J. Phyaiol. (1976), 258, pp. 631-658 With 4 plates and 12 text-ftgures Printed in Great Britain
631
STIMULUS-SECRETION COUPLING: ROLE OF CYCLIC AMP, CYCLIC GMP AND CALCIUM IN MEDIATING ENZYME (KALLIKREIN) SECRETION IN THE SUBMANDIBULAR GLAND
BY JANET ALBANO, K. D. BHOOLA, P. F. HEAP AND M. J. C. LEMON From the Departments of Pharmacology, Anatomy and Medicine, The Medical School, University of Bristol, University Walk, Bristol BS8 1TD
(Received 15 August 1975) SUMMARY
1. The role of adenosine 3':5'-phosphate (cyclic AMP) and guanosine 3': 5'-phosphate (cyclic GMP) as second messengers for the enzyme secretary response evoked by the autonomic neurotransmitters, noradrenaline and acetylcholine, is examined in this in vitro study on the guinea-pig submandibular gland. 2. Noradrenaline increased enzyme (kallikrein) secretion. The initial stimulation of enzyme release appeared to be dose-dependent. The time course of cumulative kallikrein secretion revealed a complex pattern. Isoprenaline and phenylephrine were almost as potent as noradrenaline in releasing kallikrein. Both propranolol and phentolamine were required to fully inhibit the noradrenaline-stimulated enzyme secretion. 3. The cumulative secretion of kallikrein evoked by acetylcholine was dose-dependent. The onset of secretion showed a significantly greater time-lag than that observed with noradrenaline. Atropine effectively blocked the release of kallikrein by acetylcholine. 4. Dibutyryl cyclic AMP stimulated enzyme secretion. Dibutyryl cyclic GMP caused an initial increase which was not maintained. 5. The cyclic nucleotide phosphodiesterase inhibitors, theophylline and papaverine, increased basal kallikrein secretion. The action of the cyclic phosphodiesterase inhibitors on the secretary response to noradrenaline, acetylcholine, dibutyryl cyclic AMP and dibutyryl cyclic GMP was complex. In general, the increase in enzyme release produced by the secretagogues was additively enhanced by both inhibitors. 6. Omission of calcium inhibited both acetylcholine and dibutyryl cyclic GMP stimulated kallikrein release, but to a lesser degree than that
632 JANET ALBANO AND OTHERS of noradrenaline and dibutyryl cyclic AMP. High concentrations of extracellular calcium (10 mM) appeared to enhance the action of acetylcholine. 7. Noradrenaline produced a rise in the intracellular level of cyclic AMP. The increase preceded the stimulated secretion of kallikrein. Of the various adrenergic agonists, noradrenaline and isoprenaline were the most potent, whereas phenylephrine was significantly less effective in raising basal cyclic AMP values. Acetylcholine was without effect, even in the presence of a cyclic phosphodiesterase inhibitor. 8. Acetylcholine and noradrenaline raised intracellular levels of cyclic GMP only when the tissue incubations were performed in the presence of a cyclic phosphodiesterase inhibitor. The increase in cyclic GMP produced by acetylcholine preceded enzyme secretion. 9. Morphological data substantiated the finding that the in vitro release of kallikrein evoked by the secretagogues was associated with the depletion of secretary granules and vacuolations in acinar cells of the gland slices. 10. The molecular mechanisms which control enzyme secretion in the exocrine submandibular gland are discussed. Models are presented for the role of transmitter-specific cyclic nucleotides and calcium in stimulussecretion coupling. INTRODUCTION
Hormones or first messengers are known to interact with specific receptors on the plasma membrane of cells in the target-tissue. The second messenger concept envisages that the hormone-receptor interaction induces the formation inside the cell of a chemical messenger which mediates events within the cell. Calcium, adenosine 3':5'-cyclic monophosphate (cyclic AMP) and guanosine 3' :5'-cyclic monophosphate (cyclic GMP) are considered to be second messengers of physiological and biochemical importance. The formation and turn-over of the cyclic nucleotides (cyclic AMP and cyclic GMP) in the cell is controlled by the nucleotide cyclase (adenylate and guanylate) and the cyclic nucleotide phosphodiesterases (cyclic PDE). Nearly 10 years ago, Schramm and his colleagues (Bdolah & Schramm, 1965; Babad, Ben-Zvi, Bdolah & Schramm, 1967) suggested that enzyme secretion evoked by adrenaline from the parotid gland was mediated by cyclic AMP. Using rat parotid gland slices, they demonstrated that dibutyryl cyclic AMP caused a marked increase in amylase release. Furthermore, theophylline and caffeine, which are known to inhibit cyclic nucleotide phosphodiesterase, also stimulated enzyme secretion. Taken alone, these results did not constitute convincing proof of the physiological participation of cyclic AMP in adrenaline-induced amylase secretion. The case for cyclic AMP-mediated enzyme secretion was
633 STIMULUS-SECRETION COUPLING strengthened by the demonstration of a catecholamine-activated adenylate cyclase in the rat parotid (Schramm & Naim, 1970) and guinea-pig submandibular glands (Bhoola & Lemon, 1973, 1975). One of Sutherland's criteria, by which the role of cyclic nucleotides in a biological process is assessed (Sutherland, Robinson & Butcher, 1968), namely that the tissue contains adenylate cyclase has been established (Lemon & Bhoola, 1975) and initial studies have indirectly suggested the presence ofguanylate cyclase in exocrine glands. The questions whether submandibular neurotransmitters cause a rise in cyclic AMP and cyclic GMP levels which precede the enzyme secretary response, whether inhibitors of cyclic nucleotide phosphodiesterase influence that response and whether derivatives of cyclic AMP and cyclic GMP mimic the action of the neurotransmitters form the subject of the present report. Our preliminary findings were communicated to the Fifth International Congress in Pharmacology (Bhoola & Lemon, 1972), Second International Conference on Cyclic AMP (Bhoola & Lemon, 1974) and the Physiological Society (Bhoola & Lemon, 1975; Albano, Bhoola, Croker, Heap & Lemon, 1976). METHODS
Incubation y. ocedure Male guinea-pigs (Dunkin-Hartley origin, 350-450 g) were anaesthetized by intraperitcneal injection of pentobarbitone sodium (35-40 mg/kg). The submandibular (submaxillary) glands were removed and washed in a modified Krebs-Ringer bicarbonate buffer (KRB) containing, in mM: NaCl, 121; KCl, 14-3 or 5-0; NaHCO3, 25-2; CaCl2, 2-6; MgS04, 1-2; glucose 5-6; /J-hydroxybutyric acid, 6-1 (Babad et al. 1967). The potassium concentration of the Krebs-Ringer bicarbonate solution was originally maintained at 14-3 mm in order to deplete the gland slices of endogenous transmitter (Babad et al. 1967), but subsequently 5 mm was used because the enzyme secretary response was not appreciably altered by lowering the potassium concentration (see Table 4). Glands were trimmed of the sublingual appendage, connective tissue and hilar blood vessels and cut into slices (about 1-2 mm in thickness and each slice weighing about 50 mg). The pooled slices from submandibular glands of six to nine guineapigs were initially pre-incubated for 25 min at 370 C in 25 ml. Krebs-Ringer bicarbonate solution gassed with a stream of 95 % 02-5 % CO2 in a Grant shaking water bath (160 strokes/min). The slices were removed, blotted and weighed. The tissue incubations were carried out in triplicate. Slices of known weight were placed into single Ehrlenmeyer flasks (15 ml. capacity); each flask containing 2-5 ml. (150200 mg tissue) fresh Krebs-Ringer bicarbonate solution. For the cyclic AMP and cyclic GMP experiments 100- 150 mg tissue was used. At this stage, a pre-incubation of 15 min duration was introduced for experiments in which the tissue concentration of cyclic AMP and cyclic GMP was determined. A similar pre-incubation period was used to study the effect of antagonists. Zero time was taken from the moment 50 or 100 ,ul. drug solution was added to the medium, and the incubation continued for periods ranging from 5 sec to 90 min. At the end of the incubation, the medium was frozen in solid CO2 and stored at below 00 C for subsequent assays of enzyme
634
JANET ALBANO AND OTHERS
content. For the cyclic AMP and cyclic GMP measurement experiments the tissue was removed rapidly and at once frozen in an aluminium clamp cooled in liquid nitrogen.
