The secretory response in dissociated acini from the rat submandibular gland

JOURNAL OF MORPHOLOGY 163:219-230 (1980)

The Secretory Response in Dissociated Acini From the Rat Submandi bular Gland

NORMAN FLEMING, MARK TEITELMAN, AND JENNIFER M. STURGESS Department of Pathology, The Research Institute, The Hospital for Sick Children, Toronto, M5G 1x8 Canada.

ABSTRACT Rat submandibular gland was dissociated by enzymatic digestion with collagenase and hyaluronidase, followed by mild mechanical shearing and filtration through a nylon mesh. The dissociated cell populations contained pre- dominantly groups of acinar cells which maintained their acinar arrangement. The morphological and functional viability of the cells was confirmed by electron microscopic examination and a normal secretory response to p-adrenergic or cholinergic stimulation was observed. Both isoproterenol (IPR) and carbachol caused the fusion of secretory granules into large vacuoles which were also continuous with the lumen, and into which the secretory product was released. Secretion was assessed quantitatively from the incorporation of 14C-glucosamine into the acinar cells and its subsequent release into the culture medium as labelled glycoprotein. IPR stimulated secretion to 125% of untreated controls in the concentration range 5 x M, and to 110% of controls a t 5 x M, after 40 min incubation. Carbachol stimulated secretion to 131% of controls a t 5 x M and to 115% at 5 x or 5 x lo-* M. The secretory response was blocked by the respective P-adrenergic and cholinergic antagonists, propranolol and atropine. These findings show that dissociated rat submandibular acinar cells provide a useful in vitro model for the study of mucus synthesis and secretion.

to 5 x

M but had no effect at 5 x

Most of the mucus in rat saliva is contributed by the acinar cells of the submandibular gland. These cells would provide a useful in vitro model for basic studies on mucus synthesis and secretion, and for investigation of mucus-re- lated disorders such as cystic fibrosis.

Mangos e t a1 ('75a) dispersed rat parotid glands by enzymatic dissociation with collagenase, hyaluronidase, and trypsin, coupled with mechanical disruption and selective filtration, to produce a suspension of functionally viable, single acinar cells which were used to study serous secretion (Mangos et al., '75a, b, c). We applied this technique to rat submandibular gland and separated single acinar cells which retained normal morphology, but were functionally unable to respond to adrenergic or cholinergic stimulation. We concluded that hormone receptor sites on the plasmalemma were destroyed during dissociation, a problem similar to that encountered by Kanamura and Barka (' 751, who dispersed the gland by a slightly different technique not involving trypsin digestion.

To retain active receptor sites on the sepa-

rated cells, we dissociated rat submandibular glands, using purified collagenase and crude hyaluronidase (Quissell, '78a). The resulting preparation consisted mainly of acinar cells which maintained in part their acinar arrangement but were separated from the duct cells and the gland stroma. The acinar cells retained typical morphology and their normal functional capacity to respond to stimulation with p-adrenergic and cholinergic agents. The secretory response has been characterized morphologically and biochemically.

MATERIALS AND METHODS

Reagents Collagenase (Clostridium hisolyticum), type

111, chromatographically purified; hyaluronidase (bovine testes) type I; DL-8-hydroxybu- tyric acid, sodium salt; bovine serum albumin (BSA); DL-isoproterenol hydrochloride (IPR); carbamylcholine chloride (carbachol); DL-propranolol hydrochloride, and atropine sulphate, were obtained from the Sigma Chemical Com- pany. Hanks balanced salt solution (HBSS); calcium- and magnesium-free; Eagle's

0362-252518011633-0219$01.80 0 1980 ALAN R. LISS, INC

NORMAN FLEMING, MARK TEITELMAN, AND JENNIFER M. STURGESS 220

Minimum Essential Medium amino acids (Eagle's MEM amino acids) with glutamine (50 x); HEPES buffer solution, 1 M, were obtained from the Grand Island Biological Com- pany, Grand Island, New York. D-(l-I4C)-glucosamine hydrochloride and Aquasol-2 liquid scintillation fluid were obtained from New England Nuclear, Canada. Nuclear track emulsion NTB 3 and D-19 developer were obtained from Kodak, Rochester, New York.

