Chapter 32 Development of Stimulus—Secretion Coupling in Salivary Glands

METHODS I N CELL BIOLOGY. VOLUME

23

Chapter 32 Development of Stimztlzts -Secretion Coztpling in Sulivury GZunds LESLIE S. CUTLER. CONSTANCE P. CHRISTIAN. BRIAN BOTTARO

AND

Department of Oral Diagnosis. University of Connecticut School of Dental Medicine. Farmington. Connecticut

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . .

11.Methods . . . . . . . . . . . . . . . . . . . . . A . Animals . . . . . . . . . . . . . . . . . . . . B . Secretion System . . . . . . . . . . . . . . . C . Peroxidase Assay . . . . . . . . . . . . . . . D . Preparation of Enriched Plasma Membrane Fraction .

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E . Protein Determination . . . . . . . . . . . . . . . . . . . . . . . F . Measurement of Adrenergic Binding Sites . . . . . . . . . . . . . . . G . Adenylate Cyclase Determination . . . . . . . . . . . . . . . . . . . H . Electrophysiology of Neonatal Secretion . . . . . . . . . . . . . . . . I . Catecholamine-Containing Nerves in the Developing SMG . . . . . . . . I11 . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Adenylate Cyclase in SMG Development . . . . . . . . . . . . . . . B . Secretion of Peroxidase . . . . . . . . . . . . . . . . . . . . . . . C . [3H]Dihydroalprenolo1and [3H]Dihydroergocryptine Binding . . . . . . . D . Electrophysiological Stimulation of Secretion . . . . . . . . . . . . . . E . Glyoxylic Acid Staining for Catecholamine-Containing Nerves . . . . . . . IV . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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53 1 Copynght 0 I98 I by Academic Press. lnc All rights of reproduction in any form reserved ISBN 0-12-564123-0

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I. Introduction The morphological and functional aspects of the exocrine secretory process have been extensively studied in the pancreas (Jamieson and Palade, 1967a,b, 1968a,b, 1971; Car0 and Palade, 1964; Warshawsky et al., 1963), salivary glands (Amsterdam et al., 1969, 1971; Hand, 1970, 1971, 1972; Cutler and Chaudhry, 1973a; Kim et al., 1972; Simson, 1969; Schramm, 1967), and several other exocrine systems (Oron and Bdolah, 1973; Vidic, 1973; Scott and Pease, 1959). In all these systems the basic morphology of the exocrine cells is essentially identical. All these secretory cells demonstrate the typical functional, apical to basal polarity of organelle distribution. While the exocrine cells of the rodent pancreas, parotid, and submandibular glands utilize the same morphological mechanisms for the synthesis, accumulation, and discharge of their secretory product, there are some differences in the sequence of developmental events that lead to morphologically differentiated secretory cells in each system. There are differences in the timing of the onset of secretory cell differentiation as well as differences in the rate of cell maturation (Pictet et al., 1972; Rutter et al., 1964; Redman and Sreebny, 1971; Cutler and Chaudhry, 1974). However, the general pattern of progressive accumulation of granular endoplasmic reticulum followed by the maturation of the Golgi apparatus and then the appearance of distinct zymogen granules seems to be a consistent observation in the development of most exocrine cells (Cutler and Chaudhry , 1974). Having developed t'he capability to synthesize and package proteins for export does not a priori indicate that the packaged material can be or is released by the conventional mechanisms regulating exocytosis in mature exocrine cells. The release of zymogen granules from mature secretory cells is a complex process, initiated by the interaction of specific hormonal or neurohormonal agonists with specific receptors at the cell surface. This interaction of agonist and receptor initiates a cascade of intracellular events, which leads to the fusion of secretory granules with the cell surface and the release of the granule contents into the acinar lumen. This linkage between the action of a hormonal agonist at the cell surface with the exocytotic process is referred to as stimulus-secretion coupling. There is evidence that in the developing rat pancreas and salivary glands the secretory cells develop the capability to synthesize and package secretory proteins prior to the time that these cells are capable of exocytosis in response to hormonal stimuli (Doyle and Jamieson, 1978; Grand et al., 1975; Grand and Shay, 1978; Cutler, 1977a, 1978). The present report provides evidence that the secretory cells of the developing rat submandibular gland (SMG) acquire the ability to synthesize and package secretory proteins prior to attaining the ability to release the packaged product in

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response to hormonal stimuli. This is consistent with previous observations in the pancreas and parotid gland. The report correlates the development of the secretory response with studies on hormonal activation of cell surface-associated adenylate cyclase and with direct measurements of P-adrenergic and a-adrenergic binding sites. The report thus provides a picture of the development of the stimulus-secretion coupling system in this model exocrine system. Finally, this report presents electrophysiological and morphological evidence indicating that, in this system, development of the stimulus-secretion coupling mechanism precedes the establishment of the neural connections that regulate secretion in vivo.

