Salivary Gland Secretion And Its Relation To

Salivary Gland Secretion and its Relation to Chromosomal Puffing in the Dipteran, Chironomus thummi Hans Laufer, and Yasukiyo Nakase PNAS 1965;53;511-516 doi:10.1073/pnas.53.3.511 This information is current as of May 2007. This article has been cited by other articles: www.pnas.org#otherarticles E-mail Alerts

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Hamer, D., Biol. Bull., 108, 35 (1955). Bernstein, M. H., and D. Mazia, Biochim. Biophys. Acta, 11, 59 (1953). Mz Afelius, B. A., Z. ZeUforsch., 42, 134 (1955). 6 Wilkins, M. H. F., 11th Chemistry Conference of Solvay Institute, Conference on Nucleoproteins, New York, 1955, p. 45. 7Bernstein, M. H., and D. Mazia, Biochim. Biophys. Acta, 10, 600 (1953). 8 Gall, J., Science, 139, 120 (1963). 9 Parson, D. F., J. Cell Biol., 16, 620 (1963). '0 Nass, S., and M. M. Nass, J. Cell Biol., 19, 613 (1963). 11 Kleinschmidt, A., and D. Lang, 5th Intern. Congr. for Electron Micr., Philadelphia, 1962 (Academic Press), vol. 2, pp. 0-8. 12 Hofstee, B. H. J., Biochim. Biophys. Acta, 55, 440 (1962). 13 Caulfield, J. B., J. Biophys. Biochem. Cytol., 3, 827 (1957). 14 Ris, H., Can. J. Genet. Cytol., 3, 95 (1961). 16 Kaufman, B., H. Gay, and M. R. McDonald, Intern. Rev. Cytol., 9, 77 (1960). 16 de Robertis, E., in Control of Cell Division and Cancer Induction, National Cancer Institute Monograph No. 14 (1964), p. 33. 17 de Robertis, E., J. Biophys. Biochem. Cytol., 2, 785 (1956). 18 Davies, H. G., and M. Spencer, J. Cell Biol., 14, 445 (1962). 19 Ris, H., and B. L. Chandler, in Synthesis and Structure of Macromolecules, Cold Spring Harbor Symposia on Quantitative Biology, vol. 28 (1963), p. 1. 20 DuPraw, E. J., these PROCEEDINGS, 53, 161 (1965). 21 Zubay, G., and P. Doty, J. Mol. Biol., 1, 1 (1959). 22 Commerford, S. L., M. J. Hunter, and J. L. Oncley, J. Biol. Chem., 238, 2123 (1963). 23 MacHattie, L. A., and C. A. Thomas, Science, 144, 1144 (1964). 24 Hall, C. E., and M. Litt, J. Biophys. Biochem. Cytol., 4, 1 (1958). 26 Beer, M., and R. Zobel, J. Mol. Biol., 3, 717 (1961). 26 Stoeckenius, W., J. Biophys. Biochem. Cytol., 11, 297 (1961). 27 Zubay, G., in The Nucleohistones, ed. J. Bonner and P. Ts'o (San Francisco: Holden-Day, Inc., 1964), p. 95. 28 Mazia, D., these PROCEEDINGS, 40, 521 (1954). 29 Osgood, E. E., D. P. Jenkins, R. Brooks, and R. Lawson, Ann. N. Y. Acad. Sci., 113, 717 (1964). 3 4

SALIVARY GLAND SECRETION AND ITS RELATION TO CHROMOSOMAL PUFFING IN THE DIPTERAN, CHIRONOMUS THUMMI* BY HANS LAUFER AND YASUKIYo NAKASE DEPARTMENT OF BIOLOGY, JOHNS HOPKINS UNIVERSITY

Communicated by W. D. McElroy, January 5, 1965

Puffs of the dipteran salivary gland chromosome probably reflect gene activity. Support for this belief is derived from the tissue- and stage-specificity of the puffing patterns (cf. Beermann).1 Further supporting evidence is obtained from the observations of heightened RNA synthesis in puffed loci (cf. Pelling).2 Tissuespecific puffs, particularly the Balbiani ring loci, have been correlated with secretion.3-"' In the present study we have examined the manner in which puffs are correlated with salivary secretion. Either elements of the secretion must be synthesized directly in the gland, or else their synthesis occurs elsewhere in the body and the

