Proc. NatL Acad. Sci. USAVol. 78, No. 11, pp. 6903-6906, November 1981Cell Biology

Phosphorylation of the same specific protein during amylaserelease evoked by 8-adrenergic or cholinergic agonists inrat and mouse parotid glands

(stimulus-secretion coupling/exocrine glands/protein phosphorylation)

REINHARD JAHN AND HANS-DIETER SOLINGAbteilung fir Klinische Biochemie, Medizinisehe Universitits-Klinik, Humboldtallee 1, D-3400 Gdttingen, Federal Republic of Germany

Communicated by Henry A. Lardy, July 6, 1981

ABSTRACT Stimulation of amylase secretion from the ratparotid gland by P-adrenergic agonists is associated with a specificphosphorylation of three membrane-bound proteins designatedas proteins I, H, and HI [Jahn, R., Unger, C. & S6ling, H. D.(1980) Eur. J. Biochem. 112, 345-352]. In contrast, stimulation bycarbachol induced significant phosphorylation of only protein I.This phosphorylation was low compared to isoproterenol-inducedphosphorylation but corresponded to the smaller enhancement ofamylase secretion. The mouse organ, however, is almost equallysensitive to 3-adrenergic and to cholinergic agonists. Incubationof mouse parotid gland slices with either 20 FM isoproterenol or10 FM carbachol resulted in strong and comparable releases ofamylase, which were accompanied by comparable phosphoryla-tions of protein I. Proteins H and EI were phosphorylated only inthe presence of isoproterenol. Removal of external calcium byethylene glycol bis(3-aminoethyl ether)-N,N,N',N'-tetraacetateabolished the carbachol-induced release of amylase but not thephosphorylation of protein L. Isoproterenol-induced secretion ofamylase and phosphorylation of proteins I, H, and IH were notinhibited under these conditions. Amylase release stimulated bythe ionophore A-23187 was accompanied by the phosphorylationof protein L Two-dimensional electrophoresis revealed that theradioactive spot corresponding to protein I was located at the sameposition after cholinergic and after P-adrenergic stimulation, in-dicating that both stimuli led to the phosphorylation of the samemembrane-associated protein. These findings strongly supportthe view that the phosphorylation of protein I is an important stepin the sequence of events leading from receptor activation toexocytosis.

In the parotid gland, amylase release can be stimulated inde-pendently by -adrenergic and by cholinergic agonists. Stim-ulation by ,f3agonists is mediated by cyclic AMP, is independentof extracellular Ca2", and can be mimicked by cyclic AMP de-rivatives. Cholinergic stimulation of amylase secretion appar-ently requires an increase ofthe intracellular Ca2" activity, be-cause it can be induced by the action of the Ca2" ionophore A-23187 and is inhibited by lanthanum or removal of Ca2` fromthe extracellular fluid (for a comprehensive review see ref. 1).We have previously shown (2) that /adrenergic stimulation

of amylase release from rat parotid glands is accompanied bya specific phosphorylation of three proteins located in differentcellular membrane fractions. Protein I (35,100 daltons) seemsto be localized in the smooth endoplasmic reticulum or in theplasma membrane, whereas protein II (25,700 daltons) and pro-tein III (20,400 daltons) are found in the rough endoplasmicreticulum or in the mitochondria. The cyclic AMP-dependentphosphorylation of protein I was confirmed by others with the

aid of a similar technique (3, 4). However, an involvement ofthese phosphorylations in processes other than exocytosis can-not be ruled out. In order to play an essential role in exocytosis,the observed phosphorylation ofmembrane proteins should alsobe detectable during stimulation of amylase secretion by ago-nists acting independently ofan increase ofcellular cyclic AMP.Therefore, in the present work we have compared the effect ofcholinergic with that of 1&adrenergic stimulation on amylaserelease and protein phosphorylation. Because mouse parotidglands are more sensitive to cholinergic stimulation of amylaserelease than is the rat organ (5, 6), we also studied the rela-tionship between stimulation of amylase secretion and phos-phorylation of specific proteins in mouse parotid glands.We found that stimulation ofamylase release by ,adrenergic

agonists, cholinergic agonists, or a Ca2+ ionophore is alwaysassociated with a phosphorylation ofprotein I. The stimulationof phosphorylation is proportional to the enhancement of am-ylase secretion.