11. Methods A . Animals Female Sprague-Dawley rats were singly caged in an environmentally controlled room and given rat chow and water ad libitum. At 8:00 P.M. on the appropriate day of the estrus cycle, the females were placed in breeding cages with males and left until 8:00 A.M.of the next morning. Vaginal smears were taken and examined for sperm as an evidence of copulation. The zygotes were considered zero hours old at 8:00 A.M. on the day sperm was found (Cutler and Chaudhry, 1973a,b,c, 1974, 1975, Cutler and Rodan, 1976; Cutler, 1977a,b; Mooradian and Cutler, 1978). This procedure for calculating gestational age has recently been used as the standard method for the estimation of gestational age in the study of salivary gland development (Young and van Lennep, 1978). Animals used in postnatal studies were obtained from our breeding colony in order to regulate the conditions of conception and gestation.

B . Secretion System To measure the secretory response of perinatal glands a slice system similar to that of Bogart and Picarelli (1978) was used. The incubation medium was supplemented Krebs-Ringer bicarbonate (KRB) composed of 109 mM NaCl, 13.8 mM KCl, 2.5 mM CaCl,, 1.2 mM KH2P04,25 mM NaHCO,, 1.2 mM MgS04, 5 mM P-hydroxybutyric acid, 0.5 mM adenine, 10 mM inosine, and 5.6 m M D-glucose. The medium was gassed with humidified 95% 0 2 - 5 % CO,. Slices from at least 24 rudiments (21 days of gestation) or the appropriate number of neonatal glands ( 1 and 6 days of age) were isolated in complete Krebs-Ringer buffer medium and then washed in 25 ml of buffer (37°C) for 10 minutes before the incubation was initiated. The tissue was then divided into aliquots and placed

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in nitrocellulose test tubes (25 x 80 mm) containing 4 ml of the complete Krebs-Ringer bicarbonate medium. In order to induce secretion, either L-isoproterenol, L-norepinephrine, or L-phenylephrine was added to duplicate tubes at a final concentration of M. Fresh agonist (equivalent to that added at “0” time) was added to the medium after 15 minutes of incubation to compensate for oxidation of the agonist. In some experiments the effects of the P-adrenergic antagonist propranolol or the a-adrenergic antagonist phentolamine on agonist-induced secretion were evaluated. In these experiments the respective antagonists were added to the preincubation medium and to the incubation medium at concentrations 10- to 100-fold as great as the agonist concentration. Antagonists were also added to the medium during the incubation when additional agonist was added. In addition to these adrenergic agonists, the ability of N 6 ,04-dibutyryl cyclic AMP ( M )and 8-bromo cyclic GMP ( lop3M )to induce secretion was tested in this system. Secretion was assessed by taking aliquots of the medium 30 minutes after introduction of the potential agonist and determining the amount of secretory peroxidase released into the medium.’ At the termination of the experiment the slices were homogenized in the remaining medium; the homogenate was centrifuged at 1000 g, and the resulting supernatant was assayed for peroxidase activity. The quantity of peroxidase in the homogenate plus the amount in the aliquot removed from the system during the experiment represented the total peroxidase activity in the sample at “0” time. The results were reported as the percentage of the total peroxidase in the sample at “0” time released into the medium. This release was compared to equivalent control experiments in which no agonists were added to the secretion medium.

C. Peroxidase Assay Aliquots of secretion medium were assayed by adding 0.1 ml of sample to 2.85 ml of 0.1 M sodium phosphate buffer (pH 7.0) containing 5 x M diaminobenzidine (DAB). The reaction was started by addition of 0.05 ml of 0.6% H202,and the change in absorbance was monitored at 460 nm on a Gilford 250 spectrophotometer equipped with a chart recorder. The measurements were carried out aginst a DAB-phosphate buffer blank (Herzog and Fahimi, 1973). Peroxidase activity was linear for 60-90 seconds in this system, and in our hands the assay was sensitive to less than 0.1 ng of horseradish peroxidase (HRP) (Sigma Type VI) (Cutler el al., 1977). The concentration of peroxidase in the media was calculated by comparing the AAlmin of the sample to standard values ’In the prenatal and early postnatal SMG, secretory peroxidase is present in the proacinar cells (Yamashina and Barka, 1972).