Measurement of enzyme activity Kallikrein. The esterase activity of the serine proteinase was determined by the hydrolysis of the synthetic substrate, benzoyl-1-arginine-ethyl ester (BAEe), which has been described previously (Bhoola, Dorey & Jones, 1973). The rate of hydrolysis of BAEe was followed for 3 min at 250 C in a Pye-Unicam SP 1800 recording spectrophotometer. The linear phase of the change in absorbance was used to calculate activity which was expressed as an increase in optical density units (AE at 366 nm)/min per millilitre incubation medium. Kallikrein secreted was expressed in optical density units x 106 (BAEe AE366.m)/mg wet weight of tissue. Amylase. The activity of amylase was measured by the iodine titration method as described by Bhoola & Heap (1970).
Measurement of cyclic AMP and cyclic GMP The frozen gland slices, freeze-clamped at the end of the incubation period, were plunged into glass tubes containing 0-5-1-0 ml. Tris-HCl (4 mm theophylline in 10 mM Tris-HCl; pH 7-5) and heated in a boiling-water bath for 10 min. The denatured tissue was homogenized, centrifuged at 20,000 g and the supernatant freezedried. The lyophilized sample was assayed for cyclic AMP by the competitive binding protein method of Gilman (1970) and modified by Cooper, McPherson & Schofield (1972). For the measurement of cyclic AMP, the cyclic AMP-dependent protein kinase, which specifically binds cyclic AMP, was prepared from rabbit skeletal muscle as described by Walsh, Perkins & Krebs (1968). The validity of the cyclic AMP assay was verified. Recovery of cyclic AMP added to the tissue samples before the heat denaturation step was 87-90 %. There was complete recovery of cyclic AMP added to the lyophilized samples immediately before assay. Purified beef heart cyclic nucleotide PDE (Sigma) abolished the noradrenaline-stimulated increased in cyclic AMP. For more recent measurements of cyclic AMP and the determination of cyclic GMP in the gland slices, the freeze-clamped tissue was plunged into Pyrex glass tubes containing 0-5 ml. boiling solution of 6 mM theophylline. After 10 min of boiling the tissue was frozen, subsequently thawed and homogenized. To each tube was added 0-5 ml. acidified ethanol (0-01 N-HCl in absolute ethanol). After mixing, the tubes were stored below 00 C overnight. Samples were thawed, centrifuged at 2000 g x 5 min and the supernatant decanted into small plastic tubes. The precipitate was washed with 0-5 ml. acidified alcohol. After mixing and re-centrifuging, the corresponding supernatants were combined. The extracts were taken to dryness in an oven at 500 C. Before assay, the residue was dissolved in 0-5 ml. Tris-HCl buffer (50 mM; pH 7-4). Cyclic AMP was measured by competitive protein binding utilizing the cyclic AMP binding protein from the bovine adrenal cortex (Brown, Albano, Ekins, Sgherzi & Tampion, 1971). Cyclic GMP was determined by a radioimmunoassay for which specific antibodies were raised to succinylated cyclic GMP following the procedure outlined by Steiner, Parker & Kipnis (1972). The antibody used showed a cross-reaction of less than 0-2 % with cyclic AMP at 1 p-molelml. cyclic GMP and less than 0-01 % with other cyclic nucleotides. Nucleotide mono-, di-, triphosphates showed virtually no cross-reaction up to a concentration of 104 p-mole/ ml. The standard curves for the assays were supplemented with the corresponding reagent blank (Albano, Barnes, Maudsley, Brown & Ekins, 1974). All determinations were performed in triplicate.
STIMULUS-SECRETION COUPLING
635
Morphology At the completion of the incubation, tissue for morphology was immediately immersed in a double aldehyde fixative (freshly prepared 4%, w/v, paraformaldehyde and 2-5 %, w/v, glutaraldehyde for 2 hr), and the entire fixation procedure was performed at 40 C. Tissue was washed for 15 min in sodium phosphate buffer (0-2 M, pH 7 3), post-fixed in osmium tetroxide (1% in 0-2 M sodium phosphate buffer, 4° C) for 2 hr, washed in distilled water for 30 min, dehydrated in graded ethanol and 1,2 epoxypropane and embedded in Araldite. Sections of 1 jum thickness were cut for light microscopy and stained with 1 % toluidine blue in sodium borate buffer (pH 2.4). For electron microscopy silver gold sections were stained with uranyl acetate and lead citrate and examined in a Hitachi HU-11B or HU12A electron microscope. Areas of cell vacuolation observed in the light microscopy sections of submandibular gland slices incubated with the various secretagogues were measured with a Quantimet 720 Image Analyser (Metals Research, Royston, Hertfordshire). This instrument, which is an image editing television microdensitometer, provided digital values for differing densities within a specified area. The light microscopic image of a toluidine blue-stained tissue section was monitored by an electronic scanner which displayed the image on a television screen. By activating a density contrast module the instrument was instructed to measure a specified grey level or tone of density within a required field which was delineated on the displayed image with a light sensitive pen. The area of low density (vacuolation) and the total area scanned were displayed as digital values which were subsequently converted into #m2. At least four sections of 1 jtm thickness stained with toluidine blue were examined to determine each vacuolation value. The mean area scanned was 22,000 /um2 (range 9000-49,000Ium2). We are particularly grateful to Dr M. N. Croker for making available the instrument and for assisting with the measurements. RESULTS
Secretion studies In initial experiments enzyme activity was determined in gland slices as well as the incubation medium. During a 60 min incubation period a greater proportion of the tissue kallikrein was released into the medium than that of amylase, both under conditions of basal and of stimulated secretion. Because in previous studies it had been established that kallikrein in the guinea-pig submandibular gland was sequestered within secretary granules whereas amylase was possibly localized in two pools, cytoplasmic and granular (Bhoola & Ogle, 1966; Bhoola, 1968; Bhoola & Heap, 1970), the secretion studies were confined to the measurement of kallikrein only. The release of kallikrein into the incubation medium was considered therefore to represent exocytotic secretion. Stimulation of enzyme secretion through activation of adrenoceptors Noradrenaline. In the concentration range 4 UM to 4 mM, noradrenaline caused an increase in kallikrein secretion (Text-fig. 1). At the higher concentrations (400 /tM, 4 mM) this increase was observed within the
636
JANET ALBANO AND OTHERS sampling time of 5 sec, but at the lower concentrations (40 ,M, 4/tM) the onset of stimulated secretion was not apparent until 30 see or 1 min after the addition of noradrenaline. The initial secretary response increased linearly between 5 see and 2-5 min, and the accumulation of kallikrein during this period increased with time. Between 5 and 30 min the rate of 2500
