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Transport of intravenously administered horseradish peroxidase into the rat submaxillary saliva
TRANSPORT OF INTRAVENOUSLY ADMINISTERED HORSERADISH PEROXIDASE INTO THE RAT SUBMAXILLARY SALIVA Y. YOSHIDA.S. YAMAMOTOand
Y. KAKUW
Department of Physiology, Osaka Dental University. i-47, Kyobashi, H&hi-ku. Osaka, Japan Summary-When horseradish peroxidase (HPO) was administered i.v. to rats. peroxidase activity was detected histochemically in the lumen of the striated duct and in the basal portion of the striated duct cells of the submaxillary gland. On polyacrylamide-gel electrophoresis, two bands characteristic of HP0 were identified in the submaxillary saliva. By Ouchterlony’s test and immunoelectrophoresis, an antigen specific to HP0 was detected in the saliva. It is concluded that intravenously administered HP0 is taken up by the submaxillary gland cells and then transported into the saliva.
INTRODUCTION Low concentration of serum proteins, albumin and W, p- and y-globulins, have been identified in the human parotid, submaxillary and whole saliva (Ellison, Mashimo and Mandel, 1960; Mandel and Ellison, 1961; Stoffer, Kraus and Holmes, 1962; Kraus and Sirisinha, 1962; Simons rt al., 1964) and in the rat submaxillary saliva (Junqueira, Toledo and Ferri, 1965) which suggests the possibility that the salivary glands take up macromolecules from the blood and secrete them into the saliva. Schein and Tung (1962, 1964) have shown that ’ 3’ I-labelled human albumin administered iv. to rabbits and human subjects and ’ 3‘I-labelled egg albumin or human globulin administered intravenously (i.v.) to the rabbit appeared in the whole saliva. They have also shown immunologically that human albumin which had been administered i.v. to a rabbit appeared in the whole saliva. Antibodies which react with Srrlr?lonrlltr typhosa and Shigella dysenterim have been detected in the human parotid saliva. Their titre was paralleled closely with the bactericidal activity found in the serum, thereby it was assumed that the antibodies were transported from the serum into saliva through the salivary gland (Evans and Mergenhagen, 1965). On the other hand, Tomasi et al. (1965) have reported that 13’1labelled ;‘,A-globulin administered i.v. to man was not secreted into the parotid saliva, though unlabelled IgA was detected in the saliva. They have concluded that IgA contained in the saliva is synthesized in the parotid gland. Martin and Burgen (1962), who studied the transport of sugars of various molecular weights into the cat submaxillary saliva, have reported that creatine, sucrose, raffinose and polyglycols whose molecular weight ranged from 113 to 1540 were detected in the saliva, but inulin whose molecular weight was 4500 was undetectable.
In view of these controversial results, the uptake of exogenous macromolecules by the salivary gland required further examination. The present paper deals with a study on the uptake of exogenous horseradish peroxidase, a widely used macromolecular tracer which can be visualized histochemically (Graham and Karnovsky, 1966) by the rat submaxillary gland and its transport into submaxillary saliva. METHODS A tracheal cannula (Igarashi Co., No. 35) was inserted into the incision to prevent respiratory complications during the experiment. Another polyethylene tube (Clay Adams, PE 50) was inserted into a femoral vein for the administration of drugs. After administration of heparin sodium (500 units/kg) via the i.v. cannula, 1 per cent pilocarpine in 0.9 per cent NaCl (8 mg/kg) was injected i.p. to stimulate salivary secretions. A few minutes later, 30 mg horseradish peroxidase (Sigma Chemical Co., abbreviated as HPO, and peroxidase as PO) dissolved in 0.6 ml of 0.9 per cent NaCl was administered through the venous cannula. All the saliva samples employed were collected from rats to which pilocarpine had been injected. Light microscopJ Animals were killed at various times ranging from 20 min to 3 hr after administration of HP0 to examine histochemically the uptake of HP0 by submaxillary gland cells. The gland was immersed in 5 per cent glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), quickly cut into small blocks, and fixed for 4 hr. After fixation, the blocks were washed overnight in the phosphate buffer and frozen sections cut (l&20 pm thick). The sections were incubated for 20 min at room temp. in 10 ml of 0.05 M tris+HCl buffer
SO2
Y. Yoshida.
S. Yamamoto
(pH 7.6) containing 0.1~ 0.2 ml of I per cent H,O, and 5 mg of I.?‘-diaminobenzidine (Merck) (Graham and Karnovsky. 1966). As controls, some sections were incubated in the medium of Graham and Karnovskq but without ?.i’-diaminobenzidine or H,O,. The) wcrc stained u ith Mayer’s haematoxylin. mountsd in glycerin. and cyanlined by light microscopy. The submaxillary glands of non-operated animals OI after the administration iv. of 0.9 per cent NaCl solution wcrc likewise treated to detect the cndogcnous PO acti\ it!.
Submaxillary saliva was collected from the duct cannula after administration of HP0 and stored frozen until used. Polyacrylamide gel electrophoresis was performed by the methods of Ornstein (1964) and Davis (1964) at room temperature for 2 hr at 4 mA/coiumn. Tris~glycine buffer (pH X.6) was placed in the upper and lower electrode containers. the upper being led to the cathode and lower to the anode. The gels removed from the columns were incubated for 30 min in the same medium as used for histochemial demonstration of PO activity. and then fixed in 7 per cent acetic acid. PO activity was detected by a brown colouration. The submaxillary saliva collected after administration of 0.6 ml of @9 per cent NaCl and that of 0.9 per cent NaCl-uninjected animals served as control. If necessary. the saliva pooled from three to four rats was concentrated IS-fold by means of collodion bags in a cold room.
The transport of HP0 into submaxillary saliva was also studied employing immunological techniques. The antiserum against HP0 was prepared following a series of in.iections of HP0 into three rabbits. HP0 (20 mg dissolved in I ml of 0.9 per cent NaCl)was emulsified with an equal volume of Freund’s complete adjuvant (Difco) and injected intramuscularly into each rabbit, at the beginning of the experiment and I week after. Four weeks after the second immunization. IOmg ofHP0 dissolved in 1 ml of09 per cent NaCI was injected i.v. The rabbit anti-HP0 serum was drawn 6 days after the last injection. The antibody titrc was I :32. The antisera obtained from three rabbits reacted with HP0 solution on double dithlsion plates without significant difference in the formation of precipitin lines. Agar gel-douhlc diffusion test was carried out essentially as described by Ouchterlony (1948) with 1.2 per cent agar (Difco) in phosphate buffer (pH 7.4). OhservationswercmadeforZdaysat room temperature. Immunoelectrophoresis was carried out in Verona1 buffer (pH X.6) with a constant current of 8 mA per I2 slides. applied for 90min. After the anti-HP0 serum was added. diffusion was allowed to proceed for 2 days at room temperature. The gel plates of the double diflusion test and immunoelectrophorcsis were incubated for 5 min in the
and Y. Kakudo
same medium as used for histochcmical demonstration of PO activity. If necessary. saliva to be tested was concentrated by means of collodion bags.
