells, which are of both. Under’conditions when both are present, it not known to be excitable are suggestedto resemble is possible they could interact with different subsets glia. The presenceof G-proteins in glialcellshas been of receptors and effecters.Although no selectivityhas documented.ll Like sustentacular IX1lS,their funcyet been found for Gaqor Gall, selectivityhas been tion(s) has not been elucidated. Ion channels have found for other members of the G~ family.]’5’7&7sbeen reported in glia. Neurotransmitter-sensitive Indeed, results from a variety of studies in other receptors, some linked to the apparent metabolism of In systems suggest that regulation of PIPZbreakdown inositol phosphate have also been noted.8’ls’22’a’54’w by G-protein coupled receptors is unexpectedly addition, glioma tumor cellslines have been reported to express significant levels of Gaq/Gall proteins, complex.20Furthermore, while the apical antigenitity of Gaq/Gall also corresponds to recent immuno- although under normal conditions the proteins are histochemical studies localizing putative olfactory not abundant in white matter.23’47

Gaq/Ga,, in the olfactory epitheliums

CONCLUSION A number of different G-proteins are known to be present in the olfactory neuroepithelium. However, it is not known how many GTP-binding proteins are required to mediate the varied signaling systems. Perhaps a large number of different G-proteins are utilized, resulting in the presence of numerous unique proteins dedicated to specific function. Another alternative may be a small number of the GTP-binding proteins can be adapted to multiple tasks, possibly through the actions of the ~ and y

271

subunits of different effecter systems. Our data demonstrate vthe presence of GIXq/Gall in the olfactory neuroepithelium of two phylogenetically distant vertebrates, the rat and the catfish. The results suggest that this G-protein(s) plays a role in olfactory transduction. Acknowledgements—This work was supported by NIH

Grants DC-01228 and DC-00566. BPMM acknowledges support by NSF Grant IBN-9109851,NIH RO1 DC02491, and the Smokeless TobaWo Research Countil Grant 031404.

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