muscarinic agents. A. PETERNEL VREUGDENHIL* From the Department of Oral Biochemi8try, Faculty of Denti8try, Vrije Univer8iteit,

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1 J. Phyaiol. (1978), 28, pp With 4 text-figure Printed in Grea Britain COMPARISON OF ADENYLATE CYCLASE ACTIVITY AND IN VITRO SECRETION IN THE PAROTID AND SUBLINGUAL GLANDS OF THE MOUSE BY A. V. NIEUW AMERONGEN, P. A. ROUKEMA AND A. PETERNEL VREUGDENHIL* From the Department of Oral Biochemi8try, Faculty of Denti8try, Vrije Univer8iteit, Van der Boechor8tstraat 7, P.O. Box 7161, Amnterdam, the Netherland8 SUMMARY 1. Adenylate cyclase (EC ) activity has been determined in the parotid and sublingual glands of the mouse. Optimal activity of the enzyme was obtained at a Mg2+-concentration of 8 mm at ph 8-2, using AMP-PNP as the substrate. 2. Cyclic AMP degradation during the adenylate cyclase assay was relatively high in both the homogenate and the 4, g pellet-fraction of the glands. Theophylline was effective in inhibiting this degradation only in the parotid homogenate, whereas isobutylmethylxanthine inhibited the cyclic AMP degradation in both salivary glands. Using the latter phosphodiesterase inhibitor, we observed a higher adenylate cyclase activity in the sublingual glands than in the parotid glands.. Various receptor-selective sympathetic and parasympathetic agonists and antagonists have been tested for their capacity to influence the adenylate cyclase activity and the glycoprotein secretion in the parotid and sublingual glands of the mouse, in vitro. (a) The parotid glycoprotein secretion was increased by f8-adrenergic agonists, which stimulate adenylate cyclase, and by cholinergic muscarinic drugs, which do not activate this enzyme. The adrenergic ai-agonist phenylephrine appeared to be involved neither in the glycoprotein secretion nor in the direct regulation of the adenylate cyclase activity. (b) The sublingual protein and mucin secretion was increased by cholinergic muscarinic agents. The over-all protein secretion was stimulated also by phenylephrine, but this effect could be blocked by propranolol. The adenylate cyclase activity in membrane preparations was not stimulated by these secretogogues. INTRODUCTION In the double-innervated parotid gland of the mouse, isoprenaline stimulates the adenylate cyclase activity in vitro (Durham, Galanti & Revis, 1975; Chiu, Franks, Rowe & Malamud, 1976), enhances the cyclic AMP level in vivo (Malamud, 1972; Durham, Baserga & Butcher, 1974; Vreugdenhil & Roukema, 1975) and stimulates amylase secretion in vivo (Malamud, 1972; Durham & Butcher, 1974). Moreover, * This work is part of a doctoral thesis, to be submitted by A. P. Vreugdenhil in November 1978 at the Vrije Universiteit, Amsterdam.