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established the same day against freshly prepared known concentrations of HRP (Sigma Type VI). Standards were run in DAB-gelatin medium according to the procedure of Herzog and Fahimi (1973).

D. Preparation of Enriched Plasma Membrane Fraction Glands or rudiments were minced and washed in cold 0.1 M phosphate buffer containing 0.25 M sucrose, 1 mM EDTA, and 1 mM dithiothreitol (DTT). The minced tissue was homogenized in a Teflon-glass homogenizer for 30 seconds in cold wash buffer (4"C), and the homogenate was centrifuged at 1000 g for 10 minutes in the cold to remove unbroken cells, nuclei, and other debris. The supernatant was centrifuged at 30,000 g for 15 minutes at 4°C on a Beckman J21-C centrifuge, and the resultant pellet was suspended in cold 10 mM Tris (pH 7.6) containing 1 mM DTT. This suspension was placed on a sucrose gradient with steps of 38% and 42%. The gradient was centrifuged at 100,000 g for 60 minutes on a Beckman L5-50ultracentrifuge. The material that layered at the suspension-38% sucrose interface was harvested, diluted in 10 mM Tris (pH 7.6)-1 mM DTT buffer, and then concentrated by centrifugation at 30,000 g for 15 minutes. The resulting pellet has been assayed for structure, adenylate cyclase activity, 5'-nucleotidase activity, and succinic dehydrogenase activity, and the results were compared with similar assays performed on the 1000 g pellet and the 30,000 g pellet prior to the sucrose gradient step. The material recovered from the gradient contained predominantly smooth-surfaced membrane vesicles with no mitochondria when examined by electron microscopy, showed a 5- to 10-fold increase in the specific activity of adenylate cyclase, an 18- to 20-fold increase in the specific activity of 5'-nucleotidase, but no measurable succinic dehydrogenase activity.

E.

Protein Determination

All protein determinations were made according to the procedure of Lowry et al. (1951).

F. Measurement of Adrenergic Binding Sites To measure P-adrenergic binding sites, assays were initiated by adding 100 p g of membrane protein to an incubation medium of 10 mM Tris (pH 7.6) containing 1 mM DTT and ~-[~H]dihydroalprenolol (New England Nuclear, Boston, MA) (1-25 nm) with a final volume of 500 p1 (control experiments showed that 1 mM DTT did not effect [3H]dihydroalprenolo1binding). All samples were run in duplicate. The reaction was run for 5 minutes at 37°C and was terminated by adding 6 ml of cold (4°C) 0.85% saline containing M dl-propranolol and

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immediate filtration of the sample through a Whatman GB/F filter. The filter was then washed with an additional 15 ml of saline. The filters were placed in scintillation fluid and counted in an Isocap/300 liquid scintillation counter. Duplicate tubes containing M dl-propranolol were incubated, treated as aoove, and run for each sample to determine nonspecific binding. Only those counts that could be displaced by the dl-propranolol were considered specific (Mukherjee et al., 1975a,b). In previous studies, it was found that ~-[~H]dihydroalprenolol binding to adult SMG membranes was (1) saturable at about 8 nM of ~-[~H]dihydroalprenolol, (2) rapid (saturation was reached in less than 5 minutes), (3) reversible by addition of unlabeled L-alprenolol, dl-propranolol, or L-isoproterenol, and (4) linear for concentrations of 50-400 p g of membrane protein. To measure a-adrenergic binding sites, assays were initiated by adding 50 p g of membrane protein to an incubation medium of 10 mM Tris (pH 7.6) buffer containing 1 mM DTT and [3H]dihydroergocryptine(Williams and Lefkowitz, 1976; Strittmatter et al., 1977) (New England Nuclear, Boston MA) (1-40 nM) with a final volume of 500 p1. All samples were run in duplicate. The reaction was run for 10 minutes at 37°C and was terminated by addition of 6 ml of cold saline (4°C) containing lop5 M phenotolamine and immediate filtration of the sample through a Whatman GB/F filter. The filter was washed with an additional 30 ml of saline, placed in liquid scintillation fluid, and counted as above. Duplicate tubes containing lop5M phenotolamine were incubated and treated as above and run for each sample to determine nonspecific binding. Only those counts that were displaced by phentolamine were considered specific. In preliminary studies, we have found that [3H]dihydroergocryptine binding to young adult SMG membranes to be (1) saturable at about 25 nM of [3H]dihydroergocryptine, (2) relatively rapid (saturation was reached in about 10 minutes at 37"C), (3) reversible by addition of phentolamine or norepinephrine, and (4) linear for concentrations of 50-400 p g of membrane protein. These data are consistent with studies of [3H]dihydroergocryptinebinding to membranes from rat parotid gland (Strittmatter et al., 1977). The number of binding sites and the K d were determined by Scatchard analysis and by a numerical curve-fitting procedure (Hooke and Jeeves, 1961). This procedure is based on a directed iterative search, which for a given expression, y = f ( x ) , adjusts the values of the fixed parameters to minimize C ( Y e - Y J 2 , where Ye is the experimental and Y , is the calculated value of the function.

