Proenkephalin a derivatives in lacrimal gland: Occurrence and regulation of lacrimal function

Exp. Eye Rrs. (19921 54. 829-834

Proenkephalin

A Derivatives in Lacrimal Gland: Regulation of Lacrimal Function MICHELE

Department

of Physiology, (Received

Louisiana

Baltimore

M. CRIPPS” State

26 November

AND

University

D. JEAN Medical

1990 and accepted

Occurrence

and

BENNETT

Center,

New

in revised

Orleans,

LA 70112,

form 2 April

U.S.A.

1991)

The derivatives of proenkephalin A were measured in acid extracts of rat lacrimal glands by specific radioimmunoassay. Glands from adult male rats contained all four derivatives of the opiate precursor. The content in the gland of proenkephalin A-derived peptides Met”-enkephalin. Leu”-enkephalin, Met”enkephalin ArgG-Phe’ and Mets-enkephalin Argfi-Gly’-Leu” indicates tissue specific processing with an enhancement of the heptapeptide. The effect of enkephalins on the activity of adenylate cyclase in lacrimal membranes was measured and compared with the effect of the synthetic enkephalin analogue n-ala2-methionine enkephalinamide (DALA) that inhibits both lacrimal protein secretion and lacrimal adenylate cyclase. In the presence of the peptidase inhibitors thiorphan and bestatin, the inhibition of forskolin-stimulated adenylate cyclase activity by Met”-enk, Leu”-enk. Met”-enk Arg’-Phe’ and DALA were identical. Maximum inhibition was approximately 3 5 “/( at a dose of 50 //M enkephalin. Addition of the octapeptide. Met”-enk Arg’-Gly’-Leus resulted in decreased adenylate cyclase activity; however, the effect was not statistically significant. Activation of B opioid receptors by the endogenous enkephalins is indicated by the reversal of adenylate cyclase inhibition in the presence of the J-receptor antagonist ICI 174864. The data support the physiological significance of in vitro inhibition of lacrimal secretion by DALA and indicate a possible role for endogenous enkephalins in lacrimal function. Key u?ords: lacrimal gland : adenylate cyclase : peptides : proenkephalin A : Met”-enkephalin : Leu”enkephalin : Met”-enkephalin Argfi-Phe’ ; Met”-enkephalin Arg” Gly’-Leu’.

1. Introduction The enkephalins derived from proenkephalin A include Mets-enkephalin, Leu”-enkephalin, Mets-enkephalin Args-Phe’ and Mets-enk Arg6-Gly’-Leu”. These peptides have been localized by immunohistochemical techniques in several peripheral tissues including mammalian exocrine glands (Kondo et al.. 1988 ; Yonehara et al., 1989). Recent work (Lehtosala et al., 1989) has demonstrated the presence of all four derivatives of proenkephalin A in nerve fibers innervating the extra- and intraorbital lacrimal glands of guinea-pigs. In addition, Mets-enk and Leu”-enk immunoreactivities are present in human lacrimal gland preparations (Frey et al., 1986). In the guinea-pig, enkephalin immunoreactive fibers terminate near the basal surface of the acinar epithelium and also innervate the secretory ducts of the gland (Lehtosala et al., 1989). The close association of the peptidergic fibers with the secretory structures of the gland suggest that the enkephalins are important neuromodulators of lacrimal secretion. In a recent study (Cripps and Patchen-Moor, 1989), the synthetic Met”-enk analogue D-ala’-methionineenkephalinamide (DALA) inhibited both cholinergic and VIPergic stimulation of peroxidase release by rat lacrimal gland fragments. The mechanism by which DALA inhibits stimulated secretion involves a direct

post-synaptic effect on lacrimal acinar cells. The effect of DALA on lacrimal secretion is coupled to adenylate cyclase and a decrease in intracellular levels of CAMP as evidenced by the inhibition of basal, forskolinstimulated and VIP-stimulated adenylate cyclase activity in membranes prepared from lacrimal tissues (Cripps and Bennett, 1990). The purpose of this study was to determine if the proenkephalin A derivatives are present in rat lacrimal gland, a frequently used model for the study of lacrimal secretory control mechanisms. Furthermore. the physiological significance of the enkephalins was assessed by comparison of the effects and specificity of the peptides with DALA on adenylate cyclase activity in lacrimal membrane preparations. 2. Materials and Methods

Male Sprague-Dawley rats (22 5-2 75 g) were obtained from Charles Rivers, Inc. (Wilmington, MA). The rats were housed in a temperature-controlled room with a 12-hr light/dark cycle and fed standard laboratory chow. Animals were killed by intraperitoneal injection of sodium pentobarbital 1200 mg kg-’ body weight) followed by an intracardiac bolus of the same drug (100 mg kg-’ body weight ).