2 212 A. V. NIEUW AMERONGEN AND OTHERS dibutyryl cyclic AMP is able to stimulate the glycoprotein secretion (Vreugdenhil & Roukema, 1975). These data point to the involvement of cyclic AMP in the regulation of the secretary process in the parotid glands of the mouse and the activation of adenylate cyclase through the fi-adrenoceptor. Similar results have been reported for rat parotid glands. The possible involvement of a-adrenoceptors in the enzyme secretion, however, is more controversial (Batzri, Selinger & Schramm, 1971; Butcher, Goldman & Nemerovski, 1975; Leslie, Putney & Sherman, 1976). In vitro, the mucin secretion from the sublingual glands of the mouse is stimulated mainly by acetylcholine, whereas the protein secretion from these glands is stimulated by noradrenaline also. In contrast to the parotid glands, the basal cyclic AMP level in the sublingual glands is not increased by isoprenaline, in vivo (Vreugdenhil & Roukema, 1975). Probably, in these glands the adenylate cyclase linked fl-adrenoceptor is absent. For this reason, we studied the enzymes, which regulate the cyclic AMP level in the parotid and sublingual glands of the mouse. In this paper we describe some of the conditions for the assay of adenylate cyclase in both salivary glands of the mouse using AMP-PNP as the substrate. We also present the results of a study on the receptors involved in the regulation of both the secretion and the adenylate cyclase activity. The possibility that adenylate cyclase and cyclic AMP are involved in the secretary processes of the sublingual glands is discussed. METHODS Enzyme preparation The parotid and sublingual glands were removed from female mice (Swiss-random) of 2-22 g, cleaned in an ice-cooled solution of 9 % (w/v) NaCl and homogenized gently in a -2 M-sucrose solution, buffered by 5 mm-tris-hcl at ph 7-4 in a Potter-Elvehjem homogenizer with a teflon pestle. A plasma membrane-enriched fraction was prepared according to the method of Durham et al. (1975), with the modification that the 15, and 4, g pellet-fractions were combined. After filtration through cheesecloth, the homogenate was centrifuged at 65 g for 1 min. Subsequently, the resulting supernatant was centrifuged at 15, g for 15 min. Without interruption, the centrifugation was continued at 4, g for 6 min. The pellet was resuspended in the homogenization medium by vortexing and centrifuged again at 4, g for 6 min. The washed 4, g pellet-fraction, resuspended in the homogenization medium, in portions of 1 pil., was stored in liquid nitrogen until use. When the total homogenate was used as the enzyme source, it was homogenized again, before use, like the resuspended 4, g pellet-fraction. Adenylate cyclase assay The adenylate cyclase activity was measured by the method of Maguire & Gihnan (1974), using AMP-PNP as the substrate. The assay was performed in a total volume of 1 pi., containing: 2 mm-tris-hcl ph 8-2, 8- or 9-6 mm-mgso4, -1 % bovine serum albumin, -5 mm-amp-pnp, either -1 mm-theophylline or 2 mm-isobutylmethylxanthine and 5-1I. of the enzyme preparation. After incubation at C for 1-2 min, the reaction was terminated by placing the stoppered tubes in a boiling water bath for min. After cooling on ice, the tubes were centrifuged, and 25-5 jul. of the supernatants were introduced directly into the cyclic AMP assay according to the method of Brown, Albano, Ekins, Sgherzi & Tampion (1971). The reaction mixture for the standard curve for cyclic AMP contained an appropriate concentration of AMP-PNP for each experiment (Maguire & Gilman, 1974). Before use AMP-PNP had to be purified by column chromatography, as described by Maguire & Gilman (1974). AMP-PNP was eluted from the DEAE Sephadex A 25 column (1.5 cm x cm) at a concentration of 4 mm-

3 ADENYLATE CYCLASE AND PAROTID SECRETION triethyl-ammonium bicarbonate buffer. Purity of AMP-PNP was confirmed by thin-layer chromatography, using silica gel as the solid phase and propanol-1: conc. ammonia: distilled water (6: : 1) as a solvent system. The cyclic AMP-degradation during the adenylate cyclase assay, was measured under the conditions as described above. In this case, AMP-PNP was omitted and replaced by cyclic AMP (1-5 p-mole/too ul.). Blanks for the adenylate cyclase assay contained both the substrate and the enzyme. They were placed directly in the boiling water bath. Each experiment was performed in duplicate or triplicate. Na+/K+ATPa8e assay (Na+/K+)-activated