In order to cstimatc the molecular weight of the HP0 fraction incorporated into saliva. gel-filtration was performed with a Sephadcx G-50 column. Saliva (0.6 ml) taken from a rat which had received HP0 was applied to a XI x 10.0 cm column. Small amounts 01 Blue Dextran-2000 (Pharmacia Co.) and bromophenol blue solution were added to the samples as markers. The column was eluted with distilled water at a flow rate of 34 ml ‘hr. and wjith fractions of 4 ml. Appropriatel~ pooled fractions were concentrated I S-fold with collodion bags and examined electrophorctically,
KESCl.‘I‘S N i.stochcCd
ohserl~rrtioil.\
Submaxillary glands excised from rats to which HP0 had been administered 20 min. SO min and 3 hr earlier were examined. In all cases, heavy PO reactionproduct was detected in the intraglandular connective tissue, in the basal portion of striated duct cells and in the lumen of the striated duct. but not in the acini or the other duct regions (Figs. I and 2). The PO reactionproduct was not detected when sections were incubated in the medium lacking 3.3’-diaminobenzidine or H?O, nor in any region of the glands taken from rats to which 0.9 per cent NaCl had been administered 20 min. SO min and 3 hr earlier and from non-treated rats (Figs 3 and 4).
Though the normal saliva contained a considerable amount ofP0 activity. the presence of isozymes of PO (Klapper and Hackett, 1965; Shannon, Kay and Lew. 1966: Morita cl ul., 1970) proved to be useful in differentiating the endogenous from the exogenous enzyme. First. a comparison of the electrophoretic patterns of HP0 and saliva collected from 0.9 per cent NaCluninjected rats was made. When HP0 solution in 0.9 per cent NaCl of various concentrations ranging from 1 to I x IO ’ per cent was examined, two slowly moving clear-cut bands (Bands 1. and 2 from the cathode side) and a fast moving weak band (Band 3) were identified (Fig. 5). These three bands were identified when HP0 solution at concentrations of I x IO-‘, I x 10e3 and I x IO ’ per cent was used but not at 1 x 10e5 pel cent. When the saliva collected from 09 per cent NaCIuninJectcd rat was used. one hand with PO activity was Identified at the position corresponding to that of Band 3 of HPO. but not of Bands 1 and 2 (Fig. 6). Therefore, two isozymes. Bands I and 2, which were present in HP0 but not in control saliva were followed to test the incorporation of exogenous HP0 into the saliva. In the saliva collected from rats to which HP0 had been administered, two bands characteristic of
Transport of exogenous peroxidase Into submaxillary saliva HP0 were identified in all of the 34 rats examined (Fig. 7). These bands were detected in the saliva collected within 20 min after administration of HP0 as well as in that collected 3 hr after administration. As a control, the saliva taken from eight rats to which 0.9 per cent NaCl had been administered was examined. The results were the same as those of 0.9 per cent NaCluninjected rat (Fig. 8). However, when saliva pooled from two rats which had received 0.9 per cent NaCl was concentrated 15fold, another weak band appeared at a position corresponding to that of the slowly moving bands of HPO. In an experimental saliva concentrated 15-fold. no additional band appeared in the vicinity of the two slowly moving bands. Therefore, the band which appeared in the concentrated control saliva seems to be identical with either one of the two slowly moving bands. A conspicuous difference was observed in the pattern of secretion between rats which had received HP0 and the controls with 0.9 per cent NaCl. In the latter. the steady secretion lasted twice as long and the volume of saliva collected before exhaustion was about twice that compared with the former. While the reason for this difference was unknown, it was considered possible that the synthesis of endogenous PO in the gland was affected by the physiological conditions associated with the decrease of secretion rate induced by HP0 injection. The question then arose whether the appearance of the slowly moving bands after HP0 injection was due to newly synthesized endogenous PO or to exogenous HPO. The following experiments were performed to investigate this point. When IOOmg of bovine or human albumin dissolved in I ml of 0.9 per cent NaCl were administered i.v.. the mode of secretion changed in the same way as with HP0 injection. The results of electrophoresis of the saliva of these animals were the same as those of non-treated rats, excluding the possibility described above.
The presence of HP0 in saliva was further examined by using its immunological specificity. Solutions of various concentrations of HP0 in 09 per cent NaCl ranging from 1 to I x lo- 5 per cent were subjected to a double diffusion test against anti-HPO. Two sharp precipitin lines (Lines 1 and 2, Fig. 12) were detected. Line 1 appeared between 1 per cent HP0 solution and anti-HPO, but it was not detected when moredilutedantigenswereuscd. Line 2 was detected asa sharp line with @Ol per cent HP0 solution was used while more concentrated solutions reacted so strongly as to produce a diffuse precipitating area. Both lines were positive in PO-activity but the colouration of Line I was weaker than Line 2. When saliva taken from rats which had received HP0 was subjected to double diffusion against anti-HPO, no precipitin Iine appeared. However, saliva concentrated 50-fold, which was derived from IO.8 ml saliva pooled from
x03
23 rats which had received HPO, reacted with antiHP0 to form a single precipitin line (Fig. 13) which was identical with Line 2 and gave a positive PO reaction. Two precipitin lines were detected when the serum, drawn 60 set after administration of HP0 was used, one being identical to Line 2 and the other to Line I. The former showed strong PO activity. but the latter did not. No precipitin line appeared when 50fold concentrated control saliva was tested (Fig. 13). HP0 solution at various concentration was subjected to immunoelectrophoretic analysis (Fig. 14 and 16). Four components were detected when 1 per cent HP0 solution was used, each of the components being named as A, B, C and D as shown in Fig. 15, a schematic drawing of Fig. 14. Two precipitin lines, A and D showed positive PO activity. As the concentration of HP0 solution was decreased, the number of precipitin lines decreased. At a concentration of 0.01 per cent, only Line A was observed. When the saliva taken from rats which had received HP0 was subjected to immunoelectrophoresis, no precipitin line appeared. However, experimental saliva concentrated 50-fold reacted with anti-HP0 to form a single precipitin line which seemed to be idknticdl with Line A and was PO reaction positive (Fig. 17). Two components were detected in the serum drawn 60 see after administration of HPO; one showed positive PO activity but the other did not (Fig. 18). The former seemed to be identical to Line A and a line obtained with the experimental saliva, and the latter seemed to be identical to Line D. The 50-fold concentrated control saliva (Fig. 19). and serum before administration of HPO, did not react with anti-HPO.
