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Re-evaluation of the stimulatory effect of norepinephrine on the secretion of amylase in the parotid gland of the rat
0028-3908/89$3.00+ 0.00 Copyright 0 1989Pergamon Press plc
Neurophurmaco/og.rVol. 28. No. IO. pp. 1099-I105. 1989 Printed in Great Britain, All rights reserved
RE-EVALUATION OF THE STIMULATORY EFFECT OF NOREPINEPHRINE ON THE SECRETION OF AMYLASE IN THE PAROTID GLAND OF THE RAT F. HATA and 0. YACASAKI Department of Veterinary Pharmacology, College of Agriculture, University of Osaka Prefecture, Sakai 591, Japan (Accepted 8 February 1989)
Summary-The effect of the neurotransmitter norepinephrine(NE) in stimulating the secretion of amylase from the parotid gland of rats was studied by use of selective a- and j3-adrenergic antagonists. Its secretory response, mediated through /I-adrenoceptors, was slight during a short period of incubation, but rapidly increased after incubation for 10 min, showing a supersensitization phenomenon. Norepinephrine alone did not induce this phenomenon, but it induced the phenomenon in the presence of the a-adrenergic antagonist phentolamine or the a,-antagonist prazosin. Isoproterenol-induced supersensitization was prevented by methoxamine. While, the accumulation of cyclic AMP in the tissue during incubation with isoproterenol and NE was not significantly affected by the presence of methoxamine and phentolamine, respectively. Phorbol dibutyrate did not inhibit the secretion induced by NE in the presence of phentolamine. These findings indicate that stimuli, mediated through G(-and /I-adrenoceptors, induced secretion of amylase in parotid gland of the rat but that the a-effect inhibited the B-effect when both stimuli were applied simultaneously and that the overall response of the tissue to NE resulted from the interaction of the two adrenoceptors. Key words-amylase
secretion, parotid gland, receptor interaction, supersensitivity.
The neurotransmitter norepinephrine(NE), acetylcholine (ACh) and substance-P, or a closely related
peptide, stimulate the secretion of amylase from the parotid gland, although /I-adrenergic agonists are more effective. Numerous studies have shown that stimuli, mediated through /.I-adrenoceptors, induce the secretion of amylase by elevating the intracellular level of cyclic AMP, and that stimuli, mediated through muscarinic cholinoceptors, a-adrenoceptors and substance-P receptors, induce secretion of amylase by elevating the intracellular level of calcium ions (Butcher and Putney, 1980). The functional responses of most organs, that are innervated by autonomic nerves, are regulated in two ways, by excitation and inhibition, mediated through different neurotransmitter receptors. An exceptional case is the secretion of amylase by the parotid gland, which is stimulated by all agonists noted above. Therefore, it is of interest to study the mechanism of regulation of secretion of amylase from the parotid. Another interesting subject is the interactions between two different receptors, such as u- and /I-adrenoceptors (Woodcock and Johnston, 1980). LX*and fl-adrenoceptors (Tran, Lac, Berlan and Lafontan, 1984) P-adrenoceptors and insulin receptors (Pessin, Gitmer, Oka, Oppenheimer and Czech, 1983; Lonnroth and Smith, 1983) and a variety of other receptors (Hollenberg, 1985). The inverse regulation of cq- and /I-adrenoceptors, which seems to have functional significance in many tissue preparations (Kunos and Ishac, 1987) is of special interest.
Previously, it was found that pretreatment of parotid tissue from the rat with a B-agonist induced supersensitivity of secretion of amylase to a second addition of the same agonist (Hata, Ishida, Kagawa, Kondo, Kondo and Noguchi, 1983). This supersensitivity was specific to the /I-adrenergic system and was due to a &adrenergic response without any concomitant change in accumulation of cyclic AMP in the tissue (Hata, Noguchi, Ishikawa, Koda and Ishida, 1985a). These findings raised the problem of why a p-agonist induces supersensitivity but the neurotransmitter NE does not. As secretion of amylase, induced by NE, must be mediated through both a- and /I-adrenoceptors, in this work the relationship between the secretory responses mediated through these two types of receptors was examined. Results indicated that stimulatory effects, mediated through a- and /I-adrenoceptors, were not additive and that after incubation for about 10 min, the ol-adrenergic effect of NE inhibited the B-adrenergic effect. That is, the secretory response, induced by NE, could be
regarded as an overall response resulting from the interaction of its a- and b-adrenergic effects. METHODS
Drugs
Methoxamine hydrochloride was a gift from Nippon Shinyaku Co., Kyoto, Japan. Phentolamine hydrochloride was a gift from Ciba-Geigy Ltd, Basel, Switzerland. f-Norepinephrine bitartrate, f-isoproter1099
F.
II00
HATA
and 0.
YAGASAKZ
enol bitartrate, Al-propranolol hydrochloride, prazosin hydrochloride, amylose and 4j3-phorbol 12,13dibutyrate were purchased from Sigma Chemical Co., St Louis, U.S.A. Preparation of parotid tissue from the rat
Parotid glands were obtained from male Wistar rats (250-35Og) and small pieces of the tissue were prepared as described previously (Hata et al., 1983). Before the experiments, Krebs-Ringer Tris solution, consisting (in mM) of NaCl 120, KC1 4.8, KH,P04 1.2, MgSO., 1.2, CaCI, 3.0, Tris HCI buffer (pH 7.4) 16 and glucose 5, was aerated with 0, and the pieces of parotid tissue were equilibrated with the solution for 20 min at 37’C with shaking. All incubations were carried out at 37’C. Secretion of amylase
The parotid tissues were pretreated with agonists, as described previously (Hata et al., 1983). About 30mg of pieces of tissue were incubated in 10 ml Krebs-Ringer Tris solution with an agonist for 10 min (this incubation was called the first incubation, or pre-treatment). The tissue was then washed well with 50ml Krebs-Ringer Tris solution and transferred to the solution for 10min (rest period) and then challenged by re-incubation with the same agonist for IOmin (second incubation). In other experiments, the cumulative secretion of amylase into the medium was also measured at intervals during the incubation. The activity of the amylase secreted was expressed as mg maltose per 100 mg parotid tissue. In some experiments, the secretion was also expressed as
Amylase 0
secreted 100
zi
2
so
~~1
08
I 7
I 6
-log
[drug],
I 5
M
Fig. 1. Concentration-response curves of parotid tissue from the rat to NE (0). isoproterenol (0) and methoxamine (A). Parotid tissues were incubated at 37°C for IO min. Points and bars are means and standard errors for 4-6 experiments. Details of experimental conditions are described in the Methods.