G . Adenylate Cyclase Determination An aliquot of the enriched membrane fraction (10 p g of protein) was incubated for 10 minutes in 100 p1 of assay mixture containing 25 mM Tris-HC1 (pH 7.6), 5 mM MgClz, 1 mM CAMP, 1 mM DTT, 10 mM phosphocreatine, 50 units

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phosphocreatine kinase, 2 X M GTP, and 0.1 mM ATP (3 x lo6 cpm CY-[~'P]ATP). The incubation was stopped by adding 100 p1 of stopping solution (4 mM ATP, 1.4 mM CAMP, and 2% sodium dodecyl sulfate) and then 20,000 cpm of [3H]cAMP for estimation of recovery from chromatography on Dowex Ag 50WX4 and neutral alumina columns (Salomon er al., 1974; Cutler and Rodan, 1976). Duplicate samples were counted for 10 minutes in 10 ml of Bray's solution (Bray, 1960) in an Isocap/300 liquid scintillation counter set with separate channels for 3H and '*P. The results were calculated as picomoles of CAMP produced per milligram of protein per minute. In initial experiments membrane-associated adenylate cyclase activity from prenatal glands (21 days in utero) was tested for response to M guanylylimidodiphosphate (intactness of guanyl nucleotide regulatory site) and 10 mM NaF (intactness of fluoride activation site). In subsequent studies the response of adenylate cyclase from glands of different ages ( 18 and 2 1 days in utero and 1 , 4 , 6 , and 120 days of age) to a saturating concentration ( M) and L-isoproterenol was determined.

H. Electrophysiology of Neonatal Secretion Neonatal rats ( 1 , 3 , 5, 7, 9, and 1 1 days old) were anesthetized (Ketamine HC1) and placed in supine position. The neck region was exposed and the left superior cervical ganglion (sympathetic) located. Salivation was induced by direct stimulation of the superior cervical ganglion by 10 Hz in frequency and 4-8 volts in intensity for 20 minutes using a Grass square-wave stimulator. The efficacy of this procedure was confirmed by similar stimulation of the submandibular ganglion (parasympathetic) and the observation of a clear, watery saliva from the SMG duct. The effect of this stimulation on protein secretion was determined by homogenizing the stimulated gland (glands from 3-8 animals were pooled for each assay), centrifuging the homogenate at 1000 g for 10 minutes, and assaying the 1000 g supernatant for peroxidase activity as described earlier. The amount of peroxidase secretion was inferred by comparing the amount of peroxidase remaining in the stimulated gland after 20 minutes with that found in the contralateral unstimulated gland. The data are reported as the intraglandular peroxidase found as a percentage of the control gland where the control has been normalized to 100%.

I. Catecholamine-Containing Nerves in the Developing SMG The presence of catecholamine (norepinephrine)-containing nerves in the parenchyma of the developing SMG was assessed by the method of de la Torre

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and Surgeon (1976), in which cryostat sections are exposed to a solution of sucrose-potassium phosphate-glyoxylic acid.