Animals Male Wistar rats (14@180 g) were made to

fast for 24 h, then anaesthetised with an intra- peritoneal injection of sodium pentobarbital, and the submandibular salivary glands were removed immediately. For each experiment, the glands of six animals were pooled and minced into pieces, approximately 0.5-1.0 mm:% which were then enzymatically dissociated.

Dissociation procedure The dissociation procedure was based on the

method of Quissell ('78a). The submandibular fragments were shaken for 1 h a t 37°C on a Dubnoff metabolic incubator in a dissociation solution consisting of HBSS, Ca++- and Mg++- free, 50 ml; BSA, 0.2%; collagenase, 4,200 units; and hyaluronidase, 5.0 mg. The suspension was buffered to pH 7.3 with 10 mM HEPES and maintained under normal atmospheric conditions in two loosely-capped, 50 ml Erlen- meyer flasks. The osmolality of the solution was 300 2 20 mOs. Mild mechanical shearing forces were applied after 30 min and 60 min, by gently pipetting the suspension up and down ten times in a 10 ml plastic pipette with the tip bore widened to 1.5 mm. The preparation was filtered through a double layer of Nitex nylon bolting cloth, mesh size - 320 pm, then centrifuged at 200 g for 5 min. The supernatant enzyme solution was discarded and the dispersed tissue washed in two changes of 40 ml incubation solution and centrifuged a t 200 g. The incubation solution consisted of complete HBSS, 100 ml; Eagle's MEM amino acids, 2 ml; P-hy- droxybutyrate, 5 mM; BSA, 2.0% and HEPES buffer 10 mM (pH 7.3; 300 * 20 mOs). The cells were resuspended in 8@100 ml fresh incubation solution, which was divided into 5 ml aliquots in 25 ml-Erlenmyer flasks which were allowed to equilibrate for 15 rnin before experimental treatment. All operations were carried out at 37°C and the suspensions were in free gaseous exchange with the atmosphere. All glassware was siliconized.

Treatment with secretagogues for ultrastructural studies

Aliquots of cells were incubated as follows: 1) untreated controls; 2) IPR, 5 x M; 3) propranolol, 1 x M, for 1 min followed by IPR, 5 x 10-j M; 4) carbachol, 5 x lo-' M; 5) atropine, 5 x lo-' M for 1 min followed by carbachol 5 x M. The flasks were shaken a t 37°C and samples taken at intervals from 3 to 90 min. The samples were centrifuged at 200 g for 5 min and the pelleted cells were processed for electron microscopy.

Electron microscopy The cell pellets were fixed at room tempera-

ture in 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.3, for 90 min, rinsed in three changes of 0.1 M sodium phosphate buffer, then postfixed in 1% osmium tetroxide in 0.1 M veronal acetate buffer, pH 7.3, for 60 min. The cells were dedydrated in graded ethanol solutions to a propylene oxide intermedium and embedded in an Epon-Araldite mixture. Sec- tions were stained with uranyl acetate and lead citrate and examined in a Philips EM 300 electron microscope at 60 kV.