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gland functions in their uptake, transport, and secretion. Our results indicate that the major constituents of the secretion are not synthesized directly by the gland but that this tissue mediates their transport and secretion. Materials and Methods.-Experimental animals: The experimental organisms used were Chironomus thummi reared to the fourth instar in the laboratory on a diet of nettle powder. These larvae were selected for uniformity of external morphology and size.'0 Preparation of secretion extracts: Secretion was collected from 200 or more animals in each experimental series according to the method of Defretin"2 by permitting animals to secrete onto sterile, reagent-grand sand in depression slides moistened with distilled water. The secretion was transferred to test tubes, rinsed, then extracted with 0.1 M NaCl adjusted to pH 8.5 with 0.01 M Tris (hydroxymethyl)-aminomethane ("Tris") buffer. Each mass, containing about 4-5 1g of protein, was extracted in 0.05 ml of buffered saline. Protein content of secretion and tissues was determined according to the method of Lowry et al.13 In preliminary experiments, the NaCl extract was tested for contamination by microorganisms or feces. No decrease in activity was detected in secretions produced in the presence of streptomycin and penicillin (Parke, Davis and Co.) at concentrations that inhibited more than 95% of the microorganisms. Also tests of the bacterial contaminant resistant to the antibiotics showed that these could not account for the enzyme activity of the secretion. One mg/ml of the microorganisms contained less than 1/10,000 of the hyaluronidase activity, for example, of a similar amount of salivary secretion. Contamination of the secretion by feces was eliminated by ligating the gut at the posterior end of the larva. At least four series of experiments were conducted on larvae with ligated anuses. No observable differences were detected between the secretion of these animals and that of controls. Immunochemical procedures: Precipitating antisera were produced by injecting at least 3 mg of protein antigen into rabbits; antisera were tested in agar diffusion by the method of Ouchterlony.14 Enzyme assays: Hyaluronidase, trehalase, malate dehydrogenase (MDH), and DNase were determined as in our previous publications.-, 1"5 RNase was assayed by the method of Anfinsen et al.16 Esterase and protease determinations were performed according to Laufer."7 For most assays homogenates of 2-8 pairs of glands, or 50-200 ,ug of protein, were incubated with the appropriate substrate for 30 min. Isotope tracer experiments: C'4-labeled algal protein hydrolysate (Schwartz) with an activity of 70,000 counts/min in 2.5 Mul, was injected into a posterior larval "leg" which was then ligated with nylon thread to seal the puncture. Injected larvae were exsanguinated after 10 hr and the hemolymph was pooled. The labeled blood was centrifuged free of cells at 10,000 X g. In this way a cleared supernatant of approximately 3 MAl was obtained from each animal. Pooled blood was dialyzed and concentrated for 48 hr against several changes of 50% polyvinylpyrrolidone (PVP, Sigma, av. mol. wt. 40,000) in 0.1 M NaCl. Doses of this labeled protein solution were injected into Chironomus larvae. In 11 series of experiments (each with more than 100 animals) larvae were injected with 2.5 MAl of protein solution containing 600 counts/min. The secretions from these protein-injected animals were collected by the usual procedure at designated intervals. The perchloric acid precipitate of the secretion was washed twice and plated on planchettes, dried, and assayed for radioactivity in a thin window gas flow counter with automatic readout attachment. Two series of animals were examined for the possiblity of tracer excretion via the gut. The secretion of normal animals injected with labeled blood was compared with that of others bearing a posterior gut ligature which also closed off the injection site. No hemoglobin was found to be extruded into the medium as is observed in the case of unligatured injected animals (i.e., wounded). In further experiments I"3'-human serum-albumin was injected as above. The secretion was extracted in the usual manner, concentrated, and then tested with antihuman serum albumin antibodies. Solutions of I"3'-human albumin (Squibb, Albumotope, 1 mc/ml) containing about 0.25 mg of protein were injected in 2.5-MAl doses into 4th instar Chironomus larvae. Commercial antihuman serum albumin (Nutritional Biochemical Co.) was used for the detection of the labeled albumin in the secretion of injected larvae. One-tenth ml antibody solution quantitatively precipitated 1-20 ,ug of iodinated human albumin in a microprecipitin test.'4

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Radioautographs were prepared from 10-,u sections of salivary glands from animals injected with labeled blood proteins according to the recommendations of Kopriwa and Leblond.18 Exposures were for 2-, 3-, and 4-week intervals.