the percentage of amylase activity of the total enzyme activity. The activity of amylase was measured, as described by Bernfeld (1955), with amylose as the substrate. Activity was expressed as the amount of maltose liberated into the medium in mg per 5 min at 20°C. Other methods
Cyclic AMP in tissues was measured by radioimmunoassay (Honma, Satoh, Takezawa and Ui,
(mg maltose / 100 mg tissue 200
f 300
1 St Control 2nd
0.1 (LM
1st 2nd
IPR l.OkM
1 st 2nd
10 &M
1 St 2nd 1 St
firM
2nd 1 st
NE 1OpM
1 4
2nd 1 st
IOOpM 2nd
Fig. 2. Stimulatory effects of isoproterenol (IPR) and NE on secretion of amylase from parotid tissue of the rat during the first and second incubations. Tissues were incubated without (control, spontaneous secretion) or with the indicated concentrations of drugs. Values are means and standard errors for 4-8 experiments. Significance of difference from the value for the corresponding first incubation: *+P < 0.02, ***f < 0.05. Details of experimental conditions are described in the Methods.
1101
Amylase secretion induced by NE
Amylase 0 I Control
2nd
NE + Phentolamine (IOpM) (IO /IM)
2nd
NE + Phentolamine (I00 j~M1 (IOOpM)
I St 2nd
NE + Propranolol (IOpM’ (IO/JM)
I st 2nd
NE + (lOj~M1
1st 2nd
NE + Yohimbine (IO/JM, (IO/JM)
Tissue) 300 I
I st 2nd
NE + Phentolamine QMI (IpM)
Prazosin (IO/IM)
secreted (mg Maltose/lOOmg 200 100 I I
I st I St
I st 2nd
Fig. 3. Stimulatory effects of NE, in the absence and presence of antagonists, on secretion of amylase from parotid tissue of the rat during the first and second incubations. Values are means and standard errors for 4-8 experiments. Significance of difference from the value for the corresponding first incubation:
*P < 0.01. For further details see Figure 2 and the text.
1977) with a cyclic AMP assay kit (Yamasa Shoyu Co., Chiba, Japan). Phorbol dibutyrate was dissolved in dimethyl sulfoxide. The final concentration of dimetyl sulfoxide was 0.05%, which did not have any significant effect on the secretory response. The significance of differences between values was examined by Student’s t-test.
by the a-agonist methoxamine, which had a slight stimulatory effect on secretion of amylase. It is noteworthy that methoxamine did not have an additive effect with isoproterenol but significantly inhibited the secretion during the second incubation (Fig. 4).
RESULTS
As NE acts on both a- and /?-adrenoceptors, its effects on a- and j-adrenoceptors were examined separately by measuring the amylase activity, accumulated in the medium, during incubation with selective antagonists. Amylase was secreted timedependently from parotid tissues, in response to NE, in the absence or presence of propranolol (effect on a-adrenoceptors). But the time course of secretion of the amylase, induced by NE in the presence of phentolamine (effect on b-adrenoceptors), was different from that induced by the effects of NE on
Eflects of pre-treatment of parotid tissue from the rat with agonists on secretion of amylase
Isoproterenol, NE or methoxamine induced secretion of amylase from parotid tissue from the rat in a dose-dependent manner and EC9 values of isoproterenol and NE were 0.2 and 2 PM, respectively (Fig. 1). Pre-treatment of parotid tissue from the rat with 1 or 1OpM isoproterenol for 10min (first incubation) resulted in enhancement of the secretory response during the second incubation (Fig. 2). Similar supersensitization was induced by pre-treatment with 10 or 100 p M NE, in the presence of the a-antagonist phentolamine, at the same concentration as NE (Fig. 3) but not by NE alone or NE in the presence of the b-antagonist, propranolol (Figs 2 and 3). This supersensitization was also seen when the activity of amylase secreted was expressed as a percentage of the total activity (Table 1). The effect of the al-antagonist prazosin, in inducing the supersensitization by NE, was similar to that of phentolamine, whereas the a,-antagonist yohimbine did not have this effect (Fig. 3). On the other hand isoproterenol-induced supersensitization was inhibited
Time course of secretion of amylase induced by various agonists
Table
I. Stimulatory effect of agonists on secretion of amylase during the first and second incubations Incubation First
Rest
Second
None (Control)
4.5 F 0.2 (6) 3.7 + 0.1 (6)
3.3 f 0.1 (6)
IPR, I PM NE, IOpM + Phentolamine. IOFM
8.8 f 0.5 (8) 5.9 k 0.4 (8)
14.6 + 0.7 (8).
8.7 f 0.2 (8) 5.5 * 0.5 (8)
13.6 f 0.5 (8)**
Values of amylase activity are expressed as a percentage of the total activity. Values are means f SE of means for the numbers of experiments shown in parentheses. Significantly different from the value for the corresponding first incubation, *P ~0.05; **p < 0.02. IPR = isoproterenol; NE = norepinephrine.
F. HATAand 0. YAGASAKI
II02
Amylase secreted 0
(mg maltose / 100 mg tissue)
100
200
I
I St
2nd
I
+b .
111
1 st 2nd 1 st IPR
IPR + Met~axomine
2nd 1
St
2nd
*
Fig. 4. Stimulatory effects of methoxamine and isoproterenol (IPR) on secretion of amylase. Parotid tissues from the rat were incubated with 100 PM methoxamine, I PM isoproterenol or both drugs. Values are means and standard errors for 4-8 experiments. For details see Figures 2 and 3 and Methods.
both receptors or on El-adrenoceptors only (Fig. SA) in two respects: (1) in the early period of incubation, the effect of NE on ~-adr~n~eptors was slight and less than that on a-adrenoceptors; (2) after incubation for about 10 min, the effect of NE on @-adrenoceptors was markedly increased, resulting in much greater secretion of amylase than that induced by the effect of NE on both receptors (Fig. 5). The addition of m~thoxamine increased the secretion of amylase induced by isoproterenol during incubation, for up to about 10 min, that is, the two agonists stimulated the secretion in an additive manner. However, after incubation for about 15 min, the presence of these agonists together resulted in less secretion of amylase
than that induced by isoproterenol alone (Fig. 6A). Similar results were obtained when methoxami~e was added to the parotid tissues after incubation with isoproterenol for 10 min (Fig. 6B).