* Por reprint requests at: Department of Physiology. LSU Medical Center. 1901 Perdido Street, New Orleans, LA 70112. I1.S.A. 0014-4835/92/060829+06 54

$03.00/O

0 1992 Academic Press Limited F.EK5-L

830

Exorbital lacrimal glands were removed immedately and placed into 40 mM Tris-HCl (pH 7.4). The capsules and main secretory ducts were excised : the glands were sliced into 3-5 mm? fragments. and the fragments were rinsed three times with Tris-HCI. 1,acrimal gland fragments from two animals were homogenized in 10 ml of 1 M acetic acid, containing LO mM HCI and 0.1 ‘%, /&mercaptoethanol with a Tekmar Tissumiser (Tekmar Instruments. Cincinnati, OH) at low speed for 15 min. Following centrifugation at 20000 g for 20 min, the supernatant was aliquoted, lyophilized and stored at - 70°C. Radioimmunoassay of the extracted enkephalins was performed as described by Panula and Lindberg (1987). Briefly, the lyophilized samples were reconstitued in 01 M sodium phosphate, pH 7.4. with 0.1% BSA. 0.1% sodium azide and 0.1 yj /I-mercaptoethanol (RIA buffer). The 1 5 000-g supernatant was assayed in duplicate tubes that contained 100 ~(1 of the sample or enkephalin standard (Peninsula Laboratories, Inc., Belmont. CA), 100 /rl of the iodinated peptide and 100 //I of the appropriate antiserum. Following an overnight incubation at 4”C, 100 ,~l 075 “/0 y-globulin in 0.1 M phosphate buffer, pH 7.5. was added to each tube and the bound fraction was precipitated by the addition of I ml of 2 5 % polyethylene glycol and centrifugation at 1000 g for 20 min at 4°C. The bound fraction was counted by gamma spectroscopy. Enkephalin content in the samples was calculated as pmol mg- ’ protein. with protein determined by the method of Lowry et al. ( 19 5 1). lodinated enkephalins, radiolabelled by the chloramine-T method, and antisera were the generous gift of Dr lris Lindberg (LSLI Medical Center, Department of Biochemistry, New Orleans, LA). The Met?-enk (Dandekor and Sabol. 1982 ). Met”-enk Arg”Phe: (Mocchetti et al., 1984). and Met”-enk Arg”-Gly’Leu” (Lindberg and White, 1986) antisera have been characterized previously. The Leu”-enk antiserum is a newly raised antibody and was characterized for this study. It was used in our assays at a titer of 1: 9000 with an IC,,, of 0.22 pmol. This antiserum was found to cross-react with Met”-enk by 2.1 %, with Met”-enk Arg”-Phe: by 0+12%. and with Met”-enk Arg”-Gly:kuH by O-0262,.

M. M. CRIPPS

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D J BENNETT

sc,ribed aboI,e. The supernatants from tlvo 1000 4, 10 min ccntrifugations were combined and c’entrifuged for 15 min at 27000 $1. ‘I’hc pellet M’;IS resuspended and centrifuged at 2 7000 $1for 1 5 min with a final suspension in the isolation buffer. The membranes were aliquoted. frozen in liquid nitrogen and stored at - iO”C. Membrane protein was dctermined by the method of I,owry et al. (1951). Membrane-bound adenylate cyclase activity was determined in a total volume of 100 ,~l of 40 rnbl ‘I‘ris (pH 7.5). 4 mM MgCl,. 0.5 rnhl ATP, 1 mM DTT. 0.1 rnM G’I’P. 0.1 mg ml ’ BSA. 0.1 mM ED’I’A. 0 1 mM IBMX. 10 rnlv creatine phosphate. 50 Ii ml-’ creatine phosphokinase. 30 ,uM bestatin. 0.3 ,HM thiorphan and 25 /(g membrane protein. Forskolin (Calbiochem, La Jolla, CA) was prepared as a 20 rnlw stock in 9 5 y0 ethanol. Enzyme activity was determined in triplicate samples at 37°C. Because lacrimal adenylate cyclase activity does not increase with membrane incubation beyond 10 min (Cripps and Bennett. 1990) enzyme activity was measured up to and including this time-point. The reaction was terminated by the addition of 200 /II of ice-cold 0.0 1 N HCl-9 5 % ethanol. Cyclic AMP was measured by the protein method binding assay method of Brown et al. ( 19il) with extraction of binding protein from adrenal cortices and modifications in the procedure as previously described (Cripps and Bennett. 1990). Adenylate cyclase specific activities were calculated as pmol rng-.’ membrane protein. The data were analysed by unpaired t-tests.

3. Results Radioinvnunoassay of Enkephalins The content of enkephalin-immunoreactive material in acid extracts of lacrimal glands was measured by specific radioimmunoassay (Fig. 1). Glands from adult male rats were found to contain all four derivatives of proenkephalin A. Met”-enk, Leu”-enk

Membrane preparation and Adenylate Cyclase Determination Lacrimal glands were placed in ice-cold medium containing So/, sorbitol, 0.5 mM ethylenediamnetetraacetic acid (EDTA), 5 mM histidine-imidazole buffer, pH 7.5. 9 pg ml-’ aprotinin. 3 mM dithiothereitol (DTT) and 0.2 mM phenylmethylsulfonyl fluoride (PMSF) as described by Mircheff et al. ( 198 3 ). This buffer was used throughout the membrane isolation procedure and all steps were done on ice. Fragments were prepared and homogenized as de-

Met5-,,k

Leu5-enk

Met5-enk

Met%nk

ar$‘-phe7

W+gl)!7-lel18

FK:. 1. Endogenousenkephalins in acid extracts of lacrimal glands. Derivatives of proenkephalin A were measured by specific radioimmunoassay. Values are the mean + S.E. determined from glands of six adult males.