Radiolabelling o f glycoproteins and scintillation counting

The dissociated acini were incubated for 1 5 2 h in 30 ml modified HBSS containing 1 pCiiml D-(l-'4C)-glucosamine hydrochloride (specific activity 45-60 mCiim mol), centrifuged at 200 g for 5 min, then washed in two changes of incubation solution. The cells were resuspended in 75 ml fresh solution and divided into 5 ml aliquots in 20 ml Erlenmeyer flasks. The aliquots were allowed to equilibrate at 37°C for 5 min, then incubated for 40 min with IPR (5 x to 5 x 10-5M) or carbachol (5 x to 5 x 10-5 M) alone or plus their respective antagonists, propranolol (1 x lo-' to 1 x M) and atropine sulphate (5 x lO-'to 5 x M). Controls received no treatment. After incubation the acini were centrifuged a t 500 g for 5 min and the cell pellets and supernatant culture media were separated. Each pellet was resuspended in 4 ml 5 mM Na,EDTA, pH 7.0, and the cells were disruped by sonication. The disrupted cells and the su- pernatants were precipitated with equal vol- umes of 20% trichloroacetic acid (TCA)IWO phosphotungstic acid (PTA), centrifuged at 500 g, washed with 10% TCAI1% PTA and dissolved in 1 M NaOH. Aliquots were mixed with liquid

DISSOCIATED SUBMANDIBULAR GLAND ACINI 221

scintillation fluid and I4C activity was counted in a Nuclear Chicago Mark I1 scintillation counter. Counting efficiency was determined and the counts were corrected to disintegra- tions per minute. Changes in the secretory index of stimulated cells were expressed as a percentage of the basal glycoprotein release from unstimulated controls.

A utoradiography Acinar cells which had been incubated with

14C-glucosamine hydrochloride were fixed and processed as for electron microscopy. Sections, 0.51.0 pm thick, were mounted on glass slides and coated with nuclear track emulsion by the dipping method. The emulsion was developed after 28 days exposure at 4°C. The cells were stained with toluidine blue and examined by light microscopy.

RESULTS

The cell population dissociated from the submandibular gland consisted of approximately 75% acinar cells, the remainder being epithelial cells from the duct system. Most acinar cells remained in groups of up to fifteen, which maintained their three-dimensional re- lationship around a central lumen. Assessed by trypan blue exclusion, 8590% of the cells were viable throughout the experimental period of up to 5 h.

A utoradiography After 2 h incubation with ''C-glucosamine,

most of the radioactivity was localized in the basal region of the acinar cells. Some silver grains were located over the secretorygranules and were interpreted as representing labelled glycoproteins (Fig. 1).

Radiolabelling experiments Unstimulated control cells secreted from

%13% of the total '*C-labelled glycoprotein over a 40 min incubation period. For each experiment, this level was assigned a value of 100.

Isoproterenol stimulated glycoprotein secretion from dissociated acini to 110% of control level at a concentration of 5 x lo-' M, and to 124-126% of controls at 5 x to 5 x M. This effect was blocked by propranolol (Fig. 2).

Carbachol had no effect on glycoprotein release a t concentrations of 5 x lO-'and 5 x lo-' M, but stimulated secretion to 115% of controls at a concentration of 5 x lo-', and to 131% a t 5 x lo-". The effect at 5 x and

5 x 10-5 M carbachol was blocked by atropine; the secretory response was from 7 to 11% less than controls (Fig. 3).

Ultrastructure After dissociation, the cells maintained their

acinar arrangement (Fig. 4) and displayed normal morphology and polarity (Scott and Pease, '59; Tamarin and Sreebny, '65). The nucleus, which often appeared to be double or

Fig. 1. Autoradiograph of dissociated rat submandibular acinus after 2 h incubation with Y-glucosamine. Most radioactivity is localized in the basal region of the cells with some in the secretory granules. x 600.

PROPRANOLOL CONCENTRATION (MOLAR) 1 ~ 1 0 ' lX1o6 lX1o5 l X 1 o 4

1LO

""1

D I

h

5 ~ ' i O ' 5x ' i07 5 ~ \ 0 ' Sx'iO5 J

ISOPROTERENOL CONCENTRATION (MOLAR)

Effect of concentration of isoproterenol (IPR) or isoproterenol plus propranolol (PRL) on glycoprotein secretion from dissociated rat submandibular acini over 40 min. Dotted line represents glycoprotein release from unstimulated controls.