The content of cyclic AMP of the parotid tissue was measured after incubation with isoproterenol or NE, in the absence or presence of methoxamine or phentolamine, respectively. The content of cyclic AMP increased markedly during incubation for 5min with isoproterenol or NE. Methoxamine did not have a significant effect on the accumulation of cyclic AMP induced by isoproterenol. Phentol~ine also did not affect the accumulation of cyclic AMP induced by NE (Fig. 7). In another experiment,
I-
z 35 4:
/
ix
Time fmin) Fig. 5. (A) Time course of secretion of amylase induced by NE in the absence or presence of antagonist. Parotid tissues from the rat were incubated without drugs (small dot) or with 10pM NE (O), with 10&M NE plus IO&M propranoloi (A) or with 10 pM NE plus 10 PM phentolamine (+). Values are means and standard errors for 4-8 experiments. (B) Sccrctinn in 2.5, 5 and tOmin, induced by NE plus prop~anolol (ar-effect, open cohnnns) or NE plus phentoiamine (B-effect. closed ~oiumns~. Data are replotted from (A). Significance of difference from the value for the r-effect of NE in the same period: *P -=z 0.01; **P < 0.02.
!
3
$
e
0
10
20
30
_
0
Time
10
20
30
40
lmin)
Fig. 6. Typical stimulatory effects of isoproterenol and methoxamine on secretion of amylase. Parotid tissues from the rat were incubated without (small dot) or with 1/r&d IPR (0) IOUFM methoxamine (A) or 1p RI ~soproIereno1 plus 100pC1M methoxamine (a). (B) Methoxami~e was added at the incubation time marked by an arrow. Points represent means for duplicate measurements and values are representative for 4 experiments.
I to3
Amyiase secretion induced by NE Cyclic
AMP content
tpmol/~
pfoteinf
SO I
0 1
IPR -c;-i
I
IPR + Methoxomine
NE NEt PhentGamine
Fig. 7. Accumulation of cyclic AMP in parotid tissues of the rat. Tissues were incubated for Smin with the drugs indicated in the absence of pbosphodi~terase inhibitor, and then the tissue samples were quickly frozen to determine the content of cyclic AMP. Concentrations of drugs: isoproterenol (IPR) f PM; metboxami~e, WOpM; NE and phentolamine, 1OpM. Values are means for 4-6 experiments with standard errors.
of cyclic AMP, determined after IOmin, were found to show similar tendencies to those measured after 5 min, though the stimulation was fess. the concentrations
Eflect of ~~orbo~ d~but.~r~te on secretion of ~~~I~e induced by NE in the absence at presence of~drener~~c antagonists. 4~-~horbol t 2,13-dibutyrate (phorbol dibutyrate) (I p M) had little stimulatory effect on the spontaneous secretion of amylase but significantly increased the secretion induced by NE in the presence of an LX-or B-antagonist (Figs 8A, C!, D). It had no effect, however, on NE-induced secretion (Fig. 8B). IXSCUSSION
Previously, it was found that brief treatment of the parotid tissue with a /?-agonist resulted in super-
sensitivity of the secretory response during further incubation with the same agonist (Hata ef al., 1983) and also that this phenomenon was specificaffy due to a ~~-adre~ergic response (Hata et al., 1985a). In the present study, the effect of brief pretreatment with a P-agonist was confirmed by examining the time course of secretion of amylase induced by stimulation of fi-adrenoceptors (Fig. 5). Namely, the results showed that the rate of accumulation of amylase in the medium increased after about 10min. This marked effect was similar to that observed previously with isoproterenol (Hata et af., 1983). The question was, why the neurotransmitter NE did not induce this phenomenon, even though it is an agonist of both @-adrenoceptors and a-adrenoceptors? In the present study, it was found that, in fact, NE induced this phenomenon if its cc,-effect was blocked by an al-antagonist. This finding indicated that the a-adrenergic effect of NE inhibited its @-adrenergic effect and that NE itself did not induce as great a secretion of amylase as that induced by incubation with a @-agonist, for more than IOmin. The inhibitory effect of methoxamine on isoproterenol-induced secretion, shown in Figures 4 and 6, was consistent with these findings, These results indicate that the widely accepted concept that secretion of amyfase in the parotid gland of the rat is preferentially mediated through p-adrenoceptors, which is based on the greater stimulatory effect by a p-agonist, must be reconsidered. The present results show that, in the early period of stimulation by NE, the amylase secretory response depended on a-adrenoceptors more than on p-adrenoceptors (Fig. 5). The formation of cyclic AMP, which is thought to be the second messenger of physiological responses mediated by @-adrenoceptors, may be coupled with secretion of amylase from the parotid gland (Bat& Selinger, Schramm and Robinovitch, 1973; Butcher, Goldman and N~merovski, 1975; Butcher, Thayer
500
(El
(A)
(Cl
(0)
;’
*/
n/
I
P
yi
~, 0
10
20
0
to
20
10
20
Time [mini Fig. 8. Effects of phorbol dibutyrate on the stimulatory effect of NE, in the absence and presence of antagonists. Parotid tissues were incubated without (A) or with IOrM NE(B), 10pM NE plus 10pM phentolamine [j-effect, (C)] or 10~,tM NE plus IOFM-propranolol [a-effect, (D)] in medium with (#) or without (Of 1PM phorbol dibutyrate. Points are means for 6-8 experiments with standard errors. Significance of difference from the value in medium without phorbol dibutyrate: *P c 0.01, **P < 0.05.