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JANET ALBANO AND OTHERS submandibular gland, papaverine (50 = 2 x 10-5 M) was about twenty-fivetimes more potent than theophylline (So0 = 5 x 10-4 M) in inhibiting cyclic AMP phosphodiesterase (Lemon & Bhoola, 1975). A similar degree of potency was observed in the in vitro slices experiments (Table 3). Theophylline (1-8 mM) and papaverine (0.4 mM) increased basal secretion. The secretary response to noradrenaline (0.4 mm) was increased by 1 mM theophylline and by 0 4 mm papaverine. The acetylcholine-evoked release of kallikrein was also additively increased in the presence of 4-8 mm theophylline and 0 4 mm papaverine. Whereas the secretion obtained with acetylcholine and dibutyryl cyclic GMP increased progressively when the concentration of theophylline was raised from 1 to 8 mm, that of noradrenaline and dibutyryl cyclic AMP was maximal at 1 mM theophylline and showed a relative decrease as the concentration of the methyl xanthine was increased in the incubation medium. 642
TABLE 5. Influence of calcium on kallikrein secretion evoked by transmitter substances and cyclic nucleotides from submandibular gland slices
Calcium concentration in Krebs-Ringer solution
Calcium-free ,1 mM EGTA 2-6 mM 162+24 88±13 417 ± 46 128 ± 16 610 + 78 258 + 22 469 ± 66 192 + 21 157+25 258+43
-,
5 mM
10 mm 263±66 769 ± 96 683 + 49 439 + 47 416+121 467 ± 74 425 ± 79
191+32 Basal, 15min 639 + 74 Acetylcholine 542 ± 69 Noradrenaline 468 ± 51 Isoprenaline 201+47 Basal, 30min 314 ± 31 321 + 17 350 ± 39 Dibutyryl cyclic AMP 241 + 32 312 ± 47 150 + 11 Dibutyryl cyclic GMP Submandibular gland slices were incubated with acetylcholine (4 mM), noradrenaline (4 mM) and isoprenaline (4 mM) for 15 min and dibutyryl cyclic AMP (4 mM) and dibutyryl cyclic GMP (4 mm) for 30 min in Krebs-Ringer solution containing 0 (1 mm EGTA), 2-6, 5 and 10 mm calcium. Values represent kallikrein activity expressed as BAEeAE366 x 106/mg tissue, ± S.E. of mean, n = 6-8. Each value is the mean of n (six to eight) measurements performed on n groups of slices (three to four slices; 150-200 mg); each n group was derived from pooled, pre-incubated slices prepared from both submandibular glands of nine animals.
Influence of ions on enzyme secretion Role of K+. A progressive increase in the K+ concentration of the incubation medium, was associated with increased release of kallikrein (Table 4). The peak response obtained at 75 mM-K+, with the threshold concentration between 25 and 75 mM-K+, was similar to that reported for amylase release from rat parotid slices incubated in vitro by Bdolah, Ben-zvi & Schramm (1964). The time course and magnitude of kallikrein
STIMULUS-SECRETION COUPLING 643 secretion evoked by 75 mm-K+ was similar to that obtained with 40 pM noradrenaline. Calcium dependence of the secretary response. The action of calcium on the secretion of kallikrein by the neurotransmitters and by cyclic nucleotides was investigated by altering the calcium concentration of the KrebsRinger buffer solution. When the ionic composition was altered, the concentration of NaCl was changed to maintain isotonicity. Ethylene glycol tetracetate (EGTA, 1 mM) was added to the media designated 'calcium free'. The experimental design involved a first pre-incubation period of 20 min in the standard Krebs-Ringer buffer (2.6 mm calcium), followed by a second pre-incubation period of 20 min in the specific calcium-containing Krebs-Ringer; for the 'calcium free' data both the first and second pre-incubations were carried out in the 'calcium free' Krebs-Ringer bicarbonate solution. The omission of calcium combined with the addition of EGTA to the incubation medium reduced the acetylcholine (from 2*6-fold to 1*5-fold over basal release) stimulation of enzyme secretion to a greater extent than that of noradrenaline (from 3-8-fold to 2*9-fold over basal release) and isoprenaline (from 2-9-fold to 2*2-fold over basal release). Furthermore, the action of dibutyryl cyclic GMP on enzyme release was abolished whereas that of dibutyryl cyclic AMP was only moderately affected. Of the secretagogues tested, only acetylcholine increased kallikrein secretion progressively with increasing concentrations of calcium.
Cyclic AMP and cyclic GMP levels in enzyme secreting submandibular gland slices
Cyclic AMP. Noradrenaline at a concentration of 10-4 M caused a marked increase in cyclic AMP levels in submandibular gland slices. The increase was apparent within the sampling time (5 see). Maximum values were attained between 20 see and 2 min. Subsequently there was a gradual fall in tissue concentration of cyclic AMP which returned to control values at 90 min (Text-fig. 5). The cyclic AMP concentration in control slices remained below about 0-2 p-mole/mg tissue throughout the 90 min incubation. The PDE inhibitor, 3-isobutyl-1-methyl xanthine, markedly enhanced the noradrenaline action of raising the tissue concentration of cyclic AMP (Text-fig. 6). A similar potentiation was observed with 4 mM theophylline. The noradrenaline induced increase in the tissue cyclic AMP levels was dose-dependent. With 1 /IM noradrenaline the rise in cyclic AMP level preceded the increase in kallikrein secretion. Comparison of the efficacy of various adrenergic agonists in raising the intracellular level of cyclic AMP showed that at the incubation time of 1 min, noradrenaline and isoprenaline were most potent, phenylephrine was
644 JANET ALBANO AND OTHERS significantly less active and methoxamine was ineffective (Text-fig. 7). Under similar experimental conditions, acetylcholine up to a concentration of 5 x 10-3 M, even in the presence of 4 mM theophylline or 0 5 mM isobutyl methyl xanthine, consistently failed to increase the concentration of cyclic AMP in glands incubated with the transmitter over a time course ranging from 5 sec to 60 min (see Text-fig. 7). 08
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Text-fig. 5. Time course of intracellular concentration of cyclic AMP (cAMP) in submandibular gland slices secreting kallikrein in response to noradrenaline and acetylcholine. Values for cyclic AMP are expressed as p-mole/mg tissue. Incubation times ranged from 5 sec to 90 min and are plotted on semilog. paper. Basal (x), 100 /lM noradrenaline (X), 5 mM acetylcholine (0). Each value is the mean of n (nine) measurements performed on n groups of slices (two to three slices, 75-125 mg); each n group was derived from pooled, pre-incubated slices prepared from both submandibular glands of eight animals. Bar shows 1 S.E. of mean, n = 9 (each n value was the mean of triplicate cyclic AMP measurement). Data from Bhoola & Lemon (1975), with the preliminary values for 0-5 and 0 7 min omitted.
Cyclic GMP. Both acetylcholine and noradrenaline failed to affect the intracellular concentration of cyclic GMP at several incubation times from 30 sec to 30 min. Only in the presence of isobutyl methyl xanthine (cyclic nucleotide PDE inhibitor) was a significant rise in cyclic GMP observed with acetylcholine (Text-fig. 8) and noradrenaline (Text-fig. 9). The increased levels of cyclic GMP observed after 2 min incubation with acetylcholine (0.4 mM) preceded the onset of significant kallikrein release
STIMULUS-SECRETION COUPLING
645 (5 min). However, the rise in cyclic GMP produced by noradrenaline (0.4 mM) did not appear to precede the increase in enzyme secretion.
