Nitric oxide modulates salivary amylase and fluid, but not epidermal growth factor secretion in conscious rats

No. 11, pp. 9515’63,199!4 Inc. Printed in the USA. All rights reserved om-3205/99/s-see front matter

Life Sciences, Vol. 64,

copyrisht0 1959 Elsev+er seiem

PI1 s0024-3205(!39)ooo21-1

NITRIC OXIDE MODULATES SALIVARY AMYLASE AND FLUID, BUT NOT EPIDERMAL GROWTH FACTOR SECRETION IN CONSCIOUS RATS Z. Lohinai’, B. Burghard?,

T. Zelles3 and G. Vargazs3

‘Experimental Research Department and 2nd Institute of Physiology, Semmelweis Univ. Med. School, $nstitute of Experimental Medicine, Hung. Acad. Sci. and 3Department of Oral Biology, Semmelweis Univ. Med. School, Budapest, Hungary (Received in final form December 1, 1998)

Summary The involvement of the L-arginine/NO pathway in the control of salivary fluid, amylase and epidermal growth factor (EGF) secretion was investigated in conscious rats. For the collection of saliva, an oesophageal cannula was implanted. To obtain steady secretion, submaximal carbachol background infusion was given. Different treatments included NO synthase inhibitor No-nitro-L-arginine (NOLA, with or without phentolamine, propranolol), L-arginine, D-arginine and NO donor 3morpholinosydnonimine (SIN-l) administration. Volume, amylase activity and EGF output in the secreted fluid were determined in 30 min mixed saliva samples. Carbachol infusion alone produced a modest, sustained salivary fluid and amylase secretion. NOLA (30 mg/kg) further increased both fluid (p
The neuronal localization of NOS suggests that nitric oxide (NO) may participate in the regulation of both blood flow and secretion of the salivary glands. NO is known to control the vascular tone in Correspondence: Zsolt Lohinai DMD, Semmelweis _University Medical School, Experimental Research Department and 2nd Institute of Physiology, U118i 6t 78/A, Budapest, Hungary, H-1082; Fax: +361-334-3162; E-mail: [email protected]

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salivary glands in rest, and also after autonomic nerve stimulation (5-11). There is evidence for the release of NO in human saliva where it may play a physiological role both in the antibacterial properties of saliva and in the detoxification of oral carcinogens (12). In addition, recent data suggest that NO modulates salivary fluid and protein secretion (5, 6, 13-15). However, its effects on salivary amylase and epidermal growth factor (EGF) secretion have not yet been investigated. In the present study, we investigated the role of L-arginine/NO pathway in the regulation of salivary fluid, amylase and EGF secretion using a conscious rat model. Part of the results of the current paper have been published in prelimin~ form (16, 17).

Material

and Methods

Surgical procedures Experiments were performed using 84 female Wistar Crl.(Wi)Br rats (Charles River Hungary, Budapest) weighing 280-340 g. A few days before the experiments the animals were twice habituated to Bollman cages. The rats were fed chow and water ad libitum, until 24 h before the experiment when food but not water was removed. Surgical procedures were performed according to Olsen et al. (18) and Tazi-Saad et al. (19) with slight modifications. Under ether anaesthesia, a polyethylene cannula (PE-50) was inserted into the right jugular vein for drug infusions. After laparotomy, another polyethylene catheter (PE-240) was introduced into the lower oesophagus through an incision in the stomach for collection of saliva. The catheter was secured in place with a ligature at the cardia and a purse string suture in the forestoma~h, and conducted through the abdominal wall. The muscle and the skin were sutured and the rats were placed in Bollman cages to maintain minimum restraint necessary.