Results and Discussion.-Nature of the secretion; enzyme analyses: We have shown that the salivary gland and its secretion contain the following enzyme activities: trehalase, hyaluronidase, esterase, DNase, and protease.7'-0 15 Tests of other tissues such as gut, Malpighian tubes, fat-body, and blood revealed that all contain some of the activities present in the secretion (Table 1). None of the enzymes tested was found exclusively in the salivary secretion. These enzymes are therefore shown not to be tissue-specific. TABLE 1 ENZYME ACTIVITY OF Chironomus thummi 4TH INSTA.R LARVAL TISSUES AND SALIVARY SECRETION Enzyme

secretion

5alivary

Salivary gland

Trehalase Hyaluronidase M.D.H. Esterase

9.1 7.5 0

33.4 8.9 183.6

propionate a-Naphthyl acetate Peptidase RNAse DNAse

1.9

0.8

3.7 6.2 1.2 9.7

0.8 16.2 2.5 1.6

,6-Naphthyl

Activity in OD/mg Protein Malpighian Blood tubes

Gut

Fat-body

114.2 18.7 313.6

81.4 14.5 432.3

23.8 26.7 622.5

0.5

8.1

3.8

3.7

0.5 3.2 0.07 0.5

4.3 17.0 3.1 0.5

5.4 8.3 0.5 1.3

3.2 6.8 1.4 1.5

1.24 3.4 23.8

Immunochemical analyses: Immunochemical tests revealed the existence of six antigenic components in the secretion. Immune sera prepared against either salivary gland secretion or hemolymph, when tested in agar diffusion by the Ouchterlony procedure, revealed the presence of what appear to be identical antigens in the two materials (see Fig. 1). Each line or reaction complex represents one or more antigenic components. Antisera against the secretion reacted as strongly with hemolymph as did antisera against blood. Fusion of immune precipitates between adjacent reservoirs indicates a close relationship between the components of the fused lines. Identical reactions with secretion were obtained with antibodies directed against secretion and against blood (see Fig. 1). Blood, however, was found to possess several antigens not found in the secretion. We conclude that all major antigens in the secretion are also present in blood. Uptake and secretion of protein by the salivary gland; isotope-labeled blood proteins: Within 2-5 hr after the injection of dialyzed blood proteins label appears in the secretion which becomes progressively more radioactive. A series of animals was injected with isotopically labeled insect blood protein prepared as described in the Materials and Methods section. Virtually all of the radioactivity (about 97%) of these dialyzed blood proteins was precipitable with perchloric acid. The procedures used to purify the labeled blood proteins were also found to remove added C14labeled amino acids from samples both of blood and of secretion. It seems unlikely, therefore, that the activity of the labeled blood protein was due to absorbed amino acid. Radioautographs revealed as early as 1 hr after the injection of radioactive blood proteins that the label appears in vacuoles within the cytoplasm of secretory cells of

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FIG. 1.-Agar diffusion plate showing the antigenic similarities between Chironomus thummi salivary gland secretion and blood (hemolymph). There are no antibodies detectable in secretion antisera that are not also present in antisera to blood. Central reservoir contains secretion; peripheral reservoirs starting at the top center contain: (A) antiblood; (B) anti-

secretion; (C) anti-"heterologous protein"; (D) antiblood from a

different rabbit than in (A); (E) antisecretion from the same rabbit as in (B), but from a different bleeding; (F) anti-"heterologous protein" from a different rabbit than in (C). Confluence of precipitate lines of antigen-antibody reactions emanating from adjacent peripheral reservoirs suggests antigenic identity of the reactants. In this case the injection into different rabbits of secretion or blood stimulated the formation of antibodies which are

apparently indistinguishable.