Morphological studies For the morphological data the gland slices were incubated for 1, 15 and 90 min with each of the secretagogues (control gland slices, Pls. 1(1), 1(2)). 8
044 606
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2 5 Incubation time (min)
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Text-fig. 6. Potentiation of the noradrenaline stimulated increase in cyclic AMP by the cyclic phosphodiesterase inhibitor, 3-isobutyl 1-methyl xanthine (IBMX), in submandibular gland slices secreting kallikrein. Values for cyclic AMP are expressed as p-mole/mg tissue. Incubation times: 0-5, 2, 5 and 30 min. Histograms represent basal (1]), 0-4 mM noradrenaline (M), basal with 0-5 mm IBMX (I) and 0-4 mm noradrenaline with 0-5 mm IBMX (F) values. Each value is the mean of n (three) measurements performed on n groups of slices (two to three slices, 100150 mg); each n group was derived from pooled, pre-incubated slices prepared from both submandibular glands of six animals. Bar shows 1 S.E. of mean, n = 3 (each n value was the mean of triplicate cyclic AMP measurements).
Noradrenaline and dibutyryl cyclic AMP. Even though significant secretion of kallikrein occurs within 30 sec of contact with noradrenaline, no obvious cellular changes were discernible at 1 min. However, a marked reduction in the number of secretary granules together with considerable vacuolation in the acinar cells was observed in the 15 min sections of the slices (Pls. 2(3), 2(4); Text-fig. 10). After 90 min incubation only a few granules of intermediate staining with toluidine blue remained (P1. 3) but the vacuolation was only marginally increased. Similar morphological changes of the release of secretary granules and vacuolation in acinar
JANET ALBANO AND OTHERS cells of isolated rat parotid gland slices incubated with adrenaline have been described by Batzri, Selinger & Schramm (1971). A progressive increase in vacuolation was detected between the 15 and 90 min incubations with dibutyryl cyclic AMP. The area of vacuolation induced by the cyclic nucleotide after 90 min was comparable to that measured for noradrenaline (see Text-fig. 10), whereas the number of 646
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Text-fig. 7. Comparison of the relative potency of adrenergic agonists and acetylcholine in raising cyclic AMP levels in submandibular gland slices secreting kallikrein. Values for cyclic AMP are expressed as p-mole/ mg tissue. Drug concentration 0-4 mm. Incubation times I and 30 min. Histograms represent basal (Fl), acetylcholine (E), noradrenaline (E), isoprenaline (EM), phenylephrine (a) and methoxamine (MII) values. Each value is the mean of n (three) measurements performed on n groups of slices (tw o to three slices, 100-150 mg); each n group was derived from pooled, pre-incubated slices prepared from both submandibular glands of six animals. Bar shows I S.E. of mean, n = 3 (each n value was the mean of triplicate cyclic AMP measurements).
secretary granules remaining within the acinar cells was considerably greater (PI. 4(6)). The ultrastructural organization and granule population in the intercalated and junctional ducts (Dorey &r Bhoola, 1972) appeared to be unaffected by both noradrenaline and dibutyryl cyclic AMP.
Acetylcholine and dibubtyryl cyclic GMP. The difference in the cumulative secretion of kallikrein produced by noradrenaline and acetylcholine was reflected in the morphological changes. Both the area of vacuolation and depletion of secretary granules in the acinar cells were clearly less than
647 STIMULUS-SECRETION COUPLING those observed with noradrenaline (P1. 4(7)). The full secretary picture was not apparent morphologically at 15 min but only in the 90 min sections. The main fine structural change observed with dibutyryl cyclic GMP appeared to be an increase in vacuolation in the acinar cells. No depletion of the granule population in the intercalated and junctional duct cells was observed with acetylcholine or dibutyryl cyclic GMP. 020
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Text-fig. 8. Effect of acetylcholine on cyclic GMP (cGMP) levels in the presence and absence of the cyclic nucleotide PDE inhibitor, 3-isobutyl 1-methyl xanthine (IBMX), in submandibular gland slices secreting kallikrein. Values for cyclic GMP are expressed as p-mole/mg tissue. Incubation times: 0-5, 2, 5 and 30 min. Histograms represent basal (E), 0-4 mm acetylcholine (Ed), basal with 0-5 mm IBMX (En) and 0-4 mm acetylcholine with 0-5 mm IBMX (E) values. Each value is the mean of n (three) measurements performed on n groups of slices (two to three slices; 100-150 mg); each n group was derived from pooled, pre-incubated slices prepared from both submandibular glands of six animals. Bar shows 1 s.E. of mean, n = 3 (each n value was the mean of triplicate cyclic GMP measurements).
Calcium. The morphological study was extended to gland slices incubated for 90 min in Krebs-Ringer solutions from which calcium was omitted but contained 1 mm EGTA, and in solutions which contained 10 mm calcium. Light microscopy sections from control and acetylcholinestimulated slices incubated in calcium-free solution showed increased vacuolation in the acinar cells. In addition, a reduction in granule numbers was observed in the junctional duct cells with noradrenaline and dibutyryl
JANET ALBANO AND OTHERS cyclic AMP. Gland slices incubated in 10 mm calcium showed a lesser amount of vacuolation in the acinar cells of the control and the acetylcholine and noradrenaline stimulated tissue but marginally greater in the tissue incubated with the cyclic nucleotides. 648
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Text-fig. 9. Effect of noradrenaline on cyclic GMP levels in the presence and absence of the cyclic nucleotide PDE inhibitor, 3-isobutyl 1-methyl xanthine (1BMX), in submandibular gland slices secreting kallikrein. Values for cyclic GMP are expressed as p-mole/mg tissue. Incubation times: 0-5, 2, 5 and 30 min. Histograms represent basal (D), 0-4 mM noradrenaline (M), basal with 0-5 mm IBMX (13) and 0-4 mm noradrenaline with 0-5 mm 1BMX (I) values. Each value is the mean of n (three) measurements performed on n groups of slices (two to three slices; 100-150 mg); each n group was derived from pooled, pre-incubated slices prepared from both submandibular glands of six animals. Bar shows 1 S.E. of mean, n = 3 (each n value was the mean of triplicate cyclic GMP measurements). DISCUSSION
Stimulation of enzyme secretion Adrenoceptor-mediated amylase secretion from mammalian salivary glands has been reported in a series of in vivo experiments (rat parotid, Pohto, 1968; human parotid and submandibular, Katz & Mandel, 1968; rabbit parotid, Yamamoto, Inoki & Kojima, 1968; mouse parotid, Durham & Butcher, 1974) and in vitro (rat parotid, Babad et al. 1967; guinea-pig submandibular, Carls66, Danielsson, Marklund & Stigbrand, 1974). Of the secretagogues examined in the present study, noradrenaline
STIMULUS-SECRETION COUPLING 649 was the most effective in stimulating kallikrein secretion. The response to noradrenaline contained both a- and fl-adrenoceptor-mediated components, since the presence of both adrenoceptor blockers was required to completely abolish the enzyme secretary response. The adrenoceptor components were further studied by using a limited series of agonists. Of these phenylephrine (with greater a-receptor activity) was of similar potency on a molar basis to isoprenaline (with greater fl-receptor activity; 16
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631
STIMULUS-SECRETION COUPLING: ROLE OF CYCLIC AMP, CYCLIC GMP AND CALCIUM IN MEDIATING ENZYME (KALLIKREIN) SECRETION IN THE SUBMANDIBULAR GLAND
BY JANET ALBANO, K. D. BHOOLA, P. F. HEAP AND M. J. C. LEMON From the Departments of Pharmacology, Anatomy and Medicine, The Medical School, University of Bristol, University Walk, Bristol BS8 1TD