1104
F. HATA and 0. YACASAKI
and Goldman, 1976; Wojdk, Grand and Kimberg, 1975), although there are many reports of differences between changes in the content of cyclic AMP and the secretion of amylase in the tissue (Butcher et al., 1975; Yoshimura, Nezu and Chiba, 1982; Carls66, Danielsson, Henriksson and Idhal, 1982; Henriksson, 1982; Hata et al., 1985a; Hata, Noguchi, Kondo, Koda, Ishikawa and Ishida, 1985b; Hata, Kondo and Ishida, 1988). In the present study, methoxamine inhibited P-agonist-induced supersensitization and, in the presence of phentolamine, NE induced supersensitization. Accordingly, it was of interest to examine whether methoxamine inhibited ~-agonistinduced fo~ation of cyclic AMP and whether phentolamine stimulated NE-induced formation of cyclic AMP in the tissue. Under the conditions used here, neither drug had any significant effect on the accumulation of cyclic AMP in the tissue, induced by a /I-agonist or NE. These results are compatible with the previous finding that the change in the total amount of cyclic AMP in the tissue did not play a prominent role in the induction of supersensitivity to a /I-adrenergic response (Hata et al., 1985a, b). Thus, it seems unlikely that the mechanism by which the a-adrenergic effect inhibited the ~-agonist-induced su~rsensiti~tion is related to a cyclic AMP generating system in the parotid. In the parotid gland of the rat a-adrenoceptors regulate the turnover of phosphatidylinositol by activating phospholipase C (Oron, Lowe and Selinger, 1973; Mitchell and Jones, 1974). Moreover, the turnover of phosphatidylinositol, induced by an a-agonist, is due specifically to an a,-adrenergic response (Uchida, Ito, Baum, Roth, Filburn and Sacktor, 1982). Hydrolysis of phosphatidyIinosito1 4,5-bisphosphate results in the generation of 1,2-d& acylglycerol and inositol 1,4,5-t~sphosphate. Inositol 1,4,5-t~sphosphate is known to elicit intracelIular release of calcium (Berridge, 1984; Berridge and Irvine, 1984) and its role in the stimulus-secretion coupling mechanism has been studied in submandibular gland cells of the rat (Fleming, Bilan, Sliwinski-Lis and Carvalho, 1987). 1,2-Diacylglycerol is known to activate protein kinase C (Takai, Kishimoto, Iwase, Kawahara, Mori and Nishizuka, 1979; Kishimito, Takai, Mori, Kikkawa and Nishizuka, 1980). Activation of protein kinase C by phorbol ester has been reported (Castagna, Takai, Kaibuchi, Sane, Kikkawa and Nishizuka, 1982; Niedel, Kuhn and Vandenbank, 1983). Subsequently, stimulatory effects of phorbol esters on the secretion from the parotid gland were reported; that is, 4~-phorbol-dibutyrate was shown to stimulate secretion of protein (Putney, McKinney, Aub and Leslie, 1984) and phorbol 12-myristate 13.acetate (Takuma and Ichida, 1986) and 12-o-tetradecanoylphorbol 13-acetate (Shimomura, Terada, Hashimoto and Soderling, 1988) were found to stimulate secretion of amylase. Whether activation of protein kinase C was involved in the mechanism by which the r,-adrenergic
effect inhibited ~-agonist-indu~d supersensitization was examined, From the results shown in Figure 8, it seems unlikely that activation of protein kinase C was involved in the mechanism by which the aadrenergic effect inhibited a-agonist-induced supersensitization, because phorbol dibutyrate did not inhibit the p-effect of NE. The absence of an effect of phorbol dibutyrate on NE-induced secretion was unexpected and indicated that the stimulatory effect of NE on the secretory response resulted from the interaction between its a- and /I-adrenergic effects, which more or less counteract each other. The present study showed that secretion of amylase from the parotid gland of the rat, induced by the neurotransmitter NE, was an overall response, resulting from the interaction of a-adrenergic and /I-adrenergic effects, although NE induced secretion of amylase by acting on both a,- and p-adrenoceptors. The details of this interaction are still unknown, but marked secretion. mediated through only /I-adrenoceptors, is unlikely in physiological conditions. REFERENCES
Batzri S., Selinger Z., Schramm M. and Robinovitch M. R. ( 1973) Potassium release mediated by the epineph~ne areceptor in rat parotid slices. J. bioi, Chem. 248: 361-368. Bernfeld P. f 1955) Amylase a and fi. Meth. Enzymol. 1: 149150. Berridge M. J. (1984) Inositol trisphosphate and diacylglycerol as second messengers. Biochem. J. 220: 345-360. Berridge M. J. and Irvine R. F. (1984) Inositol trisphosphate. a novel second messenger in cellular signal transduction. Nature 312: 3 IS-32
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CarIs B., Danielsson A.. Henriksson R. and Idahl L.-A. (1982) Dissociation of j?-adrenoceptor-induced effects on amylase secretion and cyclic adenosine 3’,5’-monophosphate accumulation. Br. J. Pharmac. 75: 633-638. Castagna M., Takai Y., Kaibuchi K., Sano K., Kikkawa U. and Nishizuka Y. (1982) Direct activation of calcium activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J. biol. Chem. 257: 7847-7851. Fleming N., Bilan P. T.. Siiwinski-Lis E. and Carvalho V. (1987) Muscarinic, a,-adrenergic and peptidergic agonists stimulate phosphoinositide hydrolysis and regulate mucin secretion in rat submandibular gland cells. f’@gers Arch. 409: 416-421. Hata F., Ishida H. Kagawa K., Kondo E., Kondo S. and Noguchi Y. (1983) b-Adrenoceptor alterations coupled with secretory response in rat parotid tissue. 1. Physiol. 341: 185-196.
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Henriksson R. (1982) /I,- and &adrenoceptor agonists have different effects on rat parotid acinar cells. Am. 1. Physol. 2.42: G481-G485. Hollenberg M. D. (1985) Examples of homospecific and heterospecific receptor regulation. Trends Pharmac. Sci. 6: 242-245. Honma M., Satoh T., Takezawa Z. and Ui M. (1977) An ultrasensitive method for the simultaneous dete~ination of cyclic AMP and cyclic GMP in small-volume samples from blood and tissue. Biochim. Med. 18: 257-273. Kishimoto A., Takai Y., Mori T., Kikkawa U. and Nishizuka Y. (1980) Activation of calcium and phospholipid-dependent protein kinase by diacylglycerol, its possible relation to phosphatidylinositol turnover. J. biol. Chem. 255: 2273-2276.