ouabain-sensitive ATPase was assayed as described by Bonting, Simon & Hawkins (1961). The released phosphate was measured by the method of Taussky & Shorr (195). Each determination was performed in duplicate. Incubation procedure The incubation procedure for measuring the secretion in vitro, from the parotid and sublingual glands under influence of the various sympathetic and parasympathetic agonists or antagonists, was performed as described previously (Vreugdenhil & Roukema, 1975). Twenty-four hours before decapitation, female mice were injected with reserpine (5 mg/kg), to deplete the sympathetic nerve endings. Three parotid or sublingual glands were incubated in 1.5 and 1 ml. Krebs Ringer solution, respectively. In all experiments the Krebs Ringer solution contained 2-5 mm-cacl2. 21 Analytical procedures The amylase, protein and sialic acid determinations were performed as described previously (Vreugdenhil & Roukema, 1975). Sialic acid was assayed as a marker for glycoproteins and mucins. Statietical procedure Values for statistical significance were computed using the Mann-Whitney (Wilcoxon) two sample statistic. Material Adenylyl-imidodiphosphate (AMP-PNP) was purchased from Boehringer Mannheim, - isobutyl-1-methylxanthine from Aldrich-Europe, acetylcholine iodide and atropine sulphate from Koch-Light, T,-noradrenaline and nicotine from Merck-Schuchardt, pilocarpine HCl, D,L-isoproterenol HCO, L-phenylephrine HCO, L-propranolol HCO, cyclic AMP, theophylline and aminophylline from Sigma, phenoxybenzamine HCl from Smith Kline and French Labs, hexamethonium bromide B.P.C. from May and Baker Ltd, phentolamine and reserpine from Ciba and cyclic [H]AMP from the Radiochemical Centre (Amersham). RESULTS Adenylate cyclase activity Table 1 shows that relatively high Na+/K4-ATPase and adenylate cyclase activity was present in the 4, g pellet-fraction of the parotid glands of the mouse. Though fractionation by differential centrifugation of the mucous sublingual glands was difficult, because the homogenate was very viscous, in most cases the Na+/K+- ATPase and adenylate cyclase activity was two to four fold enriched in the 4, g pellet-fraction of these glands. The optimal Mg2+ concentration (8 mm) and ph (8.2) for the adenylate cyclase assay appeared to be the same for both glands, using AMP-PNP as the substrate.

4 214 A. V. NIEUW AMERONGEN AND OTHERS However, the cyclic AMP degradation during the adenylate cyclase assay appeared to be very high in both the tissue homogenate and the 4, g pellet-fraction of the glands. 5-1 mm-theophylline inhibited this degradation more effectively in the homogenate of the parotid glands than in the sublingual glands. Also whereas aminophylline (1 mm) was able to inhibit the cyclic AMP degradation in the 4, g pellet-fraction of the parotid glands almost completely, in the sublingual glands only TABLE 1. Some characteristics of the 4, g pellet-fractions. The adenylate cyclase activity has been determined in the presence of 9-6 mnm-mgso4 and 5 mm-theophylline and is expressed as: p-mole cyclic AMP/mg protein. min. The specific activity of NA+/K+-ATPase is expressed as: molee PO'-/mg protein. hr. The data are the mean + s.e. of four to five different enzyme preparations Na+/K+-ATPase Adenylate cyclase Protein A, recovery Homo- Recovery Homo- Recovery Gland (%) genate 4,9 (%) genate 4,9 (%) Parotid ± ± Sublingual ± ± A 15 8 U X ~ ~~1M Time (min) Time (min) Fig. 1. Cyclic AMP degradation in the homogenate (A) and 4, g pellet-fraction (B) of the salivary glands of the mouse. * * parotid (A: 54-5 jug, B: 4-8 jusg of protein), x.. x sublingual (A: 5-2 jag, B: 22-1ag), both in the absence of a phosphodiesterase inhibitor, (IBMX) in the presence of 2 mii-isobutylmethylxanthine. 5 %/ of the activity was inhibited. In contrast, using 2 mm-isobutylmethylxranthine as a phosphodiesterase inhibitor, we found effective inhibition of the cyclic AMP degradation in the tissue homogenate and 4, g pellet-fraction of both glands (Fig. 1). In the presence of this inhibitor, the adenylate cyclase activity measured in the homogenate of the sublingual glands appeared to be higher than in the parotid glands (Table 2). Stimulation of the adenylate cycloase activity The enzyme activity in the 4, g pellet-fraction of the parotid and sublingual glands was linear in time up to at least and 6 min respectively and with a protein concentration up to 5 g/1oo 1. and at least 6 jug/1 Sl respectively (Fig. 2).