After surgery, a 1 mL/h saline infusion was given continuously. Following a 2 h postanaesthesia recovery, the infusion rate was increased to 3.2 mLlh. Mixed saliva was collected in graduated tubes in 30 mm periods and stored at -20 “c until assayed. In each study, basal salivary secretion was collected for two 30 min periods before treatment. Since basal salivary secretion is very low and variable, a subm~im~ background infusion of carbachol (0.03 mgfkg-h in 3.2 mL/h saline) (20) was given continuously during the course of the experiments (except in the first group of the first experiment). In the first experiment, the first group (n=8) was treated with NOLA (30 m&g, iv. bolus) alone, the second group (control rats, n=7) received only subm~im~ carbachol infusion (0.03 mg/kg-h in 3.2 n&,/h saline) for 3.5 h. After two carbachol infusion periods, the third group (n=6) received NOLA (30 mg/kg, i.v. bolus), the fourth group (n=lO) was treated with SIN-l (0.6, 2, 6 mg/kg-h, iv., each for 30 min) infusion. In the 2nd experiment, following two periods when the animals received carbachol alone, one group (n=8) was treated with NOLA (30 mg/kg, i.v. bolus), another group (n=5) received Larginine (L-arg) (300 mg/kg, i.v. bolus) immediately followed by L-arg (300 mg/kg + 30 mg&g-h, i.v.) and NOLA (30 mg/kg, iv. bolus) simultaneously, while the third group (n=8) received D arginine (D-arg) (300 mgfkg, iv. bolus) followed by simult~eous D-arg (300 m~g+30 mg&g-h) and NOLA (30 mg/kg, i.v. bolus) infusion. In the 3rd experiment, after the second carbachol infusion period, saline (0.3 ml/rat, i.v. bolus), followed by NOLA (30 m&g, iv. was treated with phentol~ne (5 mg/kg, iv. bolus) followed by while the third group (n=8> received propranolol (5 mg/kg, i.v. mg/kg, i.v. bolus) injection.

the first group (n=5) received bolus), the second group (n=8) NOLA (30 mg/kg, i.v. bolus), bolus) followed by NOLA (30

The 4th experimental group served to compare changes in amylase and EGF secretion in response to different treatments. Animals were treated either with saline (n=lO), carbachol alone (0.03 m&g-h) (n=lO), carbachol plus NOLA in low dose (3 mg/kg) (n=lO), or carbachol plus NOLA in high dose (30 mg/kg) (n=lO). For comparative purposes, one group of rats (n=6) received noradrenalin infusion (1.5 mglkg-h, iv. infusion), which is known to stimulate both amylase and EGF secretion.

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At the end of the experiments a lethal dose of sodium-pentobarbital was given to kill the animals. All animal protocols described in the present paper were approved by local Ethics Committee. Determination of volume, amylase activityand EGF concentration Saliva samples were measured to the nearest 0.1 mL. Amylase activity was determined by an enzyme activity assay (21), using starch as a substrate. One unit (U) of amylase was defined as the activity of amylase that hydrolyses 1 mg starch per min at 37’C. EGF concentration was measured by a radioreceptor assay, using a preparation of human placental syncytiotrophoblast microvillous membranes (22). One-hundred pl sample was added to 100 ~1 of human placental microvillous membranes and (lzI)-labelled human EGF (100 pl/SO,OOOcpm). After overnight incubation at room temperature, the membrane preparation was diluted in 1000 pl 5% polyethylene-glycol, centrifuged for 10 min at 1500 g and the supematant was aspirated and discarded. The radiolabel associated with the membrane was determined in a gamma counter. Using this method the binding is independent of EGF source of the species (23). Carbachol, NOLA (No-nitro-Larginine), L-arginine, D-arginine, phentolamine and propradol were purchased from Sigma Chemical Co. (St Louis, MO, USA or Eiudapest, Hungary j, SW-1 (3morpholinosydnonimine) from Cassella (Frankfurt, Germany). All drugs were f;kshly prepared, and dissolved in saline. SIN- 1 and NOLA were stored in the dark before administration. Statisticalanalysis The results are expressed as mean f standard error of mean (S.E.M.). In all experiments the 4th

collection period (that is, the second period during carbachol administration, when salivary fluid and amylase secretion were steady) was used as control. Statistical significance was calculated by analysis of variance (ANOVA) followed by Fisher’s test, using Superanova statistical software. Differences were considered significant when p
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Fluid and EGF