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Putnev J. W. Jr. McKinnev J. S.. Aub D. L. and Leslie B. A. (1984) Phorbol ester-induced protein secretion in parotid gland. Relationship to the role of inositol lipid breakdown and protein kinase C activation in stimulus-secretion coupling. Molec. Pharmac. 26: 261-266. Shimomura H., Terada A., Hashimoto Y. and Soderling T. R. (1988) The role of protein kinase C on amylase secretion from rat parotid gland. Biochem. biophys. Res. Commun. 150: 1309-1314. Takai Y., Kishimoto A., Iwase Y., Kawahara Y., Mori T. and Nishizuka Y. (1979) CaIcium-de~ndent activation of a multifunctional protein kinase by membrane phospholipids. J. biol. Chem. 254: 3692-3695. Takuma T. and Ichida T. (1986) Phorbol ester stimulates amylase secretion from rat parotid cells. FEES I_&. 199: 53-56. Tran M.-A., Lac D. T., Berlan M. and Lafontan M. (1984) Interplay of alpha-2 and beta adrenoceptors in the control of free fatty acid release from bone marrow adipose tissue. J. Pharmac. exp. Thu. 230: 228-231.
Uchida T., Ito H., Baum B. J., Roth G. S., Filburn C. R. and Sacktor B. (1982) Alpha,-adrenergic stimulation of phosphatidylinositol-phosphatidic acid turnover in rat parotid cells. Molec. Pharmac. 21: 128-132. Wojcik J. D., Grand R. J. and Kimberg D. V. (1975) Amylase secretion by rabbit parotid gland. Role of cyclic AMP and cyclic GMP. B~ochim. biophys. Acta 411: 250-262. Woodcock E. A. and Johnston C. I. (1980) a-Adrenergic receptors modulate &receptor affinity in rat kidney membranes. Nature 286,. 1599160. Yoshimura K., Nezu E. and Chiba A. (1982) Stimulation of a-amylase release and cyclic AMP accumulation by catecholamine in rat parotid slices in vitro. Jop. J. Physiol. 32: 121-135.
Neurophurmaco/og.rVol. 28. No. IO. pp. 1099-I105. 1989 Printed in Great Britain, All rights reserved
RE-EVALUATION OF THE STIMULATORY EFFECT OF NOREPINEPHRINE ON THE SECRETION OF AMYLASE IN THE PAROTID GLAND OF THE RAT F. HATA and 0. YACASAKI Department of Veterinary Pharmacology, College of Agriculture, University of Osaka Prefecture, Sakai 591, Japan (Accepted 8 February 1989)
Summary-The effect of the neurotransmitter norepinephrine(NE) in stimulating the secretion of amylase from the parotid gland of rats was studied by use of selective a- and j3-adrenergic antagonists. Its secretory response, mediated through /I-adrenoceptors, was slight during a short period of incubation, but rapidly increased after incubation for 10 min, showing a supersensitization phenomenon. Norepinephrine alone did not induce this phenomenon, but it induced the phenomenon in the presence of the a-adrenergic antagonist phentolamine or the a,-antagonist prazosin. Isoproterenol-induced supersensitization was prevented by methoxamine. While, the accumulation of cyclic AMP in the tissue during incubation with isoproterenol and NE was not significantly affected by the presence of methoxamine and phentolamine, respectively. Phorbol dibutyrate did not inhibit the secretion induced by NE in the presence of phentolamine. These findings indicate that stimuli, mediated through G(-and /I-adrenoceptors, induced secretion of amylase in parotid gland of the rat but that the a-effect inhibited the B-effect when both stimuli were applied simultaneously and that the overall response of the tissue to NE resulted from the interaction of the two adrenoceptors. Key words-amylase
secretion, parotid gland, receptor interaction, supersensitivity.
The neurotransmitter norepinephrine(NE), acetylcholine (ACh) and substance-P, or a closely related
peptide, stimulate the secretion of amylase from the parotid gland, although /I-adrenergic agonists are more effective. Numerous studies have shown that stimuli, mediated through /.I-adrenoceptors, induce the secretion of amylase by elevating the intracellular level of cyclic AMP, and that stimuli, mediated through muscarinic cholinoceptors, a-adrenoceptors and substance-P receptors, induce secretion of amylase by elevating the intracellular level of calcium ions (Butcher and Putney, 1980). The functional responses of most organs, that are innervated by autonomic nerves, are regulated in two ways, by excitation and inhibition, mediated through different neurotransmitter receptors. An exceptional case is the secretion of amylase by the parotid gland, which is stimulated by all agonists noted above. Therefore, it is of interest to study the mechanism of regulation of secretion of amylase from the parotid. Another interesting subject is the interactions between two different receptors, such as u- and /I-adrenoceptors (Woodcock and Johnston, 1980). LX*and fl-adrenoceptors (Tran, Lac, Berlan and Lafontan, 1984) P-adrenoceptors and insulin receptors (Pessin, Gitmer, Oka, Oppenheimer and Czech, 1983; Lonnroth and Smith, 1983) and a variety of other receptors (Hollenberg, 1985). The inverse regulation of cq- and /I-adrenoceptors, which seems to have functional significance in many tissue preparations (Kunos and Ishac, 1987) is of special interest.
Previously, it was found that pretreatment of parotid tissue from the rat with a B-agonist induced supersensitivity of secretion of amylase to a second addition of the same agonist (Hata, Ishida, Kagawa, Kondo, Kondo and Noguchi, 1983). This supersensitivity was specific to the /I-adrenergic system and was due to a &adrenergic response without any concomitant change in accumulation of cyclic AMP in the tissue (Hata, Noguchi, Ishikawa, Koda and Ishida, 1985a). These findings raised the problem of why a p-agonist induces supersensitivity but the neurotransmitter NE does not. As secretion of amylase, induced by NE, must be mediated through both a- and /I-adrenoceptors, in this work the relationship between the secretory responses mediated through these two types of receptors was examined. Results indicated that stimulatory effects, mediated through a- and /I-adrenoceptors, were not additive and that after incubation for about 10 min, the ol-adrenergic effect of NE inhibited the B-adrenergic effect. That is, the secretory response, induced by NE, could be
regarded as an overall response resulting from the interaction of its a- and b-adrenergic effects. METHODS
Drugs
Methoxamine hydrochloride was a gift from Nippon Shinyaku Co., Kyoto, Japan. Phentolamine hydrochloride was a gift from Ciba-Geigy Ltd, Basel, Switzerland. f-Norepinephrine bitartrate, f-isoproter1099
F.
II00
HATA
and 0.