Fig. 2.

222 NORMAN FLEMING, MARK TEITEI MAN. AND JENNIFER M. STURGESS

ATROPINE SULPHATE CONCENTRATION (MOLAR) 5x16' 5x16' 5x106 5x16'

0 CAR 1LO 0 CAR + AT

0

0

S i l o 8 5x1io7 5:lO6 5 i1o5 CARBACHOL CONCENTRATION (MOLAR)

Fig. 3. Effect of concentration of carbachol (CAR) or carbachol plus atropine (AT) on glycoprotein secretion from dissociated rat submandibular acini over 40 min. Dotted line represents glycoprotein release from unstimulated controls.

bilobed, was situated in the basal region of the cell and surrounded by parallel cisternae of rough endoplasmic reticulum (Fig. 5 ) . Mito- chondria were randomly distributed throughout the cytoplasm, and the Golgi apparatus, consisting of only a few flattened saccules and small vesicles, was located in the perinuclear region. The apical two-thirds of the cell was packed with secretory mucous granules whose contents appeared amorphous (Fig. 61, granular (Fig. 7), or lamellar (Fig. 81, all three types of granule frequently appearing in a single cell. The electron density of secretory product also varied within a single granule (Figs. 5,8). The cells were arranged around a narrow lumen (Fig. 5 , 9) and tight junctions were retained (Fig. 10). The lateral cell surfaces were folded into finger- or sheet-like processes which pro- jected into the intercellular canaliculi and intercellular spaces (Fig. 9). The small vacuoles near the basal plasmalemma (Fig. 9) were thought to be artefacts caused by the dissociation procedure, similar to those observed by others (Amsterdam and Jamieson, '72; Amakawa and Barka, '75; Kanamura and Barka, '75).

Control Cells: Untreated acinar cells generally maintained

the fine structure described above throughout the postdissociation incubation period of 90

min, and the secretory granules remained com- pact and discrete (Fig. 9). Occasionally, control cells showed a secretory response.

Cells exposed to Isoproterenol: Isoproterenol produced a rapid response in

the cells. By 3 min, the lumen was dilated with secreted material and some secretory granules had fused into larger structures (Fig. 10). At 10 min, granule fusion was greater, resulting in mucus-containing vacuoles which occupied a large portion of the cytoplasm (Fig. 11). Gran- ules at the cell apex fused with both the vacuoles and the lumen, so that the two became continuous (Fig. 11; Bogart, '75). Progressive cytoplasmic vacuolization may, therefore, be regarded as a process of luminal expansion. The release of granule contents into the enlarged lumen, either directly (Fig. 12), or via channels formed by granule fusion (Figs. 13, 141, repre- sented exocrine secretion. After 20 min, ex- tensive vacuolization sometimes resulted in deformation of the nucleus (Fig. 15). Degranu- lation was usually complete by 30 min, when empty granule ghosts were often noted (Fig. 16). At this stage, small droplets which appeared to contain lipid were occasionally observed in the perinuclear region (Fig. 16). Continued incubation of the cells for 90 min produced no further changes.

The response to IPR was inhibited by propranolol. Cells exposed to both these agents resembled untreated controls (Fig. 171, al- though an occasional secretory response was observed.

Cells exposed to carbachol: Carbachol produced a morphological change

similar to that caused by IPR. There was an initial distension of the lumen, vacuolization (or lumen enlargement) was obvious a t 3 min (Fig. 18) and by 15 min, the vacuoles had enlarged to occupy a large part of the cytoplasm (Fig. 19). The series of micrographs, Figures 20 to 22, shows a typical secretory response and indicates that some membrane is lost with the granular secretory product. Lamellar material resembling membrane fragments and vesicles was observed in degranulating cells a t 45 min (Fig. 23) and in some cells fusion continued after 90 min incubation (Fig. 24).