111. Results A.

Adenylate Cyclase in SMG Development

Adenylate cyclase activity is very low in the earliest (15-day) SMG rudiment and progressively rises to adult levels by day 18 of gestation (Table I). It is at 18 days of gestation that the first secretory granules are seen. The adenylate cyclase activity found in the membrane fraction derived from the 2 1-day embryonic SMG is intact with regard to its guanyl nucleotide and fluoride regulatory sites. A subsaturating concentration of guanylylimididophosphate( lop5M ) causes a 13to 15-fold stimulation over basal adenylate cyclase activity, while 10 mM NaF induces an 18- to 20-fold stimulation over basal activity (Table 11). There appears to be an age-related incremental increase in the responsiveness of SMG adenylate cyclase activity to a saturating concentration of isoproterenol (Table 111). Adenylate cyclase activity from SMG rudiments 18 and 21 days in utero were not responsive to isoproterenol, and enzyme activity from glands 1 and 4 days of age responded only minimally. Membrane-associated adenylate cyclase activity from the SMG of 1- and 4-day animals showed a reproducible TABLE I SUUMANDIUULAR GLAND ADENYLATE CYCLASE ACTIVITY~

Age of animal 15 days in ufero 16 days in utero 17 days in utero 18 days in utero 21 days in utero Adult

Activity (pmol cAMP/mg protein/l5 min) 3.7 ? 16.3 2 50.1 2 65.5 ? 67.4 ? 67.9 ?

0.9 2.1 6.2 5.8

3.9 4.2

“Adenylate cyclase activity was determined on a crude particulate fraction obtained by homogenization of the rudiments or glands followed by a 1000 g centrifugation step. The resulting supernatant was centrifuged at 30,000 g for 15 minutes, and the resulting pellet was resuspended and assayed for adenylate cyclase activity by using the procedure of Salomon ef al. (1974).

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TABLE II ADENYLATE CYCLASE ACTIVITY, 21-DAY EMBRYONIC SMG" Activity (pmol cAMPimg proteinhin) Basal

17.0 234.3 317.6

10-5 M G P P ( N H ) ~

10 mM NaF

"The results of a typical experiment on the effects of M Gpp(NH)p and 10 mM NaF on membrane-associated adenylate cyclase activity from 21-day embryonic SMG. The experiment was performed to evaluate the ability of guanyl nucleotides and fluoride to activate the enzyme. The partculate fraction used in this study was an enriched plasma membrane fraction derived from differential centrifugation and sucrose gradient procedures (see Methods).

25-40% stimulation in response to M isoproterenol. On the other hand, membranes from glands at 6 days of age showed full activation (2.5- to 3.5-fold) of adenylate cyclase by this dose of isoproterenol, and this was similar to the activation seen in adult membranes.

TABLE 111 HORMONAL ACTIVATION OF SMG ADENYLATE CYCLASE FUNCTION OF DEVELOPMENTAL AGE

AS A

Activity (pmol cAMP/mg proteidmin) M L-Isoproterenol

Age of animal

Basal

18daysinurero 21 days in ufero I day 4 days 6 days Adult

15.3 t 1 . 1 15.7 ? 2.0 15.2 ? 0.8 16.5 2 1.3 15.8 ? 2.5 14.9 2 1.3 ~~

15.6 t 1.8 15.1 ? 1.5 18.7 ? 1.4 (p < 0.05) 20.9 ? 1.9 (p < 0.05) 42.8 t 3.7 44.7 t 5.1 ~~~

The ability of a saturating concentration of L-isoproterenol to activate membrane-associated adenylate cyclase from the SMG as a function of age was tested. Data are reported as the mean of five experiments 2 the S.E.M.

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DEVELOPMENTAL RESPONSETO SECRETOCOCUES" Prenatal, 21 days in utero 5.4 5.8 4.9 17.8

Basal M L-Isoproterenol M L-Phenylephrine lo-" M Dibutyryl cAMP lo-' M 8-Bromo-cGMP M L-Isoproterenol M L-Phenylephrine + M L-Isoproterenol M L-Phenylephrine lo-'

+

+ +

M oL-propranolol M DL-propranolol M phentolarnine M L-phentolamine

Postnatal

I day

6 days

2.8 14.3

3.3 15.8 7. I 16.6

15.5 15.1

3.8 8.3 12.0 6.8 4.1

"Results of a typical experiment to evaluate the secretory response of SMG slices to various secretogogues and inhibitors. Data are reported as the percentage of the total peroxidase present in the slices at the start of the experiment released into the medium after 30 minutes of stimulation.

B.