ENDOGENOUS

ENKEPHALINS

IC-

o-- I

6

8

Ll LP~ IO

Time (mini

FIG. 2. Time course of basal adenylate cyclase activity. Membranes were incubated for the indicated times. Values are the mean+s.r-.. of ten experiments. Statistically significant difference at: (*) P < O-05: (**) P < 0.01.

8

6OL

I

OL’

0

’

’

2

’

’

4

Time

’

’ 6

a31

GLAND

I

I.

4

2

0

IN LACRIMAL

’

’ 8

’

’ IO

(min)

FIG. 3. Time course of inhibition of adenylate cyclase activity. Membranes were incubated for the indicated times in the absence or presence of 30 ,U~MDALA (0). Mets-enk (v). or Leu5-enk (0). Values are the meani:s.~. of the percent of basal adenylate cyclase activity determined in five to seven experiments. Statistically significant difference of:

(*)P< 0.05; (**)P < 0.01. and Met”-enk Arg6-Phe’ were easily measured: however, the level of Mets-enk Arg6-Gly’-Leu6 was near the limit of detection by radioimmunoassay and required at least a two-fold concentration of the samples in order to detect the presence of this peptide. The mean concentration of Mets-enk Arg6-Phe’ was twice that for Leu5-enk, and three times the amount of Mets-enk. The concentration of the octapeptide, Mets-enk Arg6Gly’-Leu” was less than 5% of the concentration of Met5-enk Arg6-Phe’. Adenylate Cyclnse The opioid analogue o-ala2-met-enkephalinamide (DALA) inhibits both in vitro protein secretion by perifused lacrimal gland fragments (Cripps and Patchen-Moor. 1989) and adenylate cyclase activity

Enkephalin

(PM)

FIG. 4. Effect of increasing concentrations of enkephalin on inhibition of forskolin-stimulated adenylate cyclase activity. Membranes were incubated for 10 min in the presence of 30 /(M forskolin with the indicated concentrations of DALA (0). Met”-enk (Vi. Leu”-enk (O), Met”enk arg”-phe’ (@), or Met5-enk arg”-gly7-leuY (m). Values are the mean k S.E.of percent of stimulated adenylate cyclase activity of three to five determinations per dose. Statistically significant difference of: (*) P < 0.05 : (**, P < 04) 1.

in isolated lacrimal membrane preparations (Cripps and Bennett, 1990). In order to assess the physiological role of the endogenous enkephalins in the lacrimal gland, we compared the effect of the pentapeptides with the effect of DALA on the activity of basal adenylate cyclase in lacrimal membranes. The time course of adenylate cyclase activity in lacrimal giand membrane preparations was measured under basal conditions and in the presence of DALA or the pentapeptides Mets-enk and Leus-enk. The adenylate cyclase reaction was terminated at 0, 2, 5, 7 and 10 min. Basal CAMP production (Fig. 2) was linear up to 10 min with a rate of 3.02 pmol min-’ rng-’ membrane protein. Inhibition by the opiates was calculated as a percent of basal CAMP production (Fig. 3). The inhibition of basal activity by DALA was timedependent. Significant inhibition of CAMP production was present after 10 min incubation with a decrease to 71.5 k4.2 y0 (meanf s.E.) of the control value (P < 0.01). A similar time course and percent reduction in CAMP production occurred in the presence of Met5-enk (755+3.3%: P < 0.05) and Leu5-enk (74.8*8.0x; P < 0.01). The effect of the opiates on forskolin-stimulated adenylate cyclase activity was also measured. Incubation of isolated cell membranes in the presence of 30 ,HM forskolin resulted in the production of 1 SO.6 _+ 9.1 pmol mg-’ protein per 10 min. This value was an approximate three-fold increase over basal production of 49.3 f 4.9 pmol mg-’ protein. The addition of 50 PM DALA caused a significant (P < 0.01) decrease in forskolin-stimulated activity to 104.6 110.9 pmol mg-’ protein. The inhibition of CAMP production in the presence of 50 ,!LM Mets-enk, L.euj-enk or the heptapeptide Mets-enk Arg”-Phe’ was similar to the reduction of enzyme activity by DALA. At a dose of

M. M CRIPPS

832

al :: 0 0”

60

50

1

AND

D. J BENNETT

Because adenylate cyclase activity was reduced by the addition of the antagonist by as much as 1 5 ‘j(, (e.g. in the presence of lo-’ M ICI 174864) the data are expressed as percent of control activity. The control values were determined in the presence of 30 l/&l forskolin and the appropriate dose of ICI 174864. Inhibition of adenylate cyclase resulting from the addition of 30 pM or 50 ,U,MDALA was reversed in the presence of the antagonist in a dose-dependent manner. Reversal of inhibition was nearly complete in the case of the 30 