Electrophoretic and immunological studies proved conclusively that the injected HP0 was incorporated into the submaxillary saliva. A question arises, however, whether the HP0 incorporated into the saliva retained its original molecular weight or whether it was disintegrated into relatively small molecules before being incorporated, because an enzyme with an exceptionally small molecular weight possessing PO activity does exist (Feder, Reese and Brightman, 1969). In order to test the latter possibility. an estimation of the molecular weight of HP0 recovered from the saliva was carried out using gel-filtration on a Sephadex G-50 column. Tubes No. I. 2 and 3 (Blue Dextran fraction), tubes No. 4-21 (partially excluded fraction) and tubes No. 23 and 24 (bromophenol blue fraction) were combined together concentrated l5-fold. and subjected to electrophoresis. The two bands characteristic of HP0 were identified in the Blue Dextran fraction, but not in the other fractions (Figs. 9--l I). Thus, HP0 recovered from the saliva was shown to have retained a large molecular weight of more than 9000. DISCUSSION
A reservation should be made on the conclusion drawn from the work using whole saliva as to the
X04
Y. Y’oshida. S. Yamamoto and Y. Kakudo
transport of macromolecules from the blood into salivary gland secretion. Serum proteins have been found in fluid from gingival pockets (Brill and Bronnestam, 1960); the fluid outflow from these pockets might account for the presence of some proteins in the whole saliva. Hence the present study was performed on pure submaxillary saliva carefully collected to avoid contamination with other fluids. Even if the injected HP0 was incorporated into the salivary glands, histochemical detection of exogenous HP0 in the gland seemed impractical because endogenous PO has been detected histochemically in the rat submaxillary gland (Strum and Karnovsky. 1970) and exorbital lacrimal gland (Essner. 1971). However. the present incubation in a reaction mixture for 20 min. which was much shorter than that in the above-mentioned work, successfully differentiated the exogenous HP0 from the endogenous PO; the Ireaction product was detected in the glands of HPOinjected rats. but not in control glands. The endogenous PO activity has been inhibited by glutdraldehyde fixation in Kupffer cells of the liver (Fahimi. 1970) and in the rat parotid and submaxillary gland cells (Hand, 1973). Whatever the reason of the success in the selective reaction of exogenous PO activity may be. the uptake of HP0 by the submaxillary gland. probably through the cells of the duct regions. was histochemically confirmed in the present study. On the electrophoretic studies, the two bands characteristic of HP0 were identified in the saliva taken from rats to which HP0 had been administered, but not in the control saliva. When control saliva concentrated l5-fold was examined. a band specific for HP0 appeared. But, in view of the extremely low concentration of this band. it is clear that the two bands detected in experimental saliva were incorporated exogenous HPO. Gel-filtration through Sephadex G-50 indicated that HP0 was taken up by the gland without having been disintegrated into molecules with molecular weight less than about 9000. Transport of HP0 into saliva was further confirmed by immunological techniques. Anti-HP0 was resolved into two antibodies which bind to antigens having PO activity in Ouchterlony’s test into two which bind to antigens having PO activity and two antibodies which bind to antigens having no PO activity in immunoelectrophoresis. In both cases. it was confirmed that experimental saliva contained one antigen with PO activity. while the control saliva did not react with the antiserum. The fact that the other antigens were not detected in the experimental saliva may suggest a mechanism by which the gland takes up macromolecules selectively. From these studies, we conclude that rat submaxillary gland takes up HP0 from the blood and secretes it into saliva. The types of drugs used for stimulating salivary secretion is known to affect the transport of substances from blood into saliva: Junqueria rf nl. (1965) pointed
out that the uptake was facilitated when a sympathomimetic agent was used. Thus, they showed that when adrenalin was used for stimulating salivary secretion albumin. X-, [j-, and y-globulins were detected in the rat submaxillary saliva, but albumin only was detected when carbamylcholine was used. Martin and Burgen (1962) who studied the transport of sugars into the cat submaxillary saliva, reported that the gland incorporated glucose. creatine. sucrose, rafinose and polyglycols only when the sympathetic nerve was stimulated or sympathomimetic agents were used. On the other hand. the present study. like that of Tung and Schein (I 964). has disclosed protein incorporation on the pilocarpine-stimulated gland. But whether this was due to parasympathetic stimulation of pilocarpine or not remains to be decided, because pilocarpine is known to stimulate the superior cervical ganglion (Schneyer and Hall. 1966). Evidently further analyses are needed before drawing a more generalized conclusion as to relations between the nature of the stimuli and the mode of incorporation of substances into saliva. Species difference must also bc kept in mind in dealing with this problem.
l~k~ioi~i~,~/i~c,rf~c,,ri We thank Dr. H. Ishmki.
De\>artment
of Developmental Biology. Zoological
Instltutc. College 01 Science. Kyoto Um\ersity (now at Nagoya University) for his advice and the use of his laboratory in completing this work. REFERElr;CES
Brill N. and Bronnestam
R. 1960. lmmunoelectrophoretic of tissue fluid from gingival pockets. Actu orhr. wrrd. IS, 9s 100. Daws B. J. 1964. Disc electrophoresls-2 method and application. ilrw. IV.Y. Ad. SC. 121, 404427. Ellison S. A., Mashimo P. A. and Mandel L. D. 1960. Immunochemical studies of human saliva- I. The demonstration of serum protems in whole and parotid saliv:i. .I. f&t. Rrs. 39, 892- 898. Essner E. 1971. Localizationofendogenous peroxldase In rat cxorbital lacrimal gland. J. Histocho~~. C~~~oc~/xv~~.19, 2 I h-23. Exam R. T. and Mergenhagen S. E. 1965. Occurrence of natural antibody in human parotid Ruid. Proc. Sot. cvp. Bid. Med. 119, XIS 819. Fahimi H. D. 1970. The fine structural localzation of endogenous and exogenous peroxidase activity in KupFier cells of rat liver. .I. (‘c/l Bin/. 47, 247 262. Fcder N., Reese T. S. and Brightman M. W. lY69. Microperoxidase. a new tracer of low molecular weight. A study of the interstitial compartments of the mouse brain. J. Cell Biol. 43, 35a. Graham R. C. and Karnovsky M. J. 1966. The early stage of absorption of injected horseradlsh peroxidase in the proximal tubules of mouse kidney. Ultrastructural cytochemistry by a new technique. -1. Histochrn~. C~tochrm. 14,291 302. Hand A. R. 1973. Morphologic and cytochemical indentilication of peroxisomes in the rat parotid and other exocrine glands. J. Histochem Cytochrm. 21, I3 1 141. Junqueira L. C. U.. Toledo A. M. S. and Fcrri R. G. lY65. Permeability of the stimulated rat suhmaxiilary gland to its blood serum proteins. Au&s orrrl Bid. IO, 863-~X6X. study
Transport
of exogenous
peroxidase
Klapper M. H. and Hackett D. P. 1965. Investigations on the multiple components of commercial horseradish perosidase. Eiochirn. hiopltys. acta 96, 272-282. Kraus F. W. and Sirisinha S.. 1962. Gamma globulin in saliva. Archs oral Biol. 7, 221-233. Mandel 1. D. and Ellison S. A. 1961. Characterization of salivary components separated by paper electrophoresis. Arch oral Biol. 3. 77-85. Martin K. and Burgen A. S. V. 1962. Changes in the permeability of the salivary gland caused by sympathetic stimulation and by catecholamines. J. yen. Physiol. 46, 225-243. Morita Y.. Yoshida C., Kitamura I. and Ida S. 1970. Isozymes of Japanese-radish peroxidase. Agr. biol. Chem. 34, 1191 1197. Ornstein L. 1964. Disc electrophoresis-I. Background and theory. iljlri. ‘V.Y. .A&. Sci. 121, 321-349. Ouchterlony 0. 1948. Antigen-antibody reactions in gels. Arkk Kern. Mineral Geol. B 26, 1-Y. Schein A. H. and Tung F. F. 1962. Appearance of parenterally administered proteins in saliva. Nature, Land. 196, 109221093.