The carbachol-induced response was blocked by atropine and cells exposed to both agents normally displayed no morphological altera- tions from the quiescent state during the 90 rnin period (Fig. 25).


Fig. 4. Dissociated rat submandibular gland containingpredominantly acinar cells, which remain associated in complete or incomplete acini (A). A small number of duct cells (DC) are present. x 125.

Fig. 5. Detail of dissociated acinus showing normal ultrastructure and arrangement around a narrow lumen (L). The nuclei (N) are basal and surrounded by rough endoplasmic reticulum (RER). The cell apex is packed with discrete secretory granules (SG) which vary in both the structure and electron density of their contents. x 3,070.

Figs. 6-8. Secretory granules of dissociated acinar cells contain a product that may be amorphous (Fig. 6, x 7,970); granular (Fig. 7, x 10,625); or lamellar (Fig. 8, x 14,775) in appearance. The electron density of contents may vary within a single granule (Fig. 5).

Fig. 9. Control acinar cells 90 min after dissociation showing normal morphology. The secretory granules are discrete and contain mucin. Microvilli (MV) project from the lateral plasmalemma into the intercellular canaliculi and spaces. Small vacuoles (V) close to the basal cell membrane develop during the dissociation. x 3,985.

Figs. 1G-14.

Fig. 10.

Dissociated acinar cells exposed to isoproterenol (IPR) for 3 to 20 min

3 rnin IPR: The lumen (L) has distended to the level of the tight junctions (J) and contains granular secretory product. Some fusion (F) of secretory granules is observed. X 6,080.

Fig. 11. x 6,880.

10 min IPR Large vacuoles (V) occupy much of the cytoplasm, and are continuous (arrow) with the lumen (L).

Fig. 12.

Fig. 13.

20 min IPR Single secretory granules discharge their contents into the expanded lumen (L). x 13,600

20 min IPR: Granules deep in the cell discharge their contents into the enlarged lumen (L) through channels (arrows) formed by granule fusion. x 13,840.

Fig. 14. 20 min IPR: The release of secretory product with a fibrillar structure (arrows) can be traced through a series of four fused granules into the lumen (L). x 22,080.

DISSOCIATED SUBMANDIBULAR GLAND ACINI

Fig. 15. Dissociated acinar cells exposed to IPR for 20 min: The lumen (L) has expanded into the basal region of the cells and the nuclei (N) are distorted. x 4,640.

Fig. 16. Dissociated acinar cells exposed to IPR for 30 min: Degranulation seems complete and granules (SG) appear empty. Small droplets (arrows), which appear to contain lipid, are present in the perinuclear region. x 3,200.

Fig. 17. Dissociated acinar cells exposed to IPR plus propranolol for YO min. The secretory response i s blocked, the granules (SG) remain intact and contain mucin, and the lumen or intercellular canaliculus (L) is narrow. x 7,680.

225

226 NORMAN FLEMING, MARK TEITELMAN, AND JENNIFER M. STURGESS

Figs. 18-22.

Fig. 18. 3 min carbachol: The lumen (L) is expanded and contains granular material. Degranulation occurs

Dissociated acinar cells exposed to carbachol for 3 to 20 min.

into large vacuoles (V). x 3,520.

Fig. 19. 15 min carbachol: Large vacuoles (V) occupy much of the cytoplasm and degranulation (D) continues. x 2,800.

Figs. 2CL22. 20 min carbachol: Typical sequence of degranulation. L = extended lumen.

Fig. 20. Fusion of secretory granule with plasma membrane x 32,000.

Fig. 21. Membrane rupture. x 17,600.

Fig. 22. Release of granule contents and membrane fragments into the lumen. x 26,400.


Fig. 23. Dissociated acinar cells exposed to carbachol for 45 min. Secretory granules fuse with the plasma membrane and mucus is released into the expanded lumen (L). Some products of degradat ion (D) resemble membrane fragments. x 9,600.