(Received 15 August 1975) SUMMARY
1. The role of adenosine 3':5'-phosphate (cyclic AMP) and guanosine 3': 5'-phosphate (cyclic GMP) as second messengers for the enzyme secretary response evoked by the autonomic neurotransmitters, noradrenaline and acetylcholine, is examined in this in vitro study on the guinea-pig submandibular gland. 2. Noradrenaline increased enzyme (kallikrein) secretion. The initial stimulation of enzyme release appeared to be dose-dependent. The time course of cumulative kallikrein secretion revealed a complex pattern. Isoprenaline and phenylephrine were almost as potent as noradrenaline in releasing kallikrein. Both propranolol and phentolamine were required to fully inhibit the noradrenaline-stimulated enzyme secretion. 3. The cumulative secretion of kallikrein evoked by acetylcholine was dose-dependent. The onset of secretion showed a significantly greater time-lag than that observed with noradrenaline. Atropine effectively blocked the release of kallikrein by acetylcholine. 4. Dibutyryl cyclic AMP stimulated enzyme secretion. Dibutyryl cyclic GMP caused an initial increase which was not maintained. 5. The cyclic nucleotide phosphodiesterase inhibitors, theophylline and papaverine, increased basal kallikrein secretion. The action of the cyclic phosphodiesterase inhibitors on the secretary response to noradrenaline, acetylcholine, dibutyryl cyclic AMP and dibutyryl cyclic GMP was complex. In general, the increase in enzyme release produced by the secretagogues was additively enhanced by both inhibitors. 6. Omission of calcium inhibited both acetylcholine and dibutyryl cyclic GMP stimulated kallikrein release, but to a lesser degree than that
632 JANET ALBANO AND OTHERS of noradrenaline and dibutyryl cyclic AMP. High concentrations of extracellular calcium (10 mM) appeared to enhance the action of acetylcholine. 7. Noradrenaline produced a rise in the intracellular level of cyclic AMP. The increase preceded the stimulated secretion of kallikrein. Of the various adrenergic agonists, noradrenaline and isoprenaline were the most potent, whereas phenylephrine was significantly less effective in raising basal cyclic AMP values. Acetylcholine was without effect, even in the presence of a cyclic phosphodiesterase inhibitor. 8. Acetylcholine and noradrenaline raised intracellular levels of cyclic GMP only when the tissue incubations were performed in the presence of a cyclic phosphodiesterase inhibitor. The increase in cyclic GMP produced by acetylcholine preceded enzyme secretion. 9. Morphological data substantiated the finding that the in vitro release of kallikrein evoked by the secretagogues was associated with the depletion of secretary granules and vacuolations in acinar cells of the gland slices. 10. The molecular mechanisms which control enzyme secretion in the exocrine submandibular gland are discussed. Models are presented for the role of transmitter-specific cyclic nucleotides and calcium in stimulussecretion coupling. INTRODUCTION
Hormones or first messengers are known to interact with specific receptors on the plasma membrane of cells in the target-tissue. The second messenger concept envisages that the hormone-receptor interaction induces the formation inside the cell of a chemical messenger which mediates events within the cell. Calcium, adenosine 3':5'-cyclic monophosphate (cyclic AMP) and guanosine 3' :5'-cyclic monophosphate (cyclic GMP) are considered to be second messengers of physiological and biochemical importance. The formation and turn-over of the cyclic nucleotides (cyclic AMP and cyclic GMP) in the cell is controlled by the nucleotide cyclase (adenylate and guanylate) and the cyclic nucleotide phosphodiesterases (cyclic PDE). Nearly 10 years ago, Schramm and his colleagues (Bdolah & Schramm, 1965; Babad, Ben-Zvi, Bdolah & Schramm, 1967) suggested that enzyme secretion evoked by adrenaline from the parotid gland was mediated by cyclic AMP. Using rat parotid gland slices, they demonstrated that dibutyryl cyclic AMP caused a marked increase in amylase release. Furthermore, theophylline and caffeine, which are known to inhibit cyclic nucleotide phosphodiesterase, also stimulated enzyme secretion. Taken alone, these results did not constitute convincing proof of the physiological participation of cyclic AMP in adrenaline-induced amylase secretion. The case for cyclic AMP-mediated enzyme secretion was
633 STIMULUS-SECRETION COUPLING strengthened by the demonstration of a catecholamine-activated adenylate cyclase in the rat parotid (Schramm & Naim, 1970) and guinea-pig submandibular glands (Bhoola & Lemon, 1973, 1975). One of Sutherland's criteria, by which the role of cyclic nucleotides in a biological process is assessed (Sutherland, Robinson & Butcher, 1968), namely that the tissue contains adenylate cyclase has been established (Lemon & Bhoola, 1975) and initial studies have indirectly suggested the presence ofguanylate cyclase in exocrine glands. The questions whether submandibular neurotransmitters cause a rise in cyclic AMP and cyclic GMP levels which precede the enzyme secretary response, whether inhibitors of cyclic nucleotide phosphodiesterase influence that response and whether derivatives of cyclic AMP and cyclic GMP mimic the action of the neurotransmitters form the subject of the present report. Our preliminary findings were communicated to the Fifth International Congress in Pharmacology (Bhoola & Lemon, 1972), Second International Conference on Cyclic AMP (Bhoola & Lemon, 1974) and the Physiological Society (Bhoola & Lemon, 1975; Albano, Bhoola, Croker, Heap & Lemon, 1976). METHODS
Incubation y. ocedure Male guinea-pigs (Dunkin-Hartley origin, 350-450 g) were anaesthetized by intraperitcneal injection of pentobarbitone sodium (35-40 mg/kg). The submandibular (submaxillary) glands were removed and washed in a modified Krebs-Ringer bicarbonate buffer (KRB) containing, in mM: NaCl, 121; KCl, 14-3 or 5-0; NaHCO3, 25-2; CaCl2, 2-6; MgS04, 1-2; glucose 5-6; /J-hydroxybutyric acid, 6-1 (Babad et al. 1967). The potassium concentration of the Krebs-Ringer bicarbonate solution was originally maintained at 14-3 mm in order to deplete the gland slices of endogenous transmitter (Babad et al. 1967), but subsequently 5 mm was used because the enzyme secretary response was not appreciably altered by lowering the potassium concentration (see Table 4). Glands were trimmed of the sublingual appendage, connective tissue and hilar blood vessels and cut into slices (about 1-2 mm in thickness and each slice weighing about 50 mg). The pooled slices from submandibular glands of six to nine guineapigs were initially pre-incubated for 25 min at 370 C in 25 ml. Krebs-Ringer bicarbonate solution gassed with a stream of 95 % 02-5 % CO2 in a Grant shaking water bath (160 strokes/min). The slices were removed, blotted and weighed. The tissue incubations were carried out in triplicate. Slices of known weight were placed into single Ehrlenmeyer flasks (15 ml. capacity); each flask containing 2-5 ml. (150200 mg tissue) fresh Krebs-Ringer bicarbonate solution. For the cyclic AMP and cyclic GMP experiments 100- 150 mg tissue was used. At this stage, a pre-incubation of 15 min duration was introduced for experiments in which the tissue concentration of cyclic AMP and cyclic GMP was determined. A similar pre-incubation period was used to study the effect of antagonists. Zero time was taken from the moment 50 or 100 ,ul. drug solution was added to the medium, and the incubation continued for periods ranging from 5 sec to 90 min. At the end of the incubation, the medium was frozen in solid CO2 and stored at below 00 C for subsequent assays of enzyme
634
JANET ALBANO AND OTHERS
content. For the cyclic AMP and cyclic GMP measurement experiments the tissue was removed rapidly and at once frozen in an aluminium clamp cooled in liquid nitrogen.