5 ADENYLATE CYCLASE AND PAROTID SECRETION mm-naf appeared to be the most potent stimulator of the adenylate cyclase activity in both glands (Tables and 4), as was also found by Chiu et al. (1976) and Durham et al. (1975) for the parotid glands of the mouse. Noradrenaline and isoprenaline, both potent stimulators of the protein secretion from the parotid glands (Fig. ), increased the activity of adenylate cyclase. Also phenylephrine stimulated TABLE 2. Adenylate cyclase activity in a fresh homogenate of the salivary glands, in the presence of either theophylline or isobutylmethylxanthine. The enzyme activity is determined under the optimal Mg2+- and ph-conditions and is expressed as p-mole cyclic AMP/mg protein. min. The data are the mean ± s.e. of three different enzyme preparations Gland Phosphodiesterase inhibitor Parotid Sublingual Theophylline (5 mx) 16x± ± 7*8 Isobutylmethylxanthine (2 mx) 18-± ± Pp 4 ) E a < 2 p C.) 1 SL O 5 Protein (jig) Fig. 2. Protein-dependency of the adenylate cyclase activity in the 4, g pellet-fraotion of the salivary glands of the mouse in the presence of5 mx-theophylline. P: parotid, incubation time 1 min, SL: sublingual, incubation time 2 mii. TAB.r. Effect of autonomic agonists and antagonists on the adenylate cyclase activity in the 4, g pellet-fraction of the parotid glands. Percent stimulation of the adenylate cyclase activity after 2 min incubation with 1 mm-naf, 2 4aM agonist and/or 1 #ux antagonist in the presence of 2 mm-isobutylmethylxanthine. The data are the mean + s.e. of three to eight incubations. The enzyme activity in the homogenate is 1-5 and in the 4, g pellet-fraction is 98-2 p-mole cyclic AMP/mg protein. min Prop ACh Pilo Nic Control ± 9 86± 8 84 ± NA ±7 I Pr 18 ± ± 18 1 PE 115±11 88± 15 NaF 27 ± 1

6 216 A. V. NIEUW AMERONGEN AND OTHERS CY) - V 6 o v v v (, _ U, C U C 2 c.9 T CN~ C.) a) o o.' T: I IT X,..., X T ii-futj T Ir_ - - I.q I t I - -- r - Ff1-11 :::::::: 4.C4 - V LO V o V V r U,>4 ~ C U- C.) (A ) -) CC, z: o+1 Uo +I o- 712 o V C o I/ (5 'VI IN~~~~~I 4 CC e C C. CY) (12 U) (N (n E $o > ui C+L ~ L-.C Q- CL -, +l _o -R o~ T- QOa- co C Qm - fl I Eic Q =OL + < -. m a C) ) + <.2 : tm <c Q '. Q I z L-Q + L + a-.o + < + z <Z & z _ LU L.U a_. po a--al LLO o LD ocd ) M r-l C C ' u) -C) (DO w '* C It CI T- _ c

7 A DENL YA TE C YCLASE AND PAROTID SECRETION 217 the enzyme activity slightly, but L-propranolol inhibited all these effects. Cholinergic drugs did not stimulate the adenylate cyclase activity in the parotid glands (Table ). In the sublingual glands, neither fi- and cx-adrenergic nor cholinergic agonists stimulated the adenylate cyclase activity (Table 4). TABLE 4. Effect of autonomic agonists on the adenylate cyclase activity in the 4, g pellet-fraction of the sublingual glands. Percent stimulation of the adenylate cyclase activity after min incubation with 1 mm-naf or 2 HUm agonist in the presence of 2 mii-isobutylmethylxanthine. The data are the mean + s.e. of three to six incubations. The enzyme activity in the homogenate is 1-6 and in the 4- g pellet-fraction is 24-8 p-mole cyclic AMP/mg protein. min L -Prop PB Atrop HB ACh ACh+Atrop ACh+ HB Pilo Pilo+Atrop Nic Nic+HB Control NA IPr PE % sialic acid secretion ± <-1 <-1 <-5 NaF ACh Pilo Nic 24 ± ± % protein secretion X I I n.s. <-5 <-2 <-2 n.s. _ i I i -f 6--ta I NA n.s. 4 n.s. NA+ Prop NA+PB n.s. IPr n.s. 4 n.s. i I I Pr+ Prop r I PE n.s. 4 <-5 PE+ Prop n.s. PE+PB <-1 P n p 56% 14-9 % Fig. 4. Protein-bound sialic acid and protein secretionfrom the sublingual glands in vitro, under the influence of different automonic agonists (1 vim) and antagonists (1j/M). The data are the mean + a.e. of n experiments. Values for P denote statistical significance versus controls (Wilcoxon test). n.s. is not significant. Incubation time: 6 min. In vitro secretary responses (a) Parotid glands. The protein, amylase and protein-bound sialic acid secretion from the parotid glands were stimulated by acetylcholine, and noradrenaline. These responses were inhibited by atropine, and propranolol respectively. However, the