MATERIALS AND METHODSChemicals. Acrylamide and bisacrylamide for electropho-

resis were obtained from Serva (Heidelberg). Ampholines (pH3.5-10 and pH 5-7) were from LKB (Griifelfing).Ortho[32P]phosphate was supplied by Amersham Buchler(Braunschweig); DL-isoproterenol, propranolol, atropine, andcarbamoylcholine by Sigma (Munich). A-23187 was a kind giftof Eli Lilly (Giessen). All other reagents (analytical grade) wereobtained from E. Merck (Darmstadt).

Slice System and Subcellular Fractionation. Male Wistarrats (body weight 130-180 g) and male NMRI mice (body weight16-22 g) were used. Preparation of parotid gland slices, incu-bation, and processing of the tissue after incubation have beendescribed (2). A-23187 was dissolved in ethanol. The final con-centration of ethanol during the incubation did not exceed 2%(vol/vol). Subcellular fractionation was carried out at 0-40Cafter homogenization in ice-cold stop solution [0.3 M sucrose/50 mM potassium phosphate, pH 7.0/2 mM EDTA/0.2 mMethylene glycol bis(3-aminoethyl ether)-N,N,N',N'-tetraace-tate (EGTA)]. Under these conditions no phosphorylating anddephosphorylating reactions were observed during the sepa-ration procedure. The homogenates were centrifuged at 1000X gm. for 10 min. The supernatant was spun at 15,000 X gmaxfor 10 min, and the resulting supernatant was centrifuged at100,000 x gm. for 60 min. The 15,000 and 100,000 X g pelletswere resuspended in small volumes of stop solution. In someexperiments the resuspended 100,000 x g pellet was layeredonto a gradient of 34% (wt/vol) and 43% (wt/vol) sucrose and

Abbreviation: EGTA, ethylene glycol bis(8-aminoethyl ether)N,N,N',N'-tetraacetate.

The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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6904 Cell Biology: Jahn and Soling

centrifuged for 2 hr at 62,000 rpm in a Beckman SW 65 k rotor.The material banding at the interface between the 43% and the34% layer (IF 1) and at the interface between the 34% sucroselayer and the sample (IF 2) was collected, diluted in sucrose-freestop solution, and concentrated by sedimentation at 100,000X gm.x for 60 min (for details see ref. 2). Aliquots from eachsample were denatured for electrophoresis immediately afterthe end of the isolation procedure.

Electrophoresis, Autoradiography, and Enzyme Assays. So-dium dodecyl sulfate/polyacrylamide slab gel electrophoresis,treatment of the protein samples, and staining and destainingof the gels were carried out according to Laemmli (7) with themodification of Rudolph and Krueger (8). Unless otherwisementioned, 15% gels were run with about 100 ,g ofprotein perslot. Two-dimensional electrophoresis was performed accordingto O'Farrell (9); a gradient of pH 4.5-7.0 was used in the fo-cusing dimension. The samples (about 300 jig of protein) weretreated prior to isoelectric focusing according to Ames and Ni-kaido (10). For autoradiography the dried gels were exposed for1-2 weeks to Kodak X-Omat R films. Amylase was determinedby the method of Bernfeld (11) and protein, by the dye-bindingmethod of Bradford (12).