These experiments indicate the general distribution of secretion antigens.

the recipient animals. These vacuoles, observed with phase contrast microscopy, have the dimensions and appearance of secretion granules. Several such vacuoles are visible at the basal or hemal edge of secretory cells. The appearance of some of these peripheral vacuoles suggests a communication with the outside surface of the cell. This impression is reinforced by the presence of isotopically labeled protein within such peripheral vacuoles in radioautographic preparations. Our observations to date suggest that the uptake of proteins from the blood by the salivary gland occurs by a process similar to or identical with pinocytosis. They suggest further that blood proteins may be taken up and later secreted by the salivary gland. They do not tell us, however, whether proteins pass intact or whether they are modified in passage. Uptake of I'31-human serum albumin by the insect salivary cells: To test whether proteins are altered during transfer, foreign protein was injected into the blood and then traced through the gland. Four series of experiments were conducted with human serum albumin injected into C. thummi. The results of one such experiment (parts A and B) are summarized in Table 2. They will serve to indicate the nature of the results consistently obtained in a larger series of experiments. Just as Chironomus hemolymph seems to be transported by the salivary gland, human serum albumin is also taken up and transported through the gland. The radioactivity appears in the secretion within a few hours, reaching a maximum between 1 and 10 hr. The last two columns of Table 2, indicating the number of counts of I'll elaborated into the secretion per hour by one animal, provide us with a rough estimation of secretory rate. The data are variable, in part at least, because the animals secrete intermittently. When the extract prepared from secretion is assayed in the precipitin test for human albumin with antihuman albumin antibodies, as much as 95 per cent of the radioactivity can be precipitated. This shows that the

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TABLE 2 DISTRIBUTION OF COUNTS AFTER THE INJECTION OF I'll-HUMAN SERUM ALBUMIN INTO THE BLOOD OF 4TH INSTAR Chironomus thummi Incubation time (hr)

Part A 3 5 10 20 30 40 Part B 0-1 1-3 3-5 5-10 10-20 Control: not injected 0-5 Control: not injected

5-10

Counts in blood protein/ animal

gsg Protein secreted/ animal/hr

5,422 5,684 6,069 5,634 9,059 9,880

Counts in protein/ salivary gland

Counts secreted in protein/ animal/hr

62 44 273 155 849 263

64 86 282 126 126 212

Counts ppt. by

antialbumin/ animal/hr

87 703 309 292 192

1.8 1.8 1.8 2.2 2.2

100 708 315

1.8

6

No ppt.

2.4

8

No ppt.

355 211

albumin injected into the hemolymph is transported across the gland and into the secretion intact, without major alteration or degradation. Relation between salivary gland function and chromosomal puffing: The strongest evidence supporting a close relationship between salivary gland secretory activity and chromosomal puffing at a Balbiani ring locus is provided by the work of Beermann. 19 He showed by hybridization of animal stocks differing with respect to one Balbiani ring that the presence of activity at this locus determined the appearance of a particular kind of cytoplasmic granule which was released into the secretion. In addition, other experiments in the literature also suggest such relationships.,,-" The findings of the present study amplify and extend these earlier observations. In doing so they indicate that the puffing pattern cannot be related directly to the synthesis of the major constituents of the salivary secretion. Our study of the distribution of the enzymes and antigens of the secretion demonstrates their lack of tissue-specificity. The proteins and antigens are an integral part of the salivary secretion since the gelled structure of the secretion is destroyed by the proteolytic enzyme pronase.20 Furthermore, we have been able to show by isotopic tracer techniques that constituents of the secretion can be, and probably are, derived from blood. This is true regardless of whether Chironomus blood proteins or foreign proteins are labeled and followed into, through, and out of the gland. The use of I'3l-labeled human serum albumin has further permitted us to ascertain whether the proteins were altered as they passed through the gland. These proteins are taken up and transported through the gland into the secretion without detectable loss of antigen specificity, since more than 95 per cent of the radioactivity of the secretion can be precipitated by antibodies. They appear therefore to be transferred intact. Previous findings suggested that the salivary gland secretion is dependent upon tissue-specific chromosomal puffs, particularly the Balbiani rings of the salivary gland; the present results indicate a less direct relationship exists between these than has heretofore been assumed. It may be that certain of these tissue-specific