YAGASAKZ
enol bitartrate, Al-propranolol hydrochloride, prazosin hydrochloride, amylose and 4j3-phorbol 12,13dibutyrate were purchased from Sigma Chemical Co., St Louis, U.S.A. Preparation of parotid tissue from the rat
Parotid glands were obtained from male Wistar rats (250-35Og) and small pieces of the tissue were prepared as described previously (Hata et al., 1983). Before the experiments, Krebs-Ringer Tris solution, consisting (in mM) of NaCl 120, KC1 4.8, KH,P04 1.2, MgSO., 1.2, CaCI, 3.0, Tris HCI buffer (pH 7.4) 16 and glucose 5, was aerated with 0, and the pieces of parotid tissue were equilibrated with the solution for 20 min at 37’C with shaking. All incubations were carried out at 37’C. Secretion of amylase
The parotid tissues were pretreated with agonists, as described previously (Hata et al., 1983). About 30mg of pieces of tissue were incubated in 10 ml Krebs-Ringer Tris solution with an agonist for 10 min (this incubation was called the first incubation, or pre-treatment). The tissue was then washed well with 50ml Krebs-Ringer Tris solution and transferred to the solution for 10min (rest period) and then challenged by re-incubation with the same agonist for IOmin (second incubation). In other experiments, the cumulative secretion of amylase into the medium was also measured at intervals during the incubation. The activity of the amylase secreted was expressed as mg maltose per 100 mg parotid tissue. In some experiments, the secretion was also expressed as
Amylase 0
secreted 100
zi
2
so
~~1
08
I 7
I 6
-log
[drug],
I 5
M
Fig. 1. Concentration-response curves of parotid tissue from the rat to NE (0). isoproterenol (0) and methoxamine (A). Parotid tissues were incubated at 37°C for IO min. Points and bars are means and standard errors for 4-6 experiments. Details of experimental conditions are described in the Methods.
the percentage of amylase activity of the total enzyme activity. The activity of amylase was measured, as described by Bernfeld (1955), with amylose as the substrate. Activity was expressed as the amount of maltose liberated into the medium in mg per 5 min at 20°C. Other methods
Cyclic AMP in tissues was measured by radioimmunoassay (Honma, Satoh, Takezawa and Ui,
(mg maltose / 100 mg tissue 200
f 300
1 St Control 2nd
0.1 (LM
1st 2nd
IPR l.OkM
1 st 2nd
10 &M
1 St 2nd 1 St
firM
2nd 1 st
NE 1OpM
1 4
2nd 1 st
IOOpM 2nd
Fig. 2. Stimulatory effects of isoproterenol (IPR) and NE on secretion of amylase from parotid tissue of the rat during the first and second incubations. Tissues were incubated without (control, spontaneous secretion) or with the indicated concentrations of drugs. Values are means and standard errors for 4-8 experiments. Significance of difference from the value for the corresponding first incubation: *+P < 0.02, ***f < 0.05. Details of experimental conditions are described in the Methods.
1101
Amylase secretion induced by NE
Amylase 0 I Control
2nd
NE + Phentolamine (IOpM) (IO /IM)
2nd
NE + Phentolamine (I00 j~M1 (IOOpM)
I St 2nd
NE + Propranolol (IOpM’ (IO/JM)
I st 2nd
NE + (lOj~M1
1st 2nd
NE + Yohimbine (IO/JM, (IO/JM)
Tissue) 300 I
I st 2nd
NE + Phentolamine QMI (IpM)
Prazosin (IO/IM)
secreted (mg Maltose/lOOmg 200 100 I I
I st I St
I st 2nd
Fig. 3. Stimulatory effects of NE, in the absence and presence of antagonists, on secretion of amylase from parotid tissue of the rat during the first and second incubations. Values are means and standard errors for 4-8 experiments. Significance of difference from the value for the corresponding first incubation:
*P < 0.01. For further details see Figure 2 and the text.
1977) with a cyclic AMP assay kit (Yamasa Shoyu Co., Chiba, Japan). Phorbol dibutyrate was dissolved in dimethyl sulfoxide. The final concentration of dimetyl sulfoxide was 0.05%, which did not have any significant effect on the secretory response. The significance of differences between values was examined by Student’s t-test.
by the a-agonist methoxamine, which had a slight stimulatory effect on secretion of amylase. It is noteworthy that methoxamine did not have an additive effect with isoproterenol but significantly inhibited the secretion during the second incubation (Fig. 4).
RESULTS
As NE acts on both a- and /?-adrenoceptors, its effects on a- and j-adrenoceptors were examined separately by measuring the amylase activity, accumulated in the medium, during incubation with selective antagonists. Amylase was secreted timedependently from parotid tissues, in response to NE, in the absence or presence of propranolol (effect on a-adrenoceptors). But the time course of secretion of the amylase, induced by NE in the presence of phentolamine (effect on b-adrenoceptors), was different from that induced by the effects of NE on
Eflects of pre-treatment of parotid tissue from the rat with agonists on secretion of amylase
Isoproterenol, NE or methoxamine induced secretion of amylase from parotid tissue from the rat in a dose-dependent manner and EC9 values of isoproterenol and NE were 0.2 and 2 PM, respectively (Fig. 1). Pre-treatment of parotid tissue from the rat with 1 or 1OpM isoproterenol for 10min (first incubation) resulted in enhancement of the secretory response during the second incubation (Fig. 2). Similar supersensitization was induced by pre-treatment with 10 or 100 p M NE, in the presence of the a-antagonist phentolamine, at the same concentration as NE (Fig. 3) but not by NE alone or NE in the presence of the b-antagonist, propranolol (Figs 2 and 3). This supersensitization was also seen when the activity of amylase secreted was expressed as a percentage of the total activity (Table 1). The effect of the al-antagonist prazosin, in inducing the supersensitization by NE, was similar to that of phentolamine, whereas the a,-antagonist yohimbine did not have this effect (Fig. 3). On the other hand isoproterenol-induced supersensitization was inhibited
Time course of secretion of amylase induced by various agonists
Table
I. Stimulatory effect of agonists on secretion of amylase during the first and second incubations Incubation First
Rest
Second
None (Control)
4.5 F 0.2 (6) 3.7 + 0.1 (6)
3.3 f 0.1 (6)
IPR, I PM NE, IOpM + Phentolamine. IOFM
8.8 f 0.5 (8) 5.9 k 0.4 (8)
14.6 + 0.7 (8).