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Output

(1.5 mg/kg-h) significantly increased EGF output (Table). Also, both treatments significantly increased amylase output when compared to control. The stimulatory effect of NOLA was dose dependent (Table). 2400NOLA

2000-

saline SIN-1 1600 -

0 0

2 t

4 carbachol

10.06 n&kg-h

6 background

8 infusion

I

SIN-I infusion

Fig. 1 Effect of carbachol (0.03 mglkg-h) back~ound infusion plus saline (fiued circle; bolus), carbachol (0.03 mglkg-h) plus NOLA (filled square; 30 mgikg bolus) and carbachol(O.03 mg/kg-h) plus SIN-l (open square;0.6, 2, 6 mgJcg-h for 30 min) on the time course of salivary amylase output in conscious rats. Values are mean f S.E.M; ***p
Nitric Oxide on Amyiase,

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that the availability secretion.

of endogenous

Fluid and EGF Output

L-arg is not a limiting factor in the regulation

957

of amylase

Under our experimental conditions NOLA also enhanced salivary fluid secretory response induced by carbachol. On the other hand, L-arg and SIN-l did not alter carbachol-stimulated secretory volume. Damas (29) has reported similar results on fluid secretion in anaesthetised rats: intraperitoneal injection of 35 mg/kg NOLA “per se” did not induce salivation, but substantially

-

-200

saline

NOLA

SIN-1

Fig. 2a

periods (30 rnin) NOLA

SIN-l

Fig. 2b

of saline, NOLA (30 mg/kg bolus) and SIN-l (6 mg/kg-h for 30 min) on changes of (a) salivary amylase and (b) fluid secretion over carbachol(O.03 mg/kgh) stimulated background secretion, in conscious rats. Values ate mean f S.E.M; ***p
Effect

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1700

1300

900

500

100

carbachol

carbachol + NOLA

Fig. 3a

0.60 0.50 0.40 0.30 0.20 0.10 0.00 -0.10 carbachol + NOLA

carbachol

Fig. 3b

Effect of L-arginine (L-arg; 300 mg/kg bolus), D-arginine (D-arg; 300 mg/kg bolus), NOLA (30 mg/kg bolus) and their combination on changes in (a) salivary amylase and (b) fluid secretion stimulated by a submaximal dose of carbachol(O.03 m@g-h) in conscious rats. Empty, stripped and filled columns represent wi~out or with Larginine or with D-arginine. Values are mean f S.E.M; ***p
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Nitric Oxide on Amyhe,

I

Y

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Fluid and EGF Output

saline + NOLA phentohunine + NOLA

propranolol+ NOLA

carbachol

t

background

infusion

I

4

NOLA t saline pheatolamine propranolol

Fig. 4a

-)3 9

1.2

g ‘5 u 5 t

1.0 0.8

2 S

0.6

t‘ 6 3 %

0.4

saline + NOLA

I

phentolamine + NOLA

-

propraaolol

+ NOLA

0.2 0.0

0

2

I

4

6

carbachol background

6 infusion

NOLA saline phentolamine propranolol

Fig. 4b Effect of saline (bolus) plus NOLA (30 mg/kg bolus) (filled square), phentolamine (5 mg/kg bolus) plus NOLA (30 mg/kg bolus) (open square) and propranolol (5 mgkg bolus) plus NOLA (30 mg/kg bolus) (filled circle) on the time course of salivary (a) amylase output and (b) fluid secretion stimulated by a submaximal dose of carbachol (0.03 mg/kg-h) in conscious rats. Values are mean f S.E.M; ***p
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Fluid and EGF Output

TABLE Effect of Carbachol (0.03 mg/kg-h), Noradrenalin (1.5 mg/kg-h) Alone and Carbachol (0.03 mg/kg-h) Plus Small (3 mg/kg) and Large (30 m&g) Dose of NOLA on Salivary EGF and Amylase Secretion in Conscious Rats. EGF (t&30 min)

amylase (U/30 min)

basal

63i15

44& 33

carb

79f14

Treatment

basal noradrenalin

ns

67f18 21560f478

carb

74f16

carb+NOLA (small)