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puffs function in the elaboration of a transport mechanism by which the gland selects, takes up, and concentrates a major portion of its secretion. Summary.-An analysis of the proteins of the salivary secretion of Chironomus thummi larvae has revealed the presence of a number of enzymes and antigens. These enzymes and antigens are indistinguishable from a number of constituents also present in the insect's blood and in other tissues. None of the major protein components of the secretion that we have detected are tissue-specific in the sense of being unique to the salivary gland cells. By injecting isotopically labeled proteins into the larval coelom, we could demonstrate that both hemolymph proteins and human serum albumin are taken up by the salivary gland cells and secreted in saliva. Autoradiographic techniques indicated that these proteins were taken up at the hemel border of the gland and were then incorporated into vesicles having the appearance of secretion granules. Transfer of protein seems to occur without degradation, since the human serum albumin retained its antigenic properties as well as its radioactivity. The secretory processes of the salivary gland appear to be intimately linked with the activities of tissue-specific puffs of the chromosomes, particularly with the largest of the puffs (the Balbiani rings). These findings indicate that the tissuespecific puffs are not related to the synthesis of specific constituents of the salivary secretion. The possibility is suggested that the puffs are associated with the selective uptake, concentration, and release of the secretion. The authors wish to express their gratitude to Drs. Paul Gross, Philip Hartman, Irwin Konigsberg, C. L. Markert, and M. S. Steinberg for their helpful comments on the manuscript. * Supported in part by grants from the National Science Foundation. Certain of the results and conclusions presented here were reported at the Gordon Research Conference on Regulatory Mechanisms in Biology, Tilton, New Hampshire, June 15-19, 1964, and at the 4th International Symposium of Comparative Endocrinology, Paris, France, July 20-26, 1964. 1 Beermann, W., Chromosoma, 5, 139-198 (1952). 2 Pelling, C., Chromosoma, 15, 71-122 (1964). 3 Beermann, W., Z. Naturforsch., 7b, 237-242 (1952). 4Mechelke, F., Chromosoma, 5, 511-543 (1953). 5 Vogt-Kohne, L., Chromosoma, 12, 382-397 (1961). 6 Becker, H. J., Chromosoma, 13, 341-384 (1962). 7Laufer, H., Proc. Intern. Congr. Zool., 16th, Washington, D. C., 4, 215-220 (1963). 8 Laufer, H., Y. Nakase, and J. Vanderberg, Am. Zool., 3, 386 (1963). Laufer, H., Y. Nakase, and J. Vanderberg, Biol. Bull., 125, 359 (1963). 10 Laufer, H., Y. Nakase, and J. Vanderberg, Develop. Biol., 9, 367-384 (1964). 11 Panitz, R., Biol. Zentr., 83, 197-230 (1964). 12 Defretin, R., Compt. Rend., 233, 103-105 (1951). 13 Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem., 193, 265-275 (1951). 14 Kabat, E. A., and M. M. Mayer, Experimental Immunochemistry (Springfield, Illinois: Charles C Thomas, 1961), 2nd ed. 16 Laufer, H., and Y. Nakase, J. Cell Biol., in press (1965). 16 Anfinsen, C. B., R. R. Redfield, W. L. Choate, J. Page, and W. R. Carroll, J. Biol. Chem., 207, 201-210 (1954). 17 Laufer, H., Ann. N. Y. Acad. Sci., 89, 490-515 (1960). Modified for tissue homogenates using naphthyl acetate and naphthyl propionate as esterase substrates and benzoyl-DI-arginine naphthylamide hydrochloride for protease. 18 Kopriwa, B. K., and C. P. Leblond, J. Histochem. Cytochem., 10, 269-284 (1962). 19 Beermann, W., Chromoaoma, 12, 1-25 (1961). 20 Laufer, H., and Y. Nakase, unpublished data (1964).