Measurement of enzyme activity Kallikrein. The esterase activity of the serine proteinase was determined by the hydrolysis of the synthetic substrate, benzoyl-1-arginine-ethyl ester (BAEe), which has been described previously (Bhoola, Dorey & Jones, 1973). The rate of hydrolysis of BAEe was followed for 3 min at 250 C in a Pye-Unicam SP 1800 recording spectrophotometer. The linear phase of the change in absorbance was used to calculate activity which was expressed as an increase in optical density units (AE at 366 nm)/min per millilitre incubation medium. Kallikrein secreted was expressed in optical density units x 106 (BAEe AE366.m)/mg wet weight of tissue. Amylase. The activity of amylase was measured by the iodine titration method as described by Bhoola & Heap (1970).
Measurement of cyclic AMP and cyclic GMP The frozen gland slices, freeze-clamped at the end of the incubation period, were plunged into glass tubes containing 0-5-1-0 ml. Tris-HCl (4 mm theophylline in 10 mM Tris-HCl; pH 7-5) and heated in a boiling-water bath for 10 min. The denatured tissue was homogenized, centrifuged at 20,000 g and the supernatant freezedried. The lyophilized sample was assayed for cyclic AMP by the competitive binding protein method of Gilman (1970) and modified by Cooper, McPherson & Schofield (1972). For the measurement of cyclic AMP, the cyclic AMP-dependent protein kinase, which specifically binds cyclic AMP, was prepared from rabbit skeletal muscle as described by Walsh, Perkins & Krebs (1968). The validity of the cyclic AMP assay was verified. Recovery of cyclic AMP added to the tissue samples before the heat denaturation step was 87-90 %. There was complete recovery of cyclic AMP added to the lyophilized samples immediately before assay. Purified beef heart cyclic nucleotide PDE (Sigma) abolished the noradrenaline-stimulated increased in cyclic AMP. For more recent measurements of cyclic AMP and the determination of cyclic GMP in the gland slices, the freeze-clamped tissue was plunged into Pyrex glass tubes containing 0-5 ml. boiling solution of 6 mM theophylline. After 10 min of boiling the tissue was frozen, subsequently thawed and homogenized. To each tube was added 0-5 ml. acidified ethanol (0-01 N-HCl in absolute ethanol). After mixing, the tubes were stored below 00 C overnight. Samples were thawed, centrifuged at 2000 g x 5 min and the supernatant decanted into small plastic tubes. The precipitate was washed with 0-5 ml. acidified alcohol. After mixing and re-centrifuging, the corresponding supernatants were combined. The extracts were taken to dryness in an oven at 500 C. Before assay, the residue was dissolved in 0-5 ml. Tris-HCl buffer (50 mM; pH 7-4). Cyclic AMP was measured by competitive protein binding utilizing the cyclic AMP binding protein from the bovine adrenal cortex (Brown, Albano, Ekins, Sgherzi & Tampion, 1971). Cyclic GMP was determined by a radioimmunoassay for which specific antibodies were raised to succinylated cyclic GMP following the procedure outlined by Steiner, Parker & Kipnis (1972). The antibody used showed a cross-reaction of less than 0-2 % with cyclic AMP at 1 p-molelml. cyclic GMP and less than 0-01 % with other cyclic nucleotides. Nucleotide mono-, di-, triphosphates showed virtually no cross-reaction up to a concentration of 104 p-mole/ ml. The standard curves for the assays were supplemented with the corresponding reagent blank (Albano, Barnes, Maudsley, Brown & Ekins, 1974). All determinations were performed in triplicate.
STIMULUS-SECRETION COUPLING
635
Morphology At the completion of the incubation, tissue for morphology was immediately immersed in a double aldehyde fixative (freshly prepared 4%, w/v, paraformaldehyde and 2-5 %, w/v, glutaraldehyde for 2 hr), and the entire fixation procedure was performed at 40 C. Tissue was washed for 15 min in sodium phosphate buffer (0-2 M, pH 7 3), post-fixed in osmium tetroxide (1% in 0-2 M sodium phosphate buffer, 4° C) for 2 hr, washed in distilled water for 30 min, dehydrated in graded ethanol and 1,2 epoxypropane and embedded in Araldite. Sections of 1 jum thickness were cut for light microscopy and stained with 1 % toluidine blue in sodium borate buffer (pH 2.4). For electron microscopy silver gold sections were stained with uranyl acetate and lead citrate and examined in a Hitachi HU-11B or HU12A electron microscope. Areas of cell vacuolation observed in the light microscopy sections of submandibular gland slices incubated with the various secretagogues were measured with a Quantimet 720 Image Analyser (Metals Research, Royston, Hertfordshire). This instrument, which is an image editing television microdensitometer, provided digital values for differing densities within a specified area. The light microscopic image of a toluidine blue-stained tissue section was monitored by an electronic scanner which displayed the image on a television screen. By activating a density contrast module the instrument was instructed to measure a specified grey level or tone of density within a required field which was delineated on the displayed image with a light sensitive pen. The area of low density (vacuolation) and the total area scanned were displayed as digital values which were subsequently converted into #m2. At least four sections of 1 jtm thickness stained with toluidine blue were examined to determine each vacuolation value. The mean area scanned was 22,000 /um2 (range 9000-49,000Ium2). We are particularly grateful to Dr M. N. Croker for making available the instrument and for assisting with the measurements. RESULTS
Secretion studies In initial experiments enzyme activity was determined in gland slices as well as the incubation medium. During a 60 min incubation period a greater proportion of the tissue kallikrein was released into the medium than that of amylase, both under conditions of basal and of stimulated secretion. Because in previous studies it had been established that kallikrein in the guinea-pig submandibular gland was sequestered within secretary granules whereas amylase was possibly localized in two pools, cytoplasmic and granular (Bhoola & Ogle, 1966; Bhoola, 1968; Bhoola & Heap, 1970), the secretion studies were confined to the measurement of kallikrein only. The release of kallikrein into the incubation medium was considered therefore to represent exocytotic secretion. Stimulation of enzyme secretion through activation of adrenoceptors Noradrenaline. In the concentration range 4 UM to 4 mM, noradrenaline caused an increase in kallikrein secretion (Text-fig. 1). At the higher concentrations (400 /tM, 4 mM) this increase was observed within the
636
JANET ALBANO AND OTHERS sampling time of 5 sec, but at the lower concentrations (40 ,M, 4/tM) the onset of stimulated secretion was not apparent until 30 see or 1 min after the addition of noradrenaline. The initial secretary response increased linearly between 5 see and 2-5 min, and the accumulation of kallikrein during this period increased with time. Between 5 and 30 min the rate of 2500