87f15

carb

74f16

carb+NOLA (large)

54f8

Values are mean f S.E.M; ns no significant,

652 f 78

***

53f42 ***

640f

118

***

714 +105 ns

1078f125

ns

1792 f 166

*

714 f105 ***

*p
increased salivary fluid flow after the injection of pilocarpine (29). Recent studies provided evidence that the stimulatory effect of NOS blockade is not restricted to salivary secretion: inhibition of NGS by L-NAME was found to stimulate both fluid and ion secretion from rat stomach and duodenum (30) as well as from rabbit ileum (3 1). It is interesting to note that, like NOLA, VIP alone does not induce salivary secretion, but augments salivary secretion evoked by other drugs, e.g. by carbachol (32). Edwards and his coworkers performed a series of studies investigating parallel changes in salivary blood flow and fluid secretion, using another NOS inhibitor, NG-nitro-L-arginine methyl ester (LNAME). Our data are not in full agreement with their results. In their studies, in anaesthetised cats, close intraarterial injection of a very high dose of L-NAME (100 mg/kg) given alone caused a rapid increase of the secretion of submandibular saliva which persisted for the duration of the infusion and then subsided within a few minutes (13). This secretory response was either abolished or greatly reduced by atropine (13). L-NAME, however, did not affect the fluid secretory response to close intraarterial acetylcholine injection (6) and did not modify or eventually decreased this response to chorda-lingual stimulation depending on the type of the stimulation (continuous or intermittent) and the applied dose of the NOS inhibitor (33-530 mg/kg, ia.) (5,6,14,15). They have also shown that the inhibition of fluid secretion by L-NAME following stimulation of the parasympathetic innervation of the salivary glands is accompanied by a decrease in submandibular protein secretion (15). The most recent publication from this group, however, has revealed that the effects of L-NAME on salivary fluid and protein secretion are not affected by L-arginine infusion (14). Therefore, it is highly likely that these effects of L-NAME are not related to its inhibitory effect on NO synthase. To this end, it is important to mention that L-NAME (but not NOLA, the drug used in the current study) has been described as a muscarinic receptor antagonist in vitro (33), although this observation was not confirmed later in vivo (34). The difference in the results of Edwards and coworkers (6, 13- 15) and our present observations can be explained by differences in the two experimental approaches. First, we studied a different species and used a different NOS inhibitor. Second, our experiments used conscious rats, while theirs used anaesthetised animals. But, at the same time, we cannot differentiate between local and systemic effects of NOLA, while in their studies the NOS inhibitor was given to the local artery and the venous effluent plasma was collected and discarded in order to prevent the access of LNAME into the general circulation. Furthermore, in their studies both the ascending cervical sympathetic nerve and the chorda lingual nerve were cut in order to achieve sympathetic and parasympathetic