8 218 A. V. NIEUW AMERONGEN AND OTHERS over-all protein secretion was also inhibited somewhat by phenoxybenzamine, but, this effect was not significant (Fig. ). (b) Sublingual glands. The protein and the protein-bound sialic acid secretion from the sublingual glands was stimulated by acetylcholine and pilocarpine. These effects were abolished by atropine. Neither hexamethonium bromide nor az-bungarotoxin (not shown) inhibited the stimulation by acetylcholine, though nicotine had a small, but significant enhancing influence on the mucin secretion (Fig. 4). The adrenergic drugs had no significant influence on the protein-bound sialic acid secretion from the sublingual glands. Only the c-adrenergic agonist phenylephrine appeared to stimulate the over-all protein secretion. However, propranolol inhibited this response (Fig. 4). Because there was no clear relationship between the secretogogues which stimulated sublingual secretion and adenylate cyclase activity, we incubated the sublingual glands with 1 mm-naf, the only consistent stimulator of adenylate cyclase in these glands. NaF produced a threefold stimulation of the mucin secretion. DISCUSSION Several reports have appeared in the literature on the adenylate cyclase activity in the parotid glands of the rat (Schramm & Naim, 197; Butcher et al. 1975) and of the mouse (Durham et al. 1975; Chiu et al. 1976). However, hardly anything is known about this enzyme in the sublingual glands. For the latter glands, only the localization of adenylate cyclase has been studied in the rat (Kim & Han, 1975; Kim, 1976). The % recovery of the adenylate cyclase activity in the 4,g pelletfraction of the parotid glands, measured in the presence of 5 mm-theophylline, is comparable to the data of Durham et al. (1975) (Table 1). The rather low specific activity of adenylate cyclase in this fraction may be caused by the use of AMP-PNP as the substrate instead of ATP (Maguire & Gilman, 1974; Lin, Salomon, Rendell & Rodbell, 1975). Though Chiu et al. (1976) demonstrated that the major part of the phosphodiesterase activity of the parotid glands of the mouse is found in the 1,g supernatant fraction, under our conditions, the cyclic AMP degradation was high in both the homogenate and the 4, g pellet-fraction of the parotid and sublingual glands and the sensitivity of phosphodiesterase for theophylline appeared to be quite different in the two glands. However, isobutylmethylxanthine inhibited the cyclic AMP degradation in both glands very effectively (Fig. 1). Under the latter conditions, the specific activity of adenylate cyclase was higher in the sublingual glands than in the parotid glands (Table 2). Because we were interested in the regulation of the adenylate cyclase activity in relation to the secretary processes, we have tested the influence of several agonists on both parameters. The glycoprotein secretion from the parotid glands of the mouse is stimulated by fl-adrenergic drugs in vitro, as also reported by other authors for the mouse in vivo (Malamud, 1972; Durham & Butcher, 1974) and in agreement with the results for the rat (Batzri et al. 1971; Maurs, Herman, Busson, Ovtracht & Rossignol, 1974; Butcher et al. 1975; Mangos, McSherry, Barber, Arvanitakis & Wagner, 1975;