8.7 f 0.2 (8) 5.5 * 0.5 (8)
13.6 f 0.5 (8)**
Values of amylase activity are expressed as a percentage of the total activity. Values are means f SE of means for the numbers of experiments shown in parentheses. Significantly different from the value for the corresponding first incubation, *P ~0.05; **p < 0.02. IPR = isoproterenol; NE = norepinephrine.
F. HATAand 0. YAGASAKI
II02
Amylase secreted 0
(mg maltose / 100 mg tissue)
100
200
I
I St
2nd
I
+b .
111
1 st 2nd 1 st IPR
IPR + Met~axomine
2nd 1
St
2nd
*
Fig. 4. Stimulatory effects of methoxamine and isoproterenol (IPR) on secretion of amylase. Parotid tissues from the rat were incubated with 100 PM methoxamine, I PM isoproterenol or both drugs. Values are means and standard errors for 4-8 experiments. For details see Figures 2 and 3 and Methods.
both receptors or on El-adrenoceptors only (Fig. SA) in two respects: (1) in the early period of incubation, the effect of NE on ~-adr~n~eptors was slight and less than that on a-adrenoceptors; (2) after incubation for about 10 min, the effect of NE on @-adrenoceptors was markedly increased, resulting in much greater secretion of amylase than that induced by the effect of NE on both receptors (Fig. 5). The addition of m~thoxamine increased the secretion of amylase induced by isoproterenol during incubation, for up to about 10 min, that is, the two agonists stimulated the secretion in an additive manner. However, after incubation for about 15 min, the presence of these agonists together resulted in less secretion of amylase
than that induced by isoproterenol alone (Fig. 6A). Similar results were obtained when methoxami~e was added to the parotid tissues after incubation with isoproterenol for 10 min (Fig. 6B).
The content of cyclic AMP of the parotid tissue was measured after incubation with isoproterenol or NE, in the absence or presence of methoxamine or phentolamine, respectively. The content of cyclic AMP increased markedly during incubation for 5min with isoproterenol or NE. Methoxamine did not have a significant effect on the accumulation of cyclic AMP induced by isoproterenol. Phentol~ine also did not affect the accumulation of cyclic AMP induced by NE (Fig. 7). In another experiment,
I-
z 35 4:
/
ix
Time fmin) Fig. 5. (A) Time course of secretion of amylase induced by NE in the absence or presence of antagonist. Parotid tissues from the rat were incubated without drugs (small dot) or with 10pM NE (O), with 10&M NE plus IO&M propranoloi (A) or with 10 pM NE plus 10 PM phentolamine (+). Values are means and standard errors for 4-8 experiments. (B) Sccrctinn in 2.5, 5 and tOmin, induced by NE plus prop~anolol (ar-effect, open cohnnns) or NE plus phentoiamine (B-effect. closed ~oiumns~. Data are replotted from (A). Significance of difference from the value for the r-effect of NE in the same period: *P -=z 0.01; **P < 0.02.
!
3
$
e
0
10
20
30
_
0
Time
10
20
30
40
lmin)
Fig. 6. Typical stimulatory effects of isoproterenol and methoxamine on secretion of amylase. Parotid tissues from the rat were incubated without (small dot) or with 1/r&d IPR (0) IOUFM methoxamine (A) or 1p RI ~soproIereno1 plus 100pC1M methoxamine (a). (B) Methoxami~e was added at the incubation time marked by an arrow. Points represent means for duplicate measurements and values are representative for 4 experiments.
I to3
Amyiase secretion induced by NE Cyclic
AMP content
tpmol/~
pfoteinf
SO I
0 1
IPR -c;-i
I
IPR + Methoxomine
NE NEt PhentGamine
Fig. 7. Accumulation of cyclic AMP in parotid tissues of the rat. Tissues were incubated for Smin with the drugs indicated in the absence of pbosphodi~terase inhibitor, and then the tissue samples were quickly frozen to determine the content of cyclic AMP. Concentrations of drugs: isoproterenol (IPR) f PM; metboxami~e, WOpM; NE and phentolamine, 1OpM. Values are means for 4-6 experiments with standard errors.
of cyclic AMP, determined after IOmin, were found to show similar tendencies to those measured after 5 min, though the stimulation was fess. the concentrations
Eflect of ~~orbo~ d~but.~r~te on secretion of ~~~I~e induced by NE in the absence at presence of~drener~~c antagonists. 4~-~horbol t 2,13-dibutyrate (phorbol dibutyrate) (I p M) had little stimulatory effect on the spontaneous secretion of amylase but significantly increased the secretion induced by NE in the presence of an LX-or B-antagonist (Figs 8A, C!, D). It had no effect, however, on NE-induced secretion (Fig. 8B). IXSCUSSION
Previously, it was found that brief treatment of the parotid tissue with a /?-agonist resulted in super-
sensitivity of the secretory response during further incubation with the same agonist (Hata ef al., 1983) and also that this phenomenon was specificaffy due to a ~~-adre~ergic response (Hata et al., 1985a). In the present study, the effect of brief pretreatment with a P-agonist was confirmed by examining the time course of secretion of amylase induced by stimulation of fi-adrenoceptors (Fig. 5). Namely, the results showed that the rate of accumulation of amylase in the medium increased after about 10min. This marked effect was similar to that observed previously with isoproterenol (Hata et af., 1983). The question was, why the neurotransmitter NE did not induce this phenomenon, even though it is an agonist of both @-adrenoceptors and a-adrenoceptors? In the present study, it was found that, in fact, NE induced this phenomenon if its cc,-effect was blocked by an al-antagonist. This finding indicated that the a-adrenergic effect of NE inhibited its @-adrenergic effect and that NE itself did not induce as great a secretion of amylase as that induced by incubation with a @-agonist, for more than IOmin. The inhibitory effect of methoxamine on isoproterenol-induced secretion, shown in Figures 4 and 6, was consistent with these findings, These results indicate that the widely accepted concept that secretion of amyfase in the parotid gland of the rat is preferentially mediated through p-adrenoceptors, which is based on the greater stimulatory effect by a p-agonist, must be reconsidered. The present results show that, in the early period of stimulation by NE, the amylase secretory response depended on a-adrenoceptors more than on p-adrenoceptors (Fig. 5). The formation of cyclic AMP, which is thought to be the second messenger of physiological responses mediated by @-adrenoceptors, may be coupled with secretion of amylase from the parotid gland (Bat& Selinger, Schramm and Robinovitch, 1973; Butcher, Goldman and N~merovski, 1975; Butcher, Thayer
500
(El
(A)
(Cl
(0)
;’
*/
n/
I
P
yi
~, 0
10
20
0
to
20
10
20
Time [mini Fig. 8. Effects of phorbol dibutyrate on the stimulatory effect of NE, in the absence and presence of antagonists. Parotid tissues were incubated without (A) or with IOrM NE(B), 10pM NE plus 10pM phentolamine [j-effect, (C)] or 10~,tM NE plus IOFM-propranolol [a-effect, (D)] in medium with (#) or without (Of 1PM phorbol dibutyrate. Points are means for 6-8 experiments with standard errors. Significance of difference from the value in medium without phorbol dibutyrate: *P c 0.01, **P < 0.05.