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denervation. Finally, in our investigation, the inhibition of NOS activity was prevented by L arginine while in their studies the effect of LNAME was not affected by an excessive dose of this amino acid. The mechanism by which NOLA stimulates salivary fluid and amylase secretion is not known, but this effect is probably not directly related to the effect of NO on local glandular vascular tone. Previously, we showed in anaesthetised cats that NOLA caused a marked decrease in blood flow jn the submandibular gland accompained by a tendency toward blood flow reduction in parotid gland despite a significant increase in mean arterial blood pressure (10, 11). Moreover, in the rat, NOLA increased salivary secretion despite a reduced blood content of submandibular gland after pilocarpine stimulation (29). Other experiments also suggest that there is no consequential relationship between salivary fluid secretion and glandular blood flow (32). On the other hand, carbachol decreases mean arterial blood pressure by NO release from endothelial cells, an effect which may be antagonized by NOLA by NOS inhibition (35). The increased perfusion pressure after NOLA compared to carbachol alone may increase extravasation of fluid, thus secretion. However, there is experimental evidence that variations in glandular perfusion pressure have little or no influence on acetylcholine stimulated secretion of rat submandibular gland (36). Our present experiments also support this finding, because bolus saline injection in addition to carbachol background infusion did not change either salivary fluid or amylase secretion (see Fig. 1, Fig. 4a,b). One possible explanation for the mechanism of NOLA-stimulated increase of salivary secretion is that NO synthesis is inhibited locally, in neuronal, acinar, myoepithelial and vascular cells in salivary glands. According to our data, physiologically present, continuously released NO itself could be sufficient to evoke an inhibitory tone since surplus of NO evoked by either SIN- 1 or Larg administration had no further inhibitory effect on salivary amylase secretion. This is supported by earlier observations that there is a considerable level of basal NO production in the vessels of the submandibular (7, 10, 11, 13) and parotid (10, 11) glands. In addition, NOS has been local&d by immunohistochemistry in neurones and in nerve fibres and acinar cells of the salivary glands (2-4, 37). NADPH-diaphorase activity, another marker for the presence of NOS, was also found both in neuronal structures and in endothelial and ductal epithelial cells of submandibular salivary gland (4). NO produced in these cells may act in a paracrine manner and exert an inhibitory effect on salivary amylase secretory activity. The modulatory effect of NO on fluid and amylase secretion may involve the suppression of sympathetic activity, since inhibition of peripheral sympathetic vasoconstriction by NO is an important control element of vasodilation in vivo (38). Our data suggest that the blockade of alphaadrenoceptor activation by NO is not involved in this mechanism, because the alpha-receptor antagonist phentolamine did not block the stimulatory effects of NOLA on amylase output, although it inhibited the secretory rate of salivary fluid. EGF secretion, known to be regulated by alphaadrenoceptors ( 18), is not increased after NOLA suggesting that NO is not an inhibitor of the action of the alpha-receptors. Pretreatment with the beta-adrenoreceptor antagonist propranolol significantly decreased both fluid and amylase output. Indeed, amylase output was actually suppressed far below the carbachol plateau. Our data suggest that in conscious rats secretory response to muscarinic receptor stimulation depends on parallel, physiological beta-adrenergic receptor activation. This is in line with the present knowledge that is based on a large number of investigations that the sympathetic and the parasympathetic nervous systems arc acting in synergy in salivary glands (1,39-41). Besides salivary fluid and amylase secretion, we also investigated the involvement of the La&NO pathway in the regulation of salivay EGF secretion. In rats salivary EGF is mainly released from granulated convulated tubules of the submandibular glands, although small amounts from other sources can still be measured after submandibulectomy (18.20.42). The finding that NOS reactive nerve fibers surrounding the salivary ductal branches are abundant in cat (4) raised the possibility that the EGF output might be under NO control. Our present results confirmed that muscarinic cholinergic stimulation does not effect EGF secretion. and that noradrenaline induces an enormous increase in EGF output from salivary glands (18, 20, 43). Our data also showed that NOLA is ineffective on salivary EGF secretion suggesting that NO does not inhibit EGF secretion.

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In conclusion, our data indicate that the L-argfN0 pathway plays a modulatory modulatory role in the cholinergic control of salivary fluid and amylase secretion, but not in EGF output. L-arg is not a limiting factor in this regulation. The mechanism of actions of NO remains to be identified, but it may involve inhibition of presently unidentified secretagogue mechanisms that need characterisation in further studies.