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JANET ALBANO AND OTHERS submandibular gland, papaverine (50 = 2 x 10-5 M) was about twenty-fivetimes more potent than theophylline (So0 = 5 x 10-4 M) in inhibiting cyclic AMP phosphodiesterase (Lemon & Bhoola, 1975). A similar degree of potency was observed in the in vitro slices experiments (Table 3). Theophylline (1-8 mM) and papaverine (0.4 mM) increased basal secretion. The secretary response to noradrenaline (0.4 mm) was increased by 1 mM theophylline and by 0 4 mm papaverine. The acetylcholine-evoked release of kallikrein was also additively increased in the presence of 4-8 mm theophylline and 0 4 mm papaverine. Whereas the secretion obtained with acetylcholine and dibutyryl cyclic GMP increased progressively when the concentration of theophylline was raised from 1 to 8 mm, that of noradrenaline and dibutyryl cyclic AMP was maximal at 1 mM theophylline and showed a relative decrease as the concentration of the methyl xanthine was increased in the incubation medium. 642
TABLE 5. Influence of calcium on kallikrein secretion evoked by transmitter substances and cyclic nucleotides from submandibular gland slices
Calcium concentration in Krebs-Ringer solution
Calcium-free ,1 mM EGTA 2-6 mM 162+24 88±13 417 ± 46 128 ± 16 610 + 78 258 + 22 469 ± 66 192 + 21 157+25 258+43
-,
5 mM
10 mm 263±66 769 ± 96 683 + 49 439 + 47 416+121 467 ± 74 425 ± 79
191+32 Basal, 15min 639 + 74 Acetylcholine 542 ± 69 Noradrenaline 468 ± 51 Isoprenaline 201+47 Basal, 30min 314 ± 31 321 + 17 350 ± 39 Dibutyryl cyclic AMP 241 + 32 312 ± 47 150 + 11 Dibutyryl cyclic GMP Submandibular gland slices were incubated with acetylcholine (4 mM), noradrenaline (4 mM) and isoprenaline (4 mM) for 15 min and dibutyryl cyclic AMP (4 mM) and dibutyryl cyclic GMP (4 mm) for 30 min in Krebs-Ringer solution containing 0 (1 mm EGTA), 2-6, 5 and 10 mm calcium. Values represent kallikrein activity expressed as BAEeAE366 x 106/mg tissue, ± S.E. of mean, n = 6-8. Each value is the mean of n (six to eight) measurements performed on n groups of slices (three to four slices; 150-200 mg); each n group was derived from pooled, pre-incubated slices prepared from both submandibular glands of nine animals.
Influence of ions on enzyme secretion Role of K+. A progressive increase in the K+ concentration of the incubation medium, was associated with increased release of kallikrein (Table 4). The peak response obtained at 75 mM-K+, with the threshold concentration between 25 and 75 mM-K+, was similar to that reported for amylase release from rat parotid slices incubated in vitro by Bdolah, Ben-zvi & Schramm (1964). The time course and magnitude of kallikrein
STIMULUS-SECRETION COUPLING 643 secretion evoked by 75 mm-K+ was similar to that obtained with 40 pM noradrenaline. Calcium dependence of the secretary response. The action of calcium on the secretion of kallikrein by the neurotransmitters and by cyclic nucleotides was investigated by altering the calcium concentration of the KrebsRinger buffer solution. When the ionic composition was altered, the concentration of NaCl was changed to maintain isotonicity. Ethylene glycol tetracetate (EGTA, 1 mM) was added to the media designated 'calcium free'. The experimental design involved a first pre-incubation period of 20 min in the standard Krebs-Ringer buffer (2.6 mm calcium), followed by a second pre-incubation period of 20 min in the specific calcium-containing Krebs-Ringer; for the 'calcium free' data both the first and second pre-incubations were carried out in the 'calcium free' Krebs-Ringer bicarbonate solution. The omission of calcium combined with the addition of EGTA to the incubation medium reduced the acetylcholine (from 2*6-fold to 1*5-fold over basal release) stimulation of enzyme secretion to a greater extent than that of noradrenaline (from 3-8-fold to 2*9-fold over basal release) and isoprenaline (from 2-9-fold to 2*2-fold over basal release). Furthermore, the action of dibutyryl cyclic GMP on enzyme release was abolished whereas that of dibutyryl cyclic AMP was only moderately affected. Of the secretagogues tested, only acetylcholine increased kallikrein secretion progressively with increasing concentrations of calcium.
Cyclic AMP and cyclic GMP levels in enzyme secreting submandibular gland slices
Cyclic AMP. Noradrenaline at a concentration of 10-4 M caused a marked increase in cyclic AMP levels in submandibular gland slices. The increase was apparent within the sampling time (5 see). Maximum values were attained between 20 see and 2 min. Subsequently there was a gradual fall in tissue concentration of cyclic AMP which returned to control values at 90 min (Text-fig. 5). The cyclic AMP concentration in control slices remained below about 0-2 p-mole/mg tissue throughout the 90 min incubation. The PDE inhibitor, 3-isobutyl-1-methyl xanthine, markedly enhanced the noradrenaline action of raising the tissue concentration of cyclic AMP (Text-fig. 6). A similar potentiation was observed with 4 mM theophylline. The noradrenaline induced increase in the tissue cyclic AMP levels was dose-dependent. With 1 /IM noradrenaline the rise in cyclic AMP level preceded the increase in kallikrein secretion. Comparison of the efficacy of various adrenergic agonists in raising the intracellular level of cyclic AMP showed that at the incubation time of 1 min, noradrenaline and isoprenaline were most potent, phenylephrine was
644 JANET ALBANO AND OTHERS significantly less active and methoxamine was ineffective (Text-fig. 7). Under similar experimental conditions, acetylcholine up to a concentration of 5 x 10-3 M, even in the presence of 4 mM theophylline or 0 5 mM isobutyl methyl xanthine, consistently failed to increase the concentration of cyclic AMP in glands incubated with the transmitter over a time course ranging from 5 sec to 60 min (see Text-fig. 7). 08
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Text-fig. 5. Time course of intracellular concentration of cyclic AMP (cAMP) in submandibular gland slices secreting kallikrein in response to noradrenaline and acetylcholine. Values for cyclic AMP are expressed as p-mole/mg tissue. Incubation times ranged from 5 sec to 90 min and are plotted on semilog. paper. Basal (x), 100 /lM noradrenaline (X), 5 mM acetylcholine (0). Each value is the mean of n (nine) measurements performed on n groups of slices (two to three slices, 75-125 mg); each n group was derived from pooled, pre-incubated slices prepared from both submandibular glands of eight animals. Bar shows 1 S.E. of mean, n = 9 (each n value was the mean of triplicate cyclic AMP measurement). Data from Bhoola & Lemon (1975), with the preliminary values for 0-5 and 0 7 min omitted.
Cyclic GMP. Both acetylcholine and noradrenaline failed to affect the intracellular concentration of cyclic GMP at several incubation times from 30 sec to 30 min. Only in the presence of isobutyl methyl xanthine (cyclic nucleotide PDE inhibitor) was a significant rise in cyclic GMP observed with acetylcholine (Text-fig. 8) and noradrenaline (Text-fig. 9). The increased levels of cyclic GMP observed after 2 min incubation with acetylcholine (0.4 mM) preceded the onset of significant kallikrein release
STIMULUS-SECRETION COUPLING
645 (5 min). However, the rise in cyclic GMP produced by noradrenaline (0.4 mM) did not appear to precede the increase in enzyme secretion.