Secretion of Peroxidase

The results of a typical experiment on the stimulation of secretion from SMG slices from embryos and postnatal rats shown in Table IV. Neither a(pheny1ephrine)- nor P(isoprotereno1)-adrenergic agonists induced secretion from slices from prenatal glands. Dibutyryl cAMP ( lop3 M ) induced secretion from these slices, indicating that the mechanistic properties required for secretion were present and working in these cells. By 1 day after birth the slices secreted equally well in response to either a- or P-adrenergic agonists. Inhibition of this secretory response by supposedly specific a- or P-adrenergic antagonists was ambiguous, since the a-adrenergic antagonist phentolamine was more effective at blocking isoproterenol (P-adrenergic)-induced secretion than was the P-adrenergic antagonist propranolol. Dibutyryl cAMP induced secretion from these slices, but 8-bromo-cGMP had no effect on peroxidase release. By 6 days after birth the secretory response to a-adrenergic and P-adrenergic agonists was similar to that seen in adult glands (Bogart and Picarelli, 1978), with isoproterenol inducing substantially greater peroxidase release than phenylephrine.

C.

[3H]Dihydroalprenolo1and [3H]Dihydroergocryptine Binding

The number of P-adrenergic binding sites was estimated by the binding of P-adrenergic antagonist [ 3H]dihydroalprenololto partially purified plasma membranes from the SMG of animals of various ages. At all ages saturation of binding sites was rapid (saturation reached in about 5 minutes) and saturable at

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TABLE V BINDING OF [3H]DIHYDROALPRENOLOLTO SMG M E M B R A N E S ~

Age of animal 1 day 4 days 6 days 120 days

[ 3H]DHA bound ( f m o l h g protein)

67? 67? 273 ? 417 ?

7 9 21 34

Kd

1.15 X M 1.11 x 1 0 - 9 ~ 1.42 x 10-9 M 2.80 x 10-9 M

"The amount of [3H]DHA bound and the K , , were determined by Scatchard analysis and by a numerical curve-fitting procedure based on directed iterative search, which for the given expression y = f ( x ) adjusts the values of the fixed parameters to minimize I;( Ye - Yc)z, where Ye is the experimental and Yc is the calculated value of the function. Data presented are the mean of five experiments ? the S.E.M.

about 10 mM of the antagonist. Scatchard analysis and computerized curve fitting of the binding data revealed little variation in the Kd for the various ages tested, but a 7-fold increase was found in the number of binding sites from birth to adulthood (Table V). A 4-fold increase in the number of binding sites was seen between 4 and 6 days after birth. This increase in the number of binding sites coincided with the increased responsiveness of SMG adenylate cyclase to isoproterenol stimulation and to the appearance of adult-type stimulus-secretion coupling in SMG slices. The preliminary studies on a-adrenergic binding sites reported here indicated that at birth there were about 1800 fmol of dihydroergocryptine bound per milligram of SMG membrane protein with a Kd of about 3.7 X M. Adult SMG membranes bound 1263 fmol of dihydroergocryptine per milligram of protein with a Kd of about 1.6 x M .Thus, there were roughly the same number or somewhat more a-adrenergic receptors present on SMG membranes at birth than there are in adult SMG membranes. Therefore, the development of a- and P-adrenergic receptors in this gland is apparently independently regulated.

D. Electrophysiological Stimulation of Secretion The results of electrophysiological studies on secretion by developing glands are shown in Table VI. Direct electrical stimulation of the superior cervical ganglion did not result in secretion by the SMG in animals 1 and 3 days old. During the period from 5 to 11 days of age there was a progressive increase in secretion elicited by electrical stimulation of the superior cervical ganglion. The adrenergic agonist norepinephrine was able to induce secretion at all times tested.