Fig. 24. Dissociated acinar cells exposed to carbachol for 90 min. Various stages of granule fusion (F) are observed and a large vacuole or luminal extension (L) occupies most of the cytoplasm. x 5,120.

Fig. 25. Dissociated acinar cells exposed to carbachol plus atropine for 90 min. The secretory response is inhibited. The cells are closely associated, the intercellular canaliculus (IC) is narrow and the discrete secretory granules (SG) contain mucin ( x 7,680).

228 NORMAN FLEMING, MARK TEITELMAN, AND JENNIFER M. STURGESS

DISCUSSION

Rat submandibular glands are innervated by the sympathetic and parasympathetic branches of the autonomic nervous system (Hammer and Sheridan, '78) and, in vivo, both adrenergic and cholinergic transmitters control mucus secretion (Rossignol et al., '72). The precise mechanisms of secretion of mucus and their regulation in the gland are poorly under- stood. Most investigations in this field have been carried out on animal models, where com- plex interactions between neural and humoral factors can occur. In addition, transmitters and their analogues produce a number of different responses in the submandibular gland. The effects of the /3-adrenergic agonist IPR, which are thought to be mediated by the cyclic AMP sec- ond messenger system (Butcher et al., '76; Goldberg, '751, have been extensively studied. In addition to being an effective secretagogue (Ohlin, '66; Bogart, '75), IPR stimulates DNA synthesis, protein synthesis, and mitosis in acinar cells in vivo (Barka, '65; Malamud and Baserga, '67; Ekfors and Barka, '71; Feiglin and Reade, '78) and increases amino acid up- take in submandibular slices (Barka, '71). Chronic exposure to IPR causes hypertrophy and hyperplasia of the gland (Selye et al., '61; Cataldo et al., '65). The actions of the cholinergic agonist, carbachol, on the submandibular gland are less well documented. However, Ros- sign01 et a1 (' 72) have measured an increase in the release of glycoprotein from rat submandibular after exposure to the drug. In other exocrine glands, carbachol stimulates the release of zymogen granules from guinea pig pan- creas slices (Jamieson and Palade, '71) and the eMux of potassium ions from dissociated rat parotid acinar cells (Mangos et al., '752). Stimu- lation of human bronchial explants (Sturgess and Reid, '72) and of human and canine tracheal explants (Chakrin et al., '73; Boat and Kleinerman, '75; Boat et al., '77) with cholinergic agonists increases secretion from the tracheobronchial submucosal glands. The effects of carbachol may be mediated through the cyclic GMP system (Goldberg, '75). Some of the actions of adrenergic and cholinergic drugs may be interrelated, as carbachol is known to produce a discharge of noradrenaline from sympathetic nerve endings (Axelsson, '71). This is less likely to happen in a dissociated cell system than in vivo, or in organotypic culture.

The present investigation is designed to provide an experimental model to study the direct action of stimulants on acinar cells. In the par-

tially dissociated acini, both IPR and carbachol produce a rapid secretory response which is blocked by the respective p-adrenergic and cholinergic agonists, propranolol and atropine. The duration and the morphological characteristics of the response are consistent with those observed in the submandibular acinar cells of rats, mice, and guinea pigs after exposure to secretagogues in vivo (Junqueira et al., '64; Radley, '69; Kim et al., '72; Bogart, '75; Albano et al., '76). The most obvious modification in the cells is the fusion of secretory granules, with the formation of large vacuoles into which the granules release their contents. By examining serial sections, Bogart ('75) shows that such vacuoles, produced by IPR, are extensions of the expanded acinar lumen, which penetrate deep into the cell cytoplasm. In the stimulated cells examined here, continuity between lumen (or apical extracellular space) and vacuoles is indicated also (Fig. 11). Fusion of secretory granules with these vacuoles and the release of granule contents into the vacuoles, therefore, constitute a process of exocytosis (Figs. 12-14, 2&22).