9 ADENYLATE CYCLASE AND PAROTID SECRETION 219 Leslie et al. 1976) and the rabbit (Wojcik, Grand & Kimberg, 1975). The a-adrenergic agonist phenylephrine does not increase protein secretion from these glands, in contrast to some findings for the rat (Leslie et al. 1976; Butcher, Rudich, Emler & Nemerovski, 1976) and the rabbit (Wojcik et al. 1975). Secretion from the parotid glands is stimulated also by cholinergic muscarinic agonists, in vitro, in contrast to the data from Selinger (1975) for reserpinized mice, though other authors have reported cholinergic stimulation for the rat and the rabbit (Butcher, McBride & Rudich, 1976; Leslie et al. 1976; Muir & Templeton, 1976; Wojcik et al. 1975) (Fig. ). Unlike cholinergic stimulation, in the parotid glands stimulation off-adrenoceptors results in an increase of the adenylate cyclase activity, in vitro (Table ) and the cyclic AMP level in vivo (Vreugdenhil & Roukema, 1975), as also found by other authors (Malamud, 1972; Durham et al. 1974; Durham et al. 1975; Chiu et al. 1976). Apparently, the influence of phenylephrine on the adenylate cyclase activity is owing to the small fl-adrenergic capacities of this agonist (Butcher et al. 1975). Previously, we have reported that dibutyryl cyclic AMP stimulates the parotid secretion (Vreugdenhil & Roukema, 1975). Moreover, NaF, which increases the adenylate cyclase activity, is able to induce secretion from these glands (unpublished observations). So, there is good evidence that in the parotid glands of the mouse, like other rodents, the secretion of the same products can be stimulated via different pathways: via fl-adrenoceptors in which process adenylate cyclase and cyclic AMP are involved, and via cholinergic muscarinic receptors in which Ca2+-ions (Vreugdenhil & Roukema, 1975) or cyclic GMP (Albano, Bhoola, Heap & Lemon, 1976) may be involved. In vitro, the secretion from the sublingual glands is stimulated mainly by cholinergic muscarinic agonists. (Fig. 4). The failure by noradrenaline and isoprenaline to significantly evoke secretary responses and to stimulate adenylate cyclase activity in vitro (Table 4) or to increase the cyclic AMP level in vivo (Vreugdenhil & Roukema, 1975), suggests an absence of fl-adrenoceptors in the sublingual gland of the mouse. The observation that NaF, which stimulates the adenylate cyclase activity very effectively, also induces mucin secretion in vitro, raises the possibility that adenylate cyclase and cyclic AMP may in fact be involved in the sublingual secretary process. The studies of Kim & Han (1975), Durham et al. (1975) and Kim (1976) indicate that a rather high adenylate cyclase activity is present in the luminal plasma membranes of the serous cells of both the parotid glands of the rat and the mouse and also of the sublingual glands of the rat. These observations suggest that in addition to a 'trigger' function, cyclic AMP may play other roles in the secretion processes of both glands. We are continuing the study to test this hypothesis for the sublingual glands. We would like to thank Miss A. den Hertog for her excellent technical assistance. The photographic work of Mr J. Minnaard is gratefully acknowledged.

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11 ADENYLATE CYCLASE AND PAROTID SECRETION 221 MuR, T. C. & TEMPLETON, D. (1976). The role of ',5'-adenosine monophosphate (cyclic AMP) in the ability of sympathetic nerve stimulation to enhance growth and secretion in rat salivary glands in vivo. J. Physiol. 259, SCHiM, M. & NAim, E. (197). Adenyl cyclase of rat parotid gland: activation by fluoride and norepinephrine. J. biol. Chem. 245, SELINGER, Z. (1975). Diverse functions of calcium in the rat parotid acinar cell. Calcium Transport in Contraction and Secretion, ed. CARAFOLI, E. et al., PP Amsterdam: North-Holland Publishing Company. TAUBSKY, H. H. & SHOER, E. (195). A microcolorimetric method for the determination of inorganic phsphorus. J. biol. Chem. 22, VKEUGDENHIL, A. P. & RouxslA, P. A. (1975). Comparison of the secretory processes in the parotid and sublingual glands of the mouse. I. regulation of the secretary processes. Biochim. biophys. Acta 41, Wojcmx, J. D., GRAND, R. J. & KIMBERG, D. V. (1975). Amylase secretion by rabbit parotid gland: role of cyclic AMP and cyclic GMP. Biochim. biophys. Acta 411,