into submaxillary
Schneyer
80.5
saliva
C. A. and Hall H. D. 1966. Autonomic
pathways
involved in a sympathetic-like action of pilocarpine on salivary composition. Proc. Sot. e~p. Biol. Med. 121, 96
100. Shannon L. M., Kay E. and Lew J. Y. 1966. Peroxidase isozymes from horseradish roots. J. hiol. Chem. 241, 2166 2-l 72. Simons K.. Weber T.. Stiel M. and Grasbeck R. 1964. Immunoelectrophoresis of humah saliva. 4ctu wd. ,xmd. Suppl. 412, 257-264. Strum .I. and Karnovsky M. J. 1970. Ultrastructural localization of peroxidase in submaxillary acinar cells. J. ultrustruct. Res. 31, 323-336. Stoffer H. R., Kraus F. W. and Holmes A. C. 1962. Immunochemical identification of salivary proteins. Proc. Sot. rxp. Biol. Med. 111,467-471. Tomasi T. B. Jr., Tan E. M., Solomon A. and Prendergast R. A. 1965. Characteristics of an immune system common to certain external secretions. J. exp. Med. 121, 101-124. Tung F. F. and Schein A. H. 1964. The appearance of administered proteins in saliva. J. dent. Res. 43, 4233432.
R&um&Quand la peroxydase du raifort (POR) a Cte administree aux rats par voie intraveineuse, l’activite peroxydase fut detectee histochimiquement dans la lumiere des canaux stries et dans la portion basale de cellules du canal strie de la glande sous-maxillaire. Sur l’electrophorese de gel polyacrylamidique on a identifie dans la salive sous-maxillaire deux bandes caracteristiques de POR. La detection dans la salive a Cte faite par le test de Ouchterlonys et immunoClectrophorese et par l’antigene specifique a la POR. On conclut que la POR, administree par voie intraveineuse, est assimilee par les cellules de la glande sous maxillaire et puis transportee dans la salive.
Zusammenfassung-Als Meerrettichperoxydase (HPO) Ratten i.v. verabfolgt wurde, entdeckte man Peroxydascaktivitlt histochemisch in dem Lumen des gefurchten Gangs und in dem Basalteil der gefurchten Gangzellen der submaxillaren Driise. Auf Polyacrylamid-Gel Elektrophorese wurden zwei fiir HP0 charkateristische Bander in dem submaxillaren Speichel identifiziert. Durch Ouchterlonys Test und Immunoelektrophoresc wurde ein fur HP0 eigenttimliches Antigen in dem Speichel entdeckt. Man kommt zu dem SchluB, daI.3intervenijs verabfolgtes HP0 von den submaxilllren Driisenzellen aufgenommen und dann in den Speichel iibertragen wird.
Transport
of exogenous peroxidase
Figs. I and 2. Pilocarpine-stimulated
into submaxillary
saliva
submaxillary gland excised 10 min after administration
of HPO.
Heavy PO reaction product (arrows), is seen in striated duct cells (S), but not in acinar cells (A) and convoluted glandular tubule cells (G). Counterstained with haematoxylin. x 200 Figs. 3 and 4. Pilocarpine-stimulated submaxillary gland excised 10 min after administration NaCI. No PO reaction product is present. Haematoxylin. x 200
of 0.9 “/;,
PLATE
1
A.O.B. f.p. 806
Y. Yosh ida, S. Yamamoto and Y. Kakudo
I
2
3
PLATE 2
Figs. 5-l 1. Electrophoretic patterns after staining for PO activity. Fig. 5. 0.1 per cent. HP0 sol. Fig. 6. Saliva from 0.9 per cent NaCl-uninjected rat. Fig. 7. Salivafrom a rat to which HP0 had been administered. Bands I and 2 are characteristic of HPO. Fig. 8. Saliva from a rat to which 0.9 per cent NaCI had been administered. Figs. 9-i I. Eluates from a Sephadex column. Fig. 9. Blue Dextran fraction. Fig.1 0. Partially excluded fraction. Fig. Il. Bromophenol blue fraction. Note that the bands I and 2 are identified in only the Blue Dextran fraction.
Transport
of exogenous peroxidase
into submaxillary saliva
PLATE 3
Fig. 12. Immunodiffusion of HP0 soln at various concentrations. Peripheral wells contain I per cent (l), 1 x 10-l per cent (2), 1 x 1O-2 per cent (3), 1 x 1O-3 per cent (4), I x 10e4 per cent (5), and 1 x lop5 per cent HP0 (6). Centre well contains anti-HP0 serum. The right schematic drawing. Fig. 13. Immunodiffusion showing identity of precipitin lines produced by 1 x 10e2 per cent HP0 (wells 1 and 3) and by 50-fold conc.saliva after administration of HP0 (wells 2 and 4). Wells 5 and 6 contain 0.9 per cent NaCl and 50-fold concentration of saliva after administration of 0.9 per cent NaCI, respectively. Centre well contains anti-HP0 serum.
Y. Yoshida, S. Yamamoto
W(C)
and Y. Kakudo
(A) (D)
PLATE
4
Figs. 14.-19. Immunoelectrophoretic analysis. Figures 14 and 16. HPOsoln at variousconcentrations. I per cent HP0 (14 upper), I ’ IO--’ per cent HP0 (14 lower), I < lO-‘2 per cent HP0 (I6 upper), 1 ,: lO-3 per cent HP0 (16 lower). Centre troughs contain anti-HP0 serum. Fig. 15. Schematic drawing of Fig. 14. Fig. 17. 1 per cent HP0 soln (upper) and 50-foldconc. salivaafteradministration of HP0 (lower). Note that the experimental saliva forms a single precipitm line which seems to be identical to Line A. Fig. 18. Rat serum drawn after administration of HP0 (upper) and 50-fold cont. saliva after administration of NPO (lower). Fig. 19. 50-fold cont. saliva after administration of HP0 (upper). SO-fold cont. saliva after administration of 0.9 per cent NaCl (lower).