Morphological studies For the morphological data the gland slices were incubated for 1, 15 and 90 min with each of the secretagogues (control gland slices, Pls. 1(1), 1(2)). 8
044 606
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Text-fig. 6. Potentiation of the noradrenaline stimulated increase in cyclic AMP by the cyclic phosphodiesterase inhibitor, 3-isobutyl 1-methyl xanthine (IBMX), in submandibular gland slices secreting kallikrein. Values for cyclic AMP are expressed as p-mole/mg tissue. Incubation times: 0-5, 2, 5 and 30 min. Histograms represent basal (1]), 0-4 mM noradrenaline (M), basal with 0-5 mm IBMX (I) and 0-4 mm noradrenaline with 0-5 mm IBMX (F) values. Each value is the mean of n (three) measurements performed on n groups of slices (two to three slices, 100150 mg); each n group was derived from pooled, pre-incubated slices prepared from both submandibular glands of six animals. Bar shows 1 S.E. of mean, n = 3 (each n value was the mean of triplicate cyclic AMP measurements).
Noradrenaline and dibutyryl cyclic AMP. Even though significant secretion of kallikrein occurs within 30 sec of contact with noradrenaline, no obvious cellular changes were discernible at 1 min. However, a marked reduction in the number of secretary granules together with considerable vacuolation in the acinar cells was observed in the 15 min sections of the slices (Pls. 2(3), 2(4); Text-fig. 10). After 90 min incubation only a few granules of intermediate staining with toluidine blue remained (P1. 3) but the vacuolation was only marginally increased. Similar morphological changes of the release of secretary granules and vacuolation in acinar
JANET ALBANO AND OTHERS cells of isolated rat parotid gland slices incubated with adrenaline have been described by Batzri, Selinger & Schramm (1971). A progressive increase in vacuolation was detected between the 15 and 90 min incubations with dibutyryl cyclic AMP. The area of vacuolation induced by the cyclic nucleotide after 90 min was comparable to that measured for noradrenaline (see Text-fig. 10), whereas the number of 646
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Text-fig. 7. Comparison of the relative potency of adrenergic agonists and acetylcholine in raising cyclic AMP levels in submandibular gland slices secreting kallikrein. Values for cyclic AMP are expressed as p-mole/ mg tissue. Drug concentration 0-4 mm. Incubation times I and 30 min. Histograms represent basal (Fl), acetylcholine (E), noradrenaline (E), isoprenaline (EM), phenylephrine (a) and methoxamine (MII) values. Each value is the mean of n (three) measurements performed on n groups of slices (tw o to three slices, 100-150 mg); each n group was derived from pooled, pre-incubated slices prepared from both submandibular glands of six animals. Bar shows I S.E. of mean, n = 3 (each n value was the mean of triplicate cyclic AMP measurements).
secretary granules remaining within the acinar cells was considerably greater (PI. 4(6)). The ultrastructural organization and granule population in the intercalated and junctional ducts (Dorey &r Bhoola, 1972) appeared to be unaffected by both noradrenaline and dibutyryl cyclic AMP.
Acetylcholine and dibubtyryl cyclic GMP. The difference in the cumulative secretion of kallikrein produced by noradrenaline and acetylcholine was reflected in the morphological changes. Both the area of vacuolation and depletion of secretary granules in the acinar cells were clearly less than
647 STIMULUS-SECRETION COUPLING those observed with noradrenaline (P1. 4(7)). The full secretary picture was not apparent morphologically at 15 min but only in the 90 min sections. The main fine structural change observed with dibutyryl cyclic GMP appeared to be an increase in vacuolation in the acinar cells. No depletion of the granule population in the intercalated and junctional duct cells was observed with acetylcholine or dibutyryl cyclic GMP. 020
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Text-fig. 8. Effect of acetylcholine on cyclic GMP (cGMP) levels in the presence and absence of the cyclic nucleotide PDE inhibitor, 3-isobutyl 1-methyl xanthine (IBMX), in submandibular gland slices secreting kallikrein. Values for cyclic GMP are expressed as p-mole/mg tissue. Incubation times: 0-5, 2, 5 and 30 min. Histograms represent basal (E), 0-4 mm acetylcholine (Ed), basal with 0-5 mm IBMX (En) and 0-4 mm acetylcholine with 0-5 mm IBMX (E) values. Each value is the mean of n (three) measurements performed on n groups of slices (two to three slices; 100-150 mg); each n group was derived from pooled, pre-incubated slices prepared from both submandibular glands of six animals. Bar shows 1 s.E. of mean, n = 3 (each n value was the mean of triplicate cyclic GMP measurements).
Calcium. The morphological study was extended to gland slices incubated for 90 min in Krebs-Ringer solutions from which calcium was omitted but contained 1 mm EGTA, and in solutions which contained 10 mm calcium. Light microscopy sections from control and acetylcholinestimulated slices incubated in calcium-free solution showed increased vacuolation in the acinar cells. In addition, a reduction in granule numbers was observed in the junctional duct cells with noradrenaline and dibutyryl
JANET ALBANO AND OTHERS cyclic AMP. Gland slices incubated in 10 mm calcium showed a lesser amount of vacuolation in the acinar cells of the control and the acetylcholine and noradrenaline stimulated tissue but marginally greater in the tissue incubated with the cyclic nucleotides. 648
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Text-fig. 9. Effect of noradrenaline on cyclic GMP levels in the presence and absence of the cyclic nucleotide PDE inhibitor, 3-isobutyl 1-methyl xanthine (1BMX), in submandibular gland slices secreting kallikrein. Values for cyclic GMP are expressed as p-mole/mg tissue. Incubation times: 0-5, 2, 5 and 30 min. Histograms represent basal (D), 0-4 mM noradrenaline (M), basal with 0-5 mm IBMX (13) and 0-4 mm noradrenaline with 0-5 mm 1BMX (I) values. Each value is the mean of n (three) measurements performed on n groups of slices (two to three slices; 100-150 mg); each n group was derived from pooled, pre-incubated slices prepared from both submandibular glands of six animals. Bar shows 1 S.E. of mean, n = 3 (each n value was the mean of triplicate cyclic GMP measurements). DISCUSSION
Stimulation of enzyme secretion Adrenoceptor-mediated amylase secretion from mammalian salivary glands has been reported in a series of in vivo experiments (rat parotid, Pohto, 1968; human parotid and submandibular, Katz & Mandel, 1968; rabbit parotid, Yamamoto, Inoki & Kojima, 1968; mouse parotid, Durham & Butcher, 1974) and in vitro (rat parotid, Babad et al. 1967; guinea-pig submandibular, Carls66, Danielsson, Marklund & Stigbrand, 1974). Of the secretagogues examined in the present study, noradrenaline
STIMULUS-SECRETION COUPLING 649 was the most effective in stimulating kallikrein secretion. The response to noradrenaline contained both a- and fl-adrenoceptor-mediated components, since the presence of both adrenoceptor blockers was required to completely abolish the enzyme secretary response. The adrenoceptor components were further studied by using a limited series of agonists. Of these phenylephrine (with greater a-receptor activity) was of similar potency on a molar basis to isoprenaline (with greater fl-receptor activity; 16
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