1104
F. HATA and 0. YACASAKI
and Goldman, 1976; Wojdk, Grand and Kimberg, 1975), although there are many reports of differences between changes in the content of cyclic AMP and the secretion of amylase in the tissue (Butcher et al., 1975; Yoshimura, Nezu and Chiba, 1982; Carls66, Danielsson, Henriksson and Idhal, 1982; Henriksson, 1982; Hata et al., 1985a; Hata, Noguchi, Kondo, Koda, Ishikawa and Ishida, 1985b; Hata, Kondo and Ishida, 1988). In the present study, methoxamine inhibited P-agonist-induced supersensitization and, in the presence of phentolamine, NE induced supersensitization. Accordingly, it was of interest to examine whether methoxamine inhibited ~-agonistinduced fo~ation of cyclic AMP and whether phentolamine stimulated NE-induced formation of cyclic AMP in the tissue. Under the conditions used here, neither drug had any significant effect on the accumulation of cyclic AMP in the tissue, induced by a /I-agonist or NE. These results are compatible with the previous finding that the change in the total amount of cyclic AMP in the tissue did not play a prominent role in the induction of supersensitivity to a /I-adrenergic response (Hata et al., 1985a, b). Thus, it seems unlikely that the mechanism by which the a-adrenergic effect inhibited the ~-agonist-induced su~rsensiti~tion is related to a cyclic AMP generating system in the parotid. In the parotid gland of the rat a-adrenoceptors regulate the turnover of phosphatidylinositol by activating phospholipase C (Oron, Lowe and Selinger, 1973; Mitchell and Jones, 1974). Moreover, the turnover of phosphatidylinositol, induced by an a-agonist, is due specifically to an a,-adrenergic response (Uchida, Ito, Baum, Roth, Filburn and Sacktor, 1982). Hydrolysis of phosphatidyIinosito1 4,5-bisphosphate results in the generation of 1,2-d& acylglycerol and inositol 1,4,5-t~sphosphate. Inositol 1,4,5-t~sphosphate is known to elicit intracelIular release of calcium (Berridge, 1984; Berridge and Irvine, 1984) and its role in the stimulus-secretion coupling mechanism has been studied in submandibular gland cells of the rat (Fleming, Bilan, Sliwinski-Lis and Carvalho, 1987). 1,2-Diacylglycerol is known to activate protein kinase C (Takai, Kishimoto, Iwase, Kawahara, Mori and Nishizuka, 1979; Kishimito, Takai, Mori, Kikkawa and Nishizuka, 1980). Activation of protein kinase C by phorbol ester has been reported (Castagna, Takai, Kaibuchi, Sane, Kikkawa and Nishizuka, 1982; Niedel, Kuhn and Vandenbank, 1983). Subsequently, stimulatory effects of phorbol esters on the secretion from the parotid gland were reported; that is, 4~-phorbol-dibutyrate was shown to stimulate secretion of protein (Putney, McKinney, Aub and Leslie, 1984) and phorbol 12-myristate 13.acetate (Takuma and Ichida, 1986) and 12-o-tetradecanoylphorbol 13-acetate (Shimomura, Terada, Hashimoto and Soderling, 1988) were found to stimulate secretion of amylase. Whether activation of protein kinase C was involved in the mechanism by which the r,-adrenergic
effect inhibited ~-agonist-indu~d supersensitization was examined, From the results shown in Figure 8, it seems unlikely that activation of protein kinase C was involved in the mechanism by which the aadrenergic effect inhibited a-agonist-induced supersensitization, because phorbol dibutyrate did not inhibit the p-effect of NE. The absence of an effect of phorbol dibutyrate on NE-induced secretion was unexpected and indicated that the stimulatory effect of NE on the secretory response resulted from the interaction between its a- and /I-adrenergic effects, which more or less counteract each other. The present study showed that secretion of amylase from the parotid gland of the rat, induced by the neurotransmitter NE, was an overall response, resulting from the interaction of a-adrenergic and /I-adrenergic effects, although NE induced secretion of amylase by acting on both a,- and p-adrenoceptors. The details of this interaction are still unknown, but marked secretion. mediated through only /I-adrenoceptors, is unlikely in physiological conditions. REFERENCES
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Putnev J. W. Jr. McKinnev J. S.. Aub D. L. and Leslie B. A. (1984) Phorbol ester-induced protein secretion in parotid gland. Relationship to the role of inositol lipid breakdown and protein kinase C activation in stimulus-secretion coupling. Molec. Pharmac. 26: 261-266. Shimomura H., Terada A., Hashimoto Y. and Soderling T. R. (1988) The role of protein kinase C on amylase secretion from rat parotid gland. Biochem. biophys. Res. Commun. 150: 1309-1314. Takai Y., Kishimoto A., Iwase Y., Kawahara Y., Mori T. and Nishizuka Y. (1979) CaIcium-de~ndent activation of a multifunctional protein kinase by membrane phospholipids. J. biol. Chem. 254: 3692-3695. Takuma T. and Ichida T. (1986) Phorbol ester stimulates amylase secretion from rat parotid cells. FEES I_&. 199: 53-56. Tran M.-A., Lac D. T., Berlan M. and Lafontan M. (1984) Interplay of alpha-2 and beta adrenoceptors in the control of free fatty acid release from bone marrow adipose tissue. J. Pharmac. exp. Thu. 230: 228-231.
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