Y. KAKUW
Department of Physiology, Osaka Dental University. i-47, Kyobashi, H&hi-ku. Osaka, Japan Summary-When horseradish peroxidase (HPO) was administered i.v. to rats. peroxidase activity was detected histochemically in the lumen of the striated duct and in the basal portion of the striated duct cells of the submaxillary gland. On polyacrylamide-gel electrophoresis, two bands characteristic of HP0 were identified in the submaxillary saliva. By Ouchterlony’s test and immunoelectrophoresis, an antigen specific to HP0 was detected in the saliva. It is concluded that intravenously administered HP0 is taken up by the submaxillary gland cells and then transported into the saliva.
INTRODUCTION Low concentration of serum proteins, albumin and W, p- and y-globulins, have been identified in the human parotid, submaxillary and whole saliva (Ellison, Mashimo and Mandel, 1960; Mandel and Ellison, 1961; Stoffer, Kraus and Holmes, 1962; Kraus and Sirisinha, 1962; Simons rt al., 1964) and in the rat submaxillary saliva (Junqueira, Toledo and Ferri, 1965) which suggests the possibility that the salivary glands take up macromolecules from the blood and secrete them into the saliva. Schein and Tung (1962, 1964) have shown that ’ 3’ I-labelled human albumin administered iv. to rabbits and human subjects and ’ 3‘I-labelled egg albumin or human globulin administered intravenously (i.v.) to the rabbit appeared in the whole saliva. They have also shown immunologically that human albumin which had been administered i.v. to a rabbit appeared in the whole saliva. Antibodies which react with Srrlr?lonrlltr typhosa and Shigella dysenterim have been detected in the human parotid saliva. Their titre was paralleled closely with the bactericidal activity found in the serum, thereby it was assumed that the antibodies were transported from the serum into saliva through the salivary gland (Evans and Mergenhagen, 1965). On the other hand, Tomasi et al. (1965) have reported that 13’1labelled ;‘,A-globulin administered i.v. to man was not secreted into the parotid saliva, though unlabelled IgA was detected in the saliva. They have concluded that IgA contained in the saliva is synthesized in the parotid gland. Martin and Burgen (1962), who studied the transport of sugars of various molecular weights into the cat submaxillary saliva, have reported that creatine, sucrose, raffinose and polyglycols whose molecular weight ranged from 113 to 1540 were detected in the saliva, but inulin whose molecular weight was 4500 was undetectable.
In view of these controversial results, the uptake of exogenous macromolecules by the salivary gland required further examination. The present paper deals with a study on the uptake of exogenous horseradish peroxidase, a widely used macromolecular tracer which can be visualized histochemically (Graham and Karnovsky, 1966) by the rat submaxillary gland and its transport into submaxillary saliva. METHODS A tracheal cannula (Igarashi Co., No. 35) was inserted into the incision to prevent respiratory complications during the experiment. Another polyethylene tube (Clay Adams, PE 50) was inserted into a femoral vein for the administration of drugs. After administration of heparin sodium (500 units/kg) via the i.v. cannula, 1 per cent pilocarpine in 0.9 per cent NaCl (8 mg/kg) was injected i.p. to stimulate salivary secretions. A few minutes later, 30 mg horseradish peroxidase (Sigma Chemical Co., abbreviated as HPO, and peroxidase as PO) dissolved in 0.6 ml of 0.9 per cent NaCl was administered through the venous cannula. All the saliva samples employed were collected from rats to which pilocarpine had been injected. Light microscopJ Animals were killed at various times ranging from 20 min to 3 hr after administration of HP0 to examine histochemically the uptake of HP0 by submaxillary gland cells. The gland was immersed in 5 per cent glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), quickly cut into small blocks, and fixed for 4 hr. After fixation, the blocks were washed overnight in the phosphate buffer and frozen sections cut (l&20 pm thick). The sections were incubated for 20 min at room temp. in 10 ml of 0.05 M tris+HCl buffer
SO2
Y. Yoshida.
S. Yamamoto
(pH 7.6) containing 0.1~ 0.2 ml of I per cent H,O, and 5 mg of I.?‘-diaminobenzidine (Merck) (Graham and Karnovsky. 1966). As controls, some sections were incubated in the medium of Graham and Karnovskq but without ?.i’-diaminobenzidine or H,O,. The) wcrc stained u ith Mayer’s haematoxylin. mountsd in glycerin. and cyanlined by light microscopy. The submaxillary glands of non-operated animals OI after the administration iv. of 0.9 per cent NaCl solution wcrc likewise treated to detect the cndogcnous PO acti\ it!.
Submaxillary saliva was collected from the duct cannula after administration of HP0 and stored frozen until used. Polyacrylamide gel electrophoresis was performed by the methods of Ornstein (1964) and Davis (1964) at room temperature for 2 hr at 4 mA/coiumn. Tris~glycine buffer (pH X.6) was placed in the upper and lower electrode containers. the upper being led to the cathode and lower to the anode. The gels removed from the columns were incubated for 30 min in the same medium as used for histochemial demonstration of PO activity. and then fixed in 7 per cent acetic acid. PO activity was detected by a brown colouration. The submaxillary saliva collected after administration of 0.6 ml of @9 per cent NaCl and that of 0.9 per cent NaCl-uninjected animals served as control. If necessary. the saliva pooled from three to four rats was concentrated IS-fold by means of collodion bags in a cold room.
The transport of HP0 into submaxillary saliva was also studied employing immunological techniques. The antiserum against HP0 was prepared following a series of in.iections of HP0 into three rabbits. HP0 (20 mg dissolved in I ml of 0.9 per cent NaCl)was emulsified with an equal volume of Freund’s complete adjuvant (Difco) and injected intramuscularly into each rabbit, at the beginning of the experiment and I week after. Four weeks after the second immunization. IOmg ofHP0 dissolved in 1 ml of09 per cent NaCI was injected i.v. The rabbit anti-HP0 serum was drawn 6 days after the last injection. The antibody titrc was I :32. The antisera obtained from three rabbits reacted with HP0 solution on double dithlsion plates without significant difference in the formation of precipitin lines. Agar gel-douhlc diffusion test was carried out essentially as described by Ouchterlony (1948) with 1.2 per cent agar (Difco) in phosphate buffer (pH 7.4). OhservationswercmadeforZdaysat room temperature. Immunoelectrophoresis was carried out in Verona1 buffer (pH X.6) with a constant current of 8 mA per I2 slides. applied for 90min. After the anti-HP0 serum was added. diffusion was allowed to proceed for 2 days at room temperature. The gel plates of the double diflusion test and immunoelectrophorcsis were incubated for 5 min in the
and Y. Kakudo
same medium as used for histochcmical demonstration of PO activity. If necessary. saliva to be tested was concentrated by means of collodion bags.