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RESULTSEffect of Carbachol on Protein Phosphorylation in Rat Par-

otid Gland Slices. In the rat parotid gland system the stimu-lation ofamylase release elicited by carbachol was considerablyless than release elicited by isoproterenol (Fig. 1). Atropine in-hibited the response to carbachol, whereas propranolol had noeffect (Fig. 1). In the presence of carbachol a slight but insig-nificant increase in the phosphorylation of protein I could beobserved in the 100,000 x g pellet (not shown). Therefore, thisfraction was further separated by density gradient centrifuga-f;f% o;n far1Amvr taf gxrvr;h fhda emfantle momnhrar%, G renfi#st;r%;,ntUUII 1in Uiprotein Iis shownincubaticylated diof phospmuch gr

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FIG. 2. Protein phosphorylation in rat parotid gland slices duringstimulation by P-adrenergic and cholinergic agonists. Rat parotidgland slices were incubated as described (2). The incubation wasstopped 20 min after addition of the stimulus, and the tissue was ho-mogenized and processed. Coomassie blue stain (left lane) and auto-radiographs are derived from the electrophoretically separated IF 1fraction obtained after density gradient centrifugation of the 100,000x g pellet. Lane A, unstimulated control; lane B, 20 4M isoproterenol;lane C, 10 /AM carbamoylcholine.

nUto rL11r1C.MUUU.eUirdU LrUU1 .11I.41i1 ylated to any measurable degree in the presence of carbachol..The analysis of one of the resulting interfaces (IF1 Atropine (100 ,LM) but not propranolol (100 ,M) prevented thein Fig. 2. In comparison to the unstimulated control carbachol-stimulated phosphorylation of protein I (not shown).n (lane A), protein I became significantly phosphor- An analysis of the other subfractions, including the cytosol, re-aring cholinergic stimulation (lane C, but the degree vealed that the phosphorylation of only protein I was signifi-nhorylation during stimulation by isoproterenol was candy affected by carbachol.eater (lane B). Proteins II and III were not phosphor- Protein Phosphorylation During Stimulation of Mouse Par-

otid Gland Slices with Isoproterenol and Carbachol. Becausethe degree of phosphorylation of protein I obtained after (-ad-renergic and cholinergic stimulation correlated with the degreeof amylase release in the rat parotid gland, we decided to ex-amine this relationship in the mouse parotid gland. This organexhibits a similar sensitivity to stimulation ofamylase secretionby carbachol and by isoproterenol, as indicated in Fig. 3. As inthe rat parotid gland, stimulation by isoproterenol led to a sig-nificant phosphorylation ofproteins I, II, and III (Fig. 4). Stim-ulation by carbachol was associated with phosphorylation ofonly

/timulation protein I. But, in contrast to the rat parotid system, phosphor-St/mulation ylation ofprotein I was as strong as that seen with isoproterenol

(Fig. 4). The enrichment of proteins II and III in the 15,0008t/9+1 I x g pellet with respect to protein I was even more pronounced

than in the rat, indicating a similar subcellular distribution ofthe proteins (see ref. 2).

_ Further Identification of Protein I by Two-DimensionalElectrophoresis. The appearance of the protein I band at the

0 10 20 30 4O 50 60 same position after electrophoresis following ,B-adrenergic andTime, min cholinergic stimulation does not exclude the possibility that two

different proteins with similar molecular weights are phos-Amylase release from isolated rat parotid gland slices phrltd hrfrte1000xgpleso os aoirdfferentagonists (mean ± SEM, n = 6).*, Control (unstim- p T t

, carbachol (10 ,M);m, carbachol (10A) + propranolol (100 glands were further separated by two-dimensional electropho-arbachol (10 4M) + atropine (100 44); o, isoproterenol (20 resis. As shown in Fig. 5, a labeled band appeared at almost the

same position after (3adrenergic and after cholinergic stimu-

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FIG. 3. Stimulation of amylase release ftid gland slices. (Inset) Incubation in Ca2+-fimM EGTA. Mouse parotid gland slices wereOQ AQ11vh& fnr two. V-0t TO-+;fl 01;f- CUCr+,M