In the present study, no morphological dif- ferences are detected in rat submandibular acinar cells stimulated with IPR or with carbachol. In the quantitative studies, secretion is stimulated optimally by IPR in the concentration range 5 x 10-'-5 x M. This response is reduced at 5 x lo-' M, indicating that p-adrenergic receptor sites on the acinar cell membrane are saturated at an IPR concentration between 5 x lo-' and 5 x M. This is consistent with the findings of Quissell ('78b) who reports an EC 50 of 3.9 x lo-' for IPR with respect to glycoprotein secretion in dissociated acinar cells.

In contrast, carbachol provokes a maximal secretory response at 5 x 10-5 M, this is reduced by half at 5 x M and lower concentrations have no effect. The inhibition by atropine of the carbachol-induced secretory response to levels lower than those of controls, may mean that the observed basal secretion rate from control cells is due to residual cholinergic activity (acetylcholine) in the preparations, or to the stabilization of the acinar cell membrane by some unknown mechanism.

Previous attempts to produce populations of submandibular acinar cells, which remain morphologically and functionally viable, have met with limited success. Barka and coworkers (Amakawa and Barka, '75; Kanamura and Barka, '75) have prepared single acinar cells by digestion with crude collagenase and hyaluron-

229 DISSOCIATED SUBMANDIBULAR GLAND ACINI

idase, chelation with EDTA, mechanical disruption, and centrifugation in Ficoll. The cells retain their normal morphology and the capacity to bind concanavalin A (Amakawa and Barka, '751, but do not respond to IPR stimulation (Kanamura and Barka, '75). These authors conclude that the P-adrenergic receptor sites on the plasma membrane are destroyed or per- turbed by contaminating proteolytic enzymes in their crude collagenase or hyaluronidase preparations. In isolated pancreatic acinar cells, prepared by a similar method, Amster- dam and Jamieson ('72) describe a reduced secretory response to carbachol, suggesting a disruption of the cholinergic receptor sites. We have dissociated rat submandibular glands with crude collagenase preparations, and are unable to demonstrate a secretory response in the dispersed acinar cells to either IPR or carbachol. Quissell ('78a) has prepared functionally normal acinar cells by enzymatic dissociation with purified collagenase and hyaluronidase coupled with mechanical force. These cells are viable, release potassium in response to cholinergic and adrenergic stimulation, and secrete glycoprotein on exposure to adrenergic agonists (Quissell, '78b).

In the present study, using cells prepared by a modified Quissell technique, we have confirmed the secretory response to p-adrenergic stimulation both quantitatively and qualita- tively, and have characterized the cholinergic response also. It is essential to use chromatographically purified collagenase, in which residual protease activity is extremely low, to separate acinar cells with intact hormone receptor sites on the plasmelemma. These cells retain their normal ultrastructure and functional integrity over a short term in vitro, pro- viding an experimental model for the study of mucus synthesis and secretion. Furthermore, the model has potential applications to evalu- ate the effect of various agents, such as hor- mones, cyclic nucleotides, and drugs on these processes. The dissociated cell model has con- siderable advantages over organotypic cul- tures, where several different cell types con- tribute to the overall response of the tissue, and where the release of factors from these cells may modify acinar cell responses (Mangos et al. '75a). Cells that retain their association in complete or incomplete acini may be a more suitable model than single dissociated cells. In the former, cell junctions are retained, suggesting the maintenance of electrical coupling, which may be a n important factor in propagat- ing the secretory stimulus (Hammer and

Sheridan, '78). In addition, the orientation of cells around a lumen makes possible a more direct comparison between the in vivo and in vitro secretory responses.

ACKNOWLEDGMENTS

We gratefully acknowledge the advice of Dr. John Mangos and Dr. David Quissell in devel- oping the experimental technique.

This work was supported by the Sellers Foundation and the Canadian Cystic Fibrosis Foundation.

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The secretory response in dissociated acini from the rat submandibular gland