Acknowledgements The authors express their gratitude to Agnes Gara, M&a Jacs6 Haraszti and Emese Sarkady for their excellent technical assistance. This work was supported by grants of the Hungarian National Research Funds OTKA No. T-017104, F-20469; E’lT T-02149/93, T-13 122/98; ETK 113, and by the Bolyai Foundation. References A. LERNER, M.A. ROSENTHAL, C. LIEBOW, E. LEBENTHAL, Textbook of Gastroenterology, T. Yamada (Ed), 218-233, J.B. Lippincott Company, Philadelphia, (1991). A. MODIN, Acta Phys. Stand. Suppl. 622 151-174 (1994). 2. S. CECCATELLI, J.M. LUNDBERG, X. ZHANG, K. AMAN, T. HOKFELT, Brain Res. 3. 656 381-395 (1994). Z. LOHINAI, A.D. SZl%ELY, L. SO&, E. FE&R, Neurosci. Lett. 192 9-12 (1995). 4. M. SCHACHTER, B. MATTHEWS, K.D. BHOOLA, Agents Actions Suppl. 38 Pt 2 3665. 370 (1992). A.V. EDWARDS, J.R. GARRETT, J. Physiol. 464 379-392 (1993). 6. N.P. KEREZOUDIS, L. OLGART, &: EDWALL, Eur. J. Pharmacol. 241 209-219 (1993). 7. A. MODIN, E. WEITZBERG, T. HOKFELT, J.M. LUNDBERG, Neurosci. 66 189-203 8. (,j994). A. FAZEKAS, J.L. MATHENY, G.I. ROTH, D.R. RICHARDSON, Res. Exp. Med. 194 9. 357-365 (1994). Z. LOHINAI, I. BALLA, J. MARCZIS, Z. VASS, A.G.B. KOVACH, Abstr of 3rd IBC 10 Internat. Symp. on Nitric Oxide, Philadelphia (USA), p47 (1994). 11. Z. LOI-IINAI, I. BALLA, J. MARCZIS, Z. VASS, A.G.B. KOVACH, Archs. oral Biol. 41(7) 699-704 (1996). 12. S. BODIS, A. HAREGEWOIN, Biochem. Biophys. Res. Comm. 194 347-350 (1993). 13. A.V. EDWARDS, J.R. GARRETT, J. PHYSIOL. 456 491-501 (1992). 14. A.V. EDWARDS, G. TOBIN, J. EKSTROM, S.R. BLOOM, Exp. Physiol. 8 1 349-359 (1996). 15. A.D. BUCKLE, S.J. PARKER, S.R. BLOOM, A.V. EDWARDS, Exp. Physiol. 80 10191030 (1995). 16. Z. LOHINAI, B. BURGHARDT, G. VARGA, Z. Gastroenterol. 5 321 Nr 83 (1996). 17. Z. LOHINAI, B. BURGHARDT, G. VARGA, Dig. Dis. Sci. 42(l) 217 (1997). P.S. OLSEN, P. KIRKEGAARD, S.S. YOULSEN, E. NEXO, Gut 25 1234-1240 (1984). :98. K. TAZI-SAAD, J. CHARIOT, C. ROZE, Peptides 13 233-239 (1992). 20. J. BLAZSEK, K. OFFENMULLER, B. BURGHARDT, Jr.1. KISFALVI, K. BIRKI, M. WENCZL, G. VARGA, T. ZELLES, Inflammopharmacol4 279-295 (1996). 21. P. BERNFELD, Meth. Enzymol. 1 149-158 (1955). 22. A.G. BOOTH, 0. OLANIYAN, O.A. VANDERPUYE, Placenta 1 327-336 (1980). 23. R.J. SIMPSON, J.A. SMITH, R.L. MORITZ, M.J. O’HARE, P.S. RUDLAND, J.R. MORISSONJ, C.J. LIOYD, B. GREGO, A.W. BURGESS, E.C. NICE, Eur. J. Biochem. 153 629-637 (1985). 24. K. AISALA, A. MITANI, Y. KITAJIMA, T. OHNO, T. ISHIHARA, J. Pharmacol. 56 245-248 (1991). 25. G. VARGA, T.E. ADRIAN, D.H. COY, R.D. REIDELBERGER, Peptides 15 713-718 (1994). 26. G. VARGA, D.R. CAMPBELL, L.J. BUSSJAEGER, T.E. SOLOMON, Eur. J. Pharmacol. 250 37-42 (1993). 1.

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