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lation. The apparent molecular weight corresponded to thatdetermined for protein 1. Although slight differences were ob-served between the patterns obtained after cholinergic (Fig. 5B)and f-adrenergic stimulation (probably due to somewhat dif-ferent loading of the gels), this finding indicates that the same

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FIG. 5. Two-dimensional separation of 100,000 x g pellets derivedfrom mouse parotid gland slices after 3-adrenergic and cholinergicstimulation. All incubations were stopped 20 min after addition of thestimulant. Electrophoresis was performed on 15% gels. The molecularweights were calculated from the RF values. The arrows on the auto-radiographs indicate the position of the band corresponding to proteinI. (A) Control (unstimulated), (B) 10 AM carbachol, (C) 20 pM iso-proterenol.

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FIG. 4. Phosphorylation of proteins I, II, and El[ in mouse parotidgland slices during stimulation by isoproterenol, carbachol, and A-23187. The incubations were stopped 20 min after addition of the stim-ulant. All fractions were subjected to electrophoresis and autoradi-ography (Coomassie blue stains on the left). Lanes A-C, 15,000 x gpellets: lane A, unstimulated control; lane B, 20pM isoproterenol; laneC, 10 pM carbachol. Lanes D-F, 100,000 x g pellets: lane D, unstim-ulated control; lane E, 20 pM isoproterenol; lane F, 10 am carbachol.Lanes G-I, 100,000 x g pellets: lane G, unstimulated control; lane H,10 HM carbachol; lane I, A-23187 (1 ng/mg of wet weight). The arrow-head on the right indicates an additional band phosphorylated in thepresence of the ionophore. Note the absence of proteins II and m inthe 100,000 x g pellets.

membrane-bound protein (protein I) is phosphorylated undertwo different stimuli.

Influence of Extracellular Calcium. Cholinergic stimulationofamylase release from mouse parotid glands depended on thepresence of external calcium. Incubation in Ca2--free buffercontaining 0.1 mM EGTA completely prevented the stimula-tion of amylase secretion by carbachol (Fig. 3 Inset), whereasthe P-adrenergic response remained unaffected. As expected,stimulation of phosphorylation of proteins I, II, and III by iso-proterenol was not abolished by the absence of external Ca2",but to our surprise the absence of external Ca2" did not affectthe carbachol-stimulated phosphorylation of protein I (data notshown). Incubations with the Ca2" ionophore A-23187 led toa rapid and transient amylase relase after a lag period of about10 min (Fig. 6) and to aphosphorylation ofprotein I (Fig. 4, laneI). Both the degree of phosphorylation and the stimulation ofamylase release were weaker than under cholinergic stimulation(Fig. 4, lane H). In addition, a strongly phosphorylated bandappeared in the low molecular weight region (arrowhead) thatwas not observed under any ofthe physiological stimuli. Higherconcentrations of the ionophore led to a decrease of the totalincorporation of phosphate into proteins and to alterations ofthe band pattern after Coomassie blue staining, indicating cy-totoxic effects of the ionophore.

DISCUSSIONThe results indicate a close relationship between phosphoryl-ation of protein I and stimulation of amylase release in rat andmouse parotid glands. Such a relationship exists independentlyof the kind of stimulus, be it isoproterenol, carbachol, A-23187,

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Stimulation

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that can be observed only during (3-adrenergic stimulationmight be related to this process, but such a relationship needs

* to be proven.Further studies are necessary to learn more about the prop-

erties and the exact location of protein I and about its functionin stimulus-secretion coupling. Moreover, because protein Iseems to be phosphorylated by a cyclic AMP-dependent as wellas by a Ca2'-dependent mechanism, we shall have to clarifywhether phosphorylation of protein I occurs at different sitesunder the two conditions, which types of protein kinases areinvolved, and how they are regulated.