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TABLE VI

PEROXIDASE SECRETION FROM THE SMG FOLLOWING DIRECT ELECTRICAL OF THE SUPERIOR CERVICAL GANGLION" STIMULATION Percentage of total intraglandular peroxidase remaining after 20 minutes stimulation Age of animal

Experimental

Norepinephrine

Unstimulated control

I day 3 days 5 days I days 9 days I 1 days

100 100 85 15 61 40

35 42 -

I00 100 100 100 100

45

~~~~

100 ~~~

Results of a typical experiment are shown. Secretion of peroxidase was inferred by comparing the amount of peroxidase remaining in the stimulated gland after 20 minutes to that found in the contralateral unstimulated gland. The data are reported as the intraglandular peroxidase found as a percentage of the control gland after 20 minutes of electrical stimulation. The peroxidase content of the control gland has been normalized to 100%.

These data suggested that the cells of the developing SMG were able to respond to secretory stimuli but direct neural stimulation was not able to elicit a secretory response.

E. Glyoxylic Acid Staining for Catecholamine-Containing Nerves Glyoxylic acid staining showed that each acinar unit of the adult rat SMG was surrounded by catecholamine-containing nerves. However, virtually no catecholamine fluorescent nerves were seen within the parenchyma of the 1-day or 3-day submandibular glands. The earliest evidence of catecholamine containing nerves in the parenchyma of the SMG was at 5-6 days after birth. From this point on, there was a progressive increase in the number of catecholaminecontaining nerves in the parenchyma. This progressive increase in catecholamine-containing nerves correlated directly with the development of a progressively increasing secretory response secondary to electrical stimulation of the superior cervical ganglion.

IV.

Discussion

The data presented give a comprehensive picture of the maturation of those factors that regulate protein secretion in this model exocrine system. It appears

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that differentiating SMG secretory cells first develop the structural and synthetic machinery required to produce and package their secretory product. Coincident with, or shortly after, the initiation of the synthesis and packaging of the exocrine product, the cells develop the physical capability to release the packaged product in a typical exocrine fashion. With regard to the rat SMG, zymogen granule production is first seen at 18 days of gestation, and at this point in time basal adenylate cyclase activity is at the same level that is found in adult glands. The adenylate cyclase activity in the fetal gland is intact with regard to its sodium fluoride and guanyl nucleotide regulatory sites. However, the enzyme does not respond to saturating concentrations of the known agonist L-isoproterenol. In vitro secretion studies indicate that fetal SMG slices are able to secrete peroxidase in response to millimolar concentrations of dibutyryl CAMP but will not respond to maximal doses of L-isoproterenol. Thus, while the embryonic SMG cells have the physical capability to produce and secrete their products, the typical stimulus-secretion coupling mechanisms that are present in the adult SMG cells have not yet appeared. The development of the mechanisms to produce and secrete exocrine proteins prior to the development of the typical stimulus-secretion coupling mechanisms seen in the mature cells does not seem to be a phenomenon unique to the rat submandibular gland. Doyle and Jamieson ( 1 978) have observed a similar situation in the developing rat pancreas, and Grand and his co-workers (1975; Grand and Schay, 1978) have found an analogous picture in the developing rat parotid gland. In all three systems the adult-type stimulus-secretion coupling mechanisms appear to evolve over a short period of time subsequent to the development of the synthetic and release pathways. Heretofore, the exact nature of the development of the stimulus-secretion coupling mechanisms has not been known. It was (and still is) not known if absence of stimulus-secretion coupling in the neonatal or embryonic parotid and pancreas was due to the absence of specific agonist receptors or to the absence of a coupling or transducing factor that could link the activated receptor-agonist complex to the response system. The data presented here suggest that in the developing submandibular gland the missing link in the stimulus-secretion response system is the P-adrenergic receptor. Direct accessment of receptor number and affinity indicates that adenylate cyclase response to hormone activation is directly related to an increasing number of P-adrenergic binding sites at the cell surface of the SMG cells as a function of age. Further, in vitro secretion studies suggest that the development of adult-type stimulus-secretion coupling patterns are also correlated with the developmental appearance of increased numbers of P-adrenergic binding sites at the cell surface. Thus, as the number of cell surface P-adrenergic receptors reaches a critical number such that adenylate cyclase activation is equivalent to that seen in adult membranes, the in vitro secretory response assumes a pattern similar to that seen in adult tissue. Finally, the electrophysiological and catecholamine fluorescent data indicate

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that during the period (birth to 5 or 6 days of age) when the P-adrenergic receptor sites are increasing in number and the adult stimulus-secretion coupling mechanisms are evolving there are no adrenergic neural connections in the gland. It is only after the total development of the complete synthetic and secretory pathways that neural connections are made. Thus, there is a highly organized and defined sequence of structural and biochemical differentiative steps in the total evolution of the SMG exocrine cell development. This sequence appears to have several common steps in at least three different exocrine systems and thus may represent a general developmental sequence common to all exocrine systems.

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