In order to cstimatc the molecular weight of the HP0 fraction incorporated into saliva. gel-filtration was performed with a Sephadcx G-50 column. Saliva (0.6 ml) taken from a rat which had received HP0 was applied to a XI x 10.0 cm column. Small amounts 01 Blue Dextran-2000 (Pharmacia Co.) and bromophenol blue solution were added to the samples as markers. The column was eluted with distilled water at a flow rate of 34 ml ‘hr. and wjith fractions of 4 ml. Appropriatel~ pooled fractions were concentrated I S-fold with collodion bags and examined electrophorctically,
KESCl.‘I‘S N i.stochcCd
ohserl~rrtioil.\
Submaxillary glands excised from rats to which HP0 had been administered 20 min. SO min and 3 hr earlier were examined. In all cases, heavy PO reactionproduct was detected in the intraglandular connective tissue, in the basal portion of striated duct cells and in the lumen of the striated duct. but not in the acini or the other duct regions (Figs. I and 2). The PO reactionproduct was not detected when sections were incubated in the medium lacking 3.3’-diaminobenzidine or H?O, nor in any region of the glands taken from rats to which 0.9 per cent NaCl had been administered 20 min. SO min and 3 hr earlier and from non-treated rats (Figs 3 and 4).
Though the normal saliva contained a considerable amount ofP0 activity. the presence of isozymes of PO (Klapper and Hackett, 1965; Shannon, Kay and Lew. 1966: Morita cl ul., 1970) proved to be useful in differentiating the endogenous from the exogenous enzyme. First. a comparison of the electrophoretic patterns of HP0 and saliva collected from 0.9 per cent NaCluninjected rats was made. When HP0 solution in 0.9 per cent NaCl of various concentrations ranging from 1 to I x IO ’ per cent was examined, two slowly moving clear-cut bands (Bands 1. and 2 from the cathode side) and a fast moving weak band (Band 3) were identified (Fig. 5). These three bands were identified when HP0 solution at concentrations of I x IO-‘, I x 10e3 and I x IO ’ per cent was used but not at 1 x 10e5 pel cent. When the saliva collected from 09 per cent NaCIuninJectcd rat was used. one hand with PO activity was Identified at the position corresponding to that of Band 3 of HPO. but not of Bands 1 and 2 (Fig. 6). Therefore, two isozymes. Bands I and 2, which were present in HP0 but not in control saliva were followed to test the incorporation of exogenous HP0 into the saliva. In the saliva collected from rats to which HP0 had been administered, two bands characteristic of
Transport of exogenous peroxidase Into submaxillary saliva HP0 were identified in all of the 34 rats examined (Fig. 7). These bands were detected in the saliva collected within 20 min after administration of HP0 as well as in that collected 3 hr after administration. As a control, the saliva taken from eight rats to which 0.9 per cent NaCl had been administered was examined. The results were the same as those of 0.9 per cent NaCluninjected rat (Fig. 8). However, when saliva pooled from two rats which had received 0.9 per cent NaCl was concentrated 15fold, another weak band appeared at a position corresponding to that of the slowly moving bands of HPO. In an experimental saliva concentrated 15-fold. no additional band appeared in the vicinity of the two slowly moving bands. Therefore, the band which appeared in the concentrated control saliva seems to be identical with either one of the two slowly moving bands. A conspicuous difference was observed in the pattern of secretion between rats which had received HP0 and the controls with 0.9 per cent NaCl. In the latter. the steady secretion lasted twice as long and the volume of saliva collected before exhaustion was about twice that compared with the former. While the reason for this difference was unknown, it was considered possible that the synthesis of endogenous PO in the gland was affected by the physiological conditions associated with the decrease of secretion rate induced by HP0 injection. The question then arose whether the appearance of the slowly moving bands after HP0 injection was due to newly synthesized endogenous PO or to exogenous HPO. The following experiments were performed to investigate this point. When IOOmg of bovine or human albumin dissolved in I ml of 0.9 per cent NaCl were administered i.v.. the mode of secretion changed in the same way as with HP0 injection. The results of electrophoresis of the saliva of these animals were the same as those of non-treated rats, excluding the possibility described above.
The presence of HP0 in saliva was further examined by using its immunological specificity. Solutions of various concentrations of HP0 in 09 per cent NaCl ranging from 1 to I x lo- 5 per cent were subjected to a double diffusion test against anti-HPO. Two sharp precipitin lines (Lines 1 and 2, Fig. 12) were detected. Line 1 appeared between 1 per cent HP0 solution and anti-HPO, but it was not detected when moredilutedantigenswereuscd. Line 2 was detected asa sharp line with @Ol per cent HP0 solution was used while more concentrated solutions reacted so strongly as to produce a diffuse precipitating area. Both lines were positive in PO-activity but the colouration of Line I was weaker than Line 2. When saliva taken from rats which had received HP0 was subjected to double diffusion against anti-HPO, no precipitin Iine appeared. However, saliva concentrated 50-fold, which was derived from IO.8 ml saliva pooled from
x03
23 rats which had received HPO, reacted with antiHP0 to form a single precipitin line (Fig. 13) which was identical with Line 2 and gave a positive PO reaction. Two precipitin lines were detected when the serum, drawn 60 set after administration of HP0 was used, one being identical to Line 2 and the other to Line I. The former showed strong PO activity. but the latter did not. No precipitin line appeared when 50fold concentrated control saliva was tested (Fig. 13). HP0 solution at various concentration was subjected to immunoelectrophoretic analysis (Fig. 14 and 16). Four components were detected when 1 per cent HP0 solution was used, each of the components being named as A, B, C and D as shown in Fig. 15, a schematic drawing of Fig. 14. Two precipitin lines, A and D showed positive PO activity. As the concentration of HP0 solution was decreased, the number of precipitin lines decreased. At a concentration of 0.01 per cent, only Line A was observed. When the saliva taken from rats which had received HP0 was subjected to immunoelectrophoresis, no precipitin line appeared. However, experimental saliva concentrated 50-fold reacted with anti-HP0 to form a single precipitin line which seemed to be idknticdl with Line A and was PO reaction positive (Fig. 17). Two components were detected in the serum drawn 60 see after administration of HPO; one showed positive PO activity but the other did not (Fig. 18). The former seemed to be identical to Line A and a line obtained with the experimental saliva, and the latter seemed to be identical to Line D. The 50-fold concentrated control saliva (Fig. 19). and serum before administration of HPO, did not react with anti-HPO.
Electrophoretic and immunological studies proved conclusively that the injected HP0 was incorporated into the submaxillary saliva. A question arises, however, whether the HP0 incorporated into the saliva retained its original molecular weight or whether it was disintegrated into relatively small molecules before being incorporated, because an enzyme with an exceptionally small molecular weight possessing PO activity does exist (Feder, Reese and Brightman, 1969). In order to test the latter possibility. an estimation of the molecular weight of HP0 recovered from the saliva was carried out using gel-filtration on a Sephadex G-50 column. Tubes No. I. 2 and 3 (Blue Dextran fraction), tubes No. 4-21 (partially excluded fraction) and tubes No. 23 and 24 (bromophenol blue fraction) were combined together concentrated l5-fold. and subjected to electrophoresis. The two bands characteristic of HP0 were identified in the Blue Dextran fraction, but not in the other fractions (Figs. 9--l I). Thus, HP0 recovered from the saliva was shown to have retained a large molecular weight of more than 9000. DISCUSSION
A reservation should be made on the conclusion drawn from the work using whole saliva as to the
X04
Y. Y’oshida. S. Yamamoto and Y. Kakudo
transport of macromolecules from the blood into salivary gland secretion. Serum proteins have been found in fluid from gingival pockets (Brill and Bronnestam, 1960); the fluid outflow from these pockets might account for the presence of some proteins in the whole saliva. Hence the present study was performed on pure submaxillary saliva carefully collected to avoid contamination with other fluids. Even if the injected HP0 was incorporated into the salivary glands, histochemical detection of exogenous HP0 in the gland seemed impractical because endogenous PO has been detected histochemically in the rat submaxillary gland (Strum and Karnovsky. 1970) and exorbital lacrimal gland (Essner. 1971). However. the present incubation in a reaction mixture for 20 min. which was much shorter than that in the above-mentioned work, successfully differentiated the exogenous HP0 from the endogenous PO; the Ireaction product was detected in the glands of HPOinjected rats. but not in control glands. The endogenous PO activity has been inhibited by glutdraldehyde fixation in Kupffer cells of the liver (Fahimi. 1970) and in the rat parotid and submaxillary gland cells (Hand, 1973). Whatever the reason of the success in the selective reaction of exogenous PO activity may be. the uptake of HP0 by the submaxillary gland. probably through the cells of the duct regions. was histochemically confirmed in the present study. On the electrophoretic studies, the two bands characteristic of HP0 were identified in the saliva taken from rats to which HP0 had been administered, but not in the control saliva. When control saliva concentrated l5-fold was examined. a band specific for HP0 appeared. But, in view of the extremely low concentration of this band. it is clear that the two bands detected in experimental saliva were incorporated exogenous HPO. Gel-filtration through Sephadex G-50 indicated that HP0 was taken up by the gland without having been disintegrated into molecules with molecular weight less than about 9000. Transport of HP0 into saliva was further confirmed by immunological techniques. Anti-HP0 was resolved into two antibodies which bind to antigens having PO activity in Ouchterlony’s test into two which bind to antigens having PO activity and two antibodies which bind to antigens having no PO activity in immunoelectrophoresis. In both cases. it was confirmed that experimental saliva contained one antigen with PO activity. while the control saliva did not react with the antiserum. The fact that the other antigens were not detected in the experimental saliva may suggest a mechanism by which the gland takes up macromolecules selectively. From these studies, we conclude that rat submaxillary gland takes up HP0 from the blood and secretes it into saliva. The types of drugs used for stimulating salivary secretion is known to affect the transport of substances from blood into saliva: Junqueria rf nl. (1965) pointed
out that the uptake was facilitated when a sympathomimetic agent was used. Thus, they showed that when adrenalin was used for stimulating salivary secretion albumin. X-, [j-, and y-globulins were detected in the rat submaxillary saliva, but albumin only was detected when carbamylcholine was used. Martin and Burgen (1962) who studied the transport of sugars into the cat submaxillary saliva, reported that the gland incorporated glucose. creatine. sucrose, rafinose and polyglycols only when the sympathetic nerve was stimulated or sympathomimetic agents were used. On the other hand. the present study. like that of Tung and Schein (I 964). has disclosed protein incorporation on the pilocarpine-stimulated gland. But whether this was due to parasympathetic stimulation of pilocarpine or not remains to be decided, because pilocarpine is known to stimulate the superior cervical ganglion (Schneyer and Hall. 1966). Evidently further analyses are needed before drawing a more generalized conclusion as to relations between the nature of the stimuli and the mode of incorporation of substances into saliva. Species difference must also bc kept in mind in dealing with this problem.
l~k~ioi~i~,~/i~c,rf~c,,ri We thank Dr. H. Ishmki.
De\>artment
of Developmental Biology. Zoological
Instltutc. College 01 Science. Kyoto Um\ersity (now at Nagoya University) for his advice and the use of his laboratory in completing this work. REFERElr;CES
Brill N. and Bronnestam
R. 1960. lmmunoelectrophoretic of tissue fluid from gingival pockets. Actu orhr. wrrd. IS, 9s 100. Daws B. J. 1964. Disc electrophoresls-2 method and application. ilrw. IV.Y. Ad. SC. 121, 404427. Ellison S. A., Mashimo P. A. and Mandel L. D. 1960. Immunochemical studies of human saliva- I. The demonstration of serum protems in whole and parotid saliv:i. .I. f&t. Rrs. 39, 892- 898. Essner E. 1971. Localizationofendogenous peroxldase In rat cxorbital lacrimal gland. J. Histocho~~. C~~~oc~/xv~~.19, 2 I h-23. Exam R. T. and Mergenhagen S. E. 1965. Occurrence of natural antibody in human parotid Ruid. Proc. Sot. cvp. Bid. Med. 119, XIS 819. Fahimi H. D. 1970. The fine structural localzation of endogenous and exogenous peroxidase activity in KupFier cells of rat liver. .I. (‘c/l Bin/. 47, 247 262. Fcder N., Reese T. S. and Brightman M. W. lY69. Microperoxidase. a new tracer of low molecular weight. A study of the interstitial compartments of the mouse brain. J. Cell Biol. 43, 35a. Graham R. C. and Karnovsky M. J. 1966. The early stage of absorption of injected horseradlsh peroxidase in the proximal tubules of mouse kidney. Ultrastructural cytochemistry by a new technique. -1. Histochrn~. C~tochrm. 14,291 302. Hand A. R. 1973. Morphologic and cytochemical indentilication of peroxisomes in the rat parotid and other exocrine glands. J. Histochem Cytochrm. 21, I3 1 141. Junqueira L. C. U.. Toledo A. M. S. and Fcrri R. G. lY65. Permeability of the stimulated rat suhmaxiilary gland to its blood serum proteins. Au&s orrrl Bid. IO, 863-~X6X. study
Transport
of exogenous
peroxidase
Klapper M. H. and Hackett D. P. 1965. Investigations on the multiple components of commercial horseradish perosidase. Eiochirn. hiopltys. acta 96, 272-282. Kraus F. W. and Sirisinha S.. 1962. Gamma globulin in saliva. Archs oral Biol. 7, 221-233. Mandel 1. D. and Ellison S. A. 1961. Characterization of salivary components separated by paper electrophoresis. Arch oral Biol. 3. 77-85. Martin K. and Burgen A. S. V. 1962. Changes in the permeability of the salivary gland caused by sympathetic stimulation and by catecholamines. J. yen. Physiol. 46, 225-243. Morita Y.. Yoshida C., Kitamura I. and Ida S. 1970. Isozymes of Japanese-radish peroxidase. Agr. biol. Chem. 34, 1191 1197. Ornstein L. 1964. Disc electrophoresis-I. Background and theory. iljlri. ‘V.Y. .A&. Sci. 121, 321-349. Ouchterlony 0. 1948. Antigen-antibody reactions in gels. Arkk Kern. Mineral Geol. B 26, 1-Y. Schein A. H. and Tung F. F. 1962. Appearance of parenterally administered proteins in saliva. Nature, Land. 196, 109221093.
into submaxillary
Schneyer
80.5
saliva
C. A. and Hall H. D. 1966. Autonomic
pathways
involved in a sympathetic-like action of pilocarpine on salivary composition. Proc. Sot. e~p. Biol. Med. 121, 96
100. Shannon L. M., Kay E. and Lew J. Y. 1966. Peroxidase isozymes from horseradish roots. J. hiol. Chem. 241, 2166 2-l 72. Simons K.. Weber T.. Stiel M. and Grasbeck R. 1964. Immunoelectrophoresis of humah saliva. 4ctu wd. ,xmd. Suppl. 412, 257-264. Strum .I. and Karnovsky M. J. 1970. Ultrastructural localization of peroxidase in submaxillary acinar cells. J. ultrustruct. Res. 31, 323-336. Stoffer H. R., Kraus F. W. and Holmes A. C. 1962. Immunochemical identification of salivary proteins. Proc. Sot. rxp. Biol. Med. 111,467-471. Tomasi T. B. Jr., Tan E. M., Solomon A. and Prendergast R. A. 1965. Characteristics of an immune system common to certain external secretions. J. exp. Med. 121, 101-124. Tung F. F. and Schein A. H. 1964. The appearance of administered proteins in saliva. J. dent. Res. 43, 4233432.
R&um&Quand la peroxydase du raifort (POR) a Cte administree aux rats par voie intraveineuse, l’activite peroxydase fut detectee histochimiquement dans la lumiere des canaux stries et dans la portion basale de cellules du canal strie de la glande sous-maxillaire. Sur l’electrophorese de gel polyacrylamidique on a identifie dans la salive sous-maxillaire deux bandes caracteristiques de POR. La detection dans la salive a Cte faite par le test de Ouchterlonys et immunoClectrophorese et par l’antigene specifique a la POR. On conclut que la POR, administree par voie intraveineuse, est assimilee par les cellules de la glande sous maxillaire et puis transportee dans la salive.
Zusammenfassung-Als Meerrettichperoxydase (HPO) Ratten i.v. verabfolgt wurde, entdeckte man Peroxydascaktivitlt histochemisch in dem Lumen des gefurchten Gangs und in dem Basalteil der gefurchten Gangzellen der submaxillaren Driise. Auf Polyacrylamid-Gel Elektrophorese wurden zwei fiir HP0 charkateristische Bander in dem submaxillaren Speichel identifiziert. Durch Ouchterlonys Test und Immunoelektrophoresc wurde ein fur HP0 eigenttimliches Antigen in dem Speichel entdeckt. Man kommt zu dem SchluB, daI.3intervenijs verabfolgtes HP0 von den submaxilllren Driisenzellen aufgenommen und dann in den Speichel iibertragen wird.
Transport
of exogenous peroxidase
Figs. I and 2. Pilocarpine-stimulated
into submaxillary
saliva
submaxillary gland excised 10 min after administration
of HPO.
Heavy PO reaction product (arrows), is seen in striated duct cells (S), but not in acinar cells (A) and convoluted glandular tubule cells (G). Counterstained with haematoxylin. x 200 Figs. 3 and 4. Pilocarpine-stimulated submaxillary gland excised 10 min after administration NaCI. No PO reaction product is present. Haematoxylin. x 200
of 0.9 “/;,
PLATE
1
A.O.B. f.p. 806
Y. Yosh ida, S. Yamamoto and Y. Kakudo
I
2
3
PLATE 2
Figs. 5-l 1. Electrophoretic patterns after staining for PO activity. Fig. 5. 0.1 per cent. HP0 sol. Fig. 6. Saliva from 0.9 per cent NaCl-uninjected rat. Fig. 7. Salivafrom a rat to which HP0 had been administered. Bands I and 2 are characteristic of HPO. Fig. 8. Saliva from a rat to which 0.9 per cent NaCI had been administered. Figs. 9-i I. Eluates from a Sephadex column. Fig. 9. Blue Dextran fraction. Fig.1 0. Partially excluded fraction. Fig. Il. Bromophenol blue fraction. Note that the bands I and 2 are identified in only the Blue Dextran fraction.
Transport
of exogenous peroxidase
into submaxillary saliva
PLATE 3
Fig. 12. Immunodiffusion of HP0 soln at various concentrations. Peripheral wells contain I per cent (l), 1 x 10-l per cent (2), 1 x 1O-2 per cent (3), 1 x 1O-3 per cent (4), I x 10e4 per cent (5), and 1 x lop5 per cent HP0 (6). Centre well contains anti-HP0 serum. The right schematic drawing. Fig. 13. Immunodiffusion showing identity of precipitin lines produced by 1 x 10e2 per cent HP0 (wells 1 and 3) and by 50-fold conc.saliva after administration of HP0 (wells 2 and 4). Wells 5 and 6 contain 0.9 per cent NaCl and 50-fold concentration of saliva after administration of 0.9 per cent NaCI, respectively. Centre well contains anti-HP0 serum.
Y. Yoshida, S. Yamamoto
W(C)
and Y. Kakudo
(A) (D)
PLATE
4
Figs. 14.-19. Immunoelectrophoretic analysis. Figures 14 and 16. HPOsoln at variousconcentrations. I per cent HP0 (14 upper), I ’ IO--’ per cent HP0 (14 lower), I < lO-‘2 per cent HP0 (I6 upper), 1 ,: lO-3 per cent HP0 (16 lower). Centre troughs contain anti-HP0 serum. Fig. 15. Schematic drawing of Fig. 14. Fig. 17. 1 per cent HP0 soln (upper) and 50-foldconc. salivaafteradministration of HP0 (lower). Note that the experimental saliva forms a single precipitm line which seems to be identical to Line A. Fig. 18. Rat serum drawn after administration of HP0 (upper) and 50-fold cont. saliva after administration of NPO (lower). Fig. 19. 50-fold cont. saliva after administration of HP0 (upper). SO-fold cont. saliva after administration of 0.9 per cent NaCl (lower).