We thank Thomas Jarchau, Max-Planck-Institut fir BiophysikalischeChemie, G6ttingen, for carrying out the two-dimensional electropho-resis and introducing us to this technique, and Ellen Rasenberger for

40 50 60 skillful technical assistance. This work was supported by a grant fromthe Deutsche Forschungsgemeinschaft (So 43/30).

FIG. 6. Effect of A-23187 on amylase release from isolated mouseparotid gland slices. A, Unstimulated control; *, addition of A-23187(1 ng/mg of wet weight).

or dibutyryl cyclic AMP. In line with such a relationship is theobservation that the degree of phosphorylation parallels thedegree of stimulation of amylase release. It remains to be ex-

plained, however, why a removal of external calcium abolishescarbachol-induced amylase secretion but not carbachol-inducedphosphorylation of protein I. There is some evidence that car-

bachol releases calcium from a pool that is not depleted by mildEGTA treatment (13-15) and may be (at least in the exocrinepancreas) associated with the plasma membrane (14, 16, 17).Therefore, it is possible that in our experiments calcium-de-pendent processes involved in the fusion of secretory granuleswith the apical plasma membrane are blocked while sufficientcalcium is released from the plasma membrane-associated poolto induce the phosphorylation of protein I, probably by acti-vation of a Ca2'-dependent protein kinase.

Inhibition ofamylase release induced by 3-agonists requiresa more rigorous and prolonged EGTA treatment than that ap-

plied in our experiments (18-20). The lower sensitivity of (-

adrenergic stimulation to Ca2+ depletion may be explained bya cyclic AMP-mediated release ofCa2" from intracellular stores(microsomes or mitochondria) that are more resistant to theeffects of EGTA. Proposals along these lines have been madeby others (19, 21). The phosphorylation of proteins II and III

1. Butcher, F. R. & Putney, J. W. (1980) Adv. Cyclic NucleotideRes. 13, 215-249.

2. Jahn, R., Unger, C. & S6ling, H. D. (1980) Eur.J. Biochem. 112,345-352.

3. Kanamori, T. & Hayakawa, T. (1980) Biochem. Int. 1, 395-402.4. Kanamori, T., Mineda, T., Oikawa, M. & Hayakawa, T. (1981)

Adv. Physiol. Sci. 28, 207-212.5. Vreugdenhil, A. P. & Roukema, P. A. (1975) Biochim. Biophys.

Acta 413, 79-94.6. Watson, E. L., Williams, J. A. & Siegel, I. A. (1979) Am.J. Phys-

iol 236, C233-C237.7. Laemmli, U. K. (1970) Nature (London) 227, 680-685.8. Rudolph, S. A. & Krueger, B. K. (1979) Adv. Cyclic Nucleotide

Res. 10, 107-133.9. O'Farrell, P. H. (1975) J. Biol Chem. 250, 4007-4021.

10. Ames, G. F. L. & Nikaido, K. (1976) Biochemistry 15, 616-623.11. Bernfeld, P. (1955) Methods Enzymol. 1, 149-158.12. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254.13. Putney, J. W. (1977)J. Physiol. 268, 139-149.14. Putney, J. W. (1979) Pharmacol Rev. 30, 209-245.15. Butcher, F. F. (1979) Life Sci. 24, 1979-1982.16. Shelby, H. T., Gross, L. P., Lichty, P. & Gardner, J. D. (1976)

J.. Clin. Invest. 58, 1482-1493.17. Stolze, H. & Schulz, I. (1980) Am. J. Physiol 238, G338-G348.18. Selinger, Z. & Naim, E. (1970) Biochim. Biophys. Acta 203,

335-337.19. Putney, J. W., Weiss, S. J., Leslie, B. A. & Mairier, S. H. (1977)

i. Pharmacol Exp. Ther. 203, 144-155.20. Butcher, F. R. (1978) Adv. Cyclic Nucleotide Res. 9, 707-721.21. Rasmussen, H. & Goodman, D. B. P. (1977) Physiol Rev. 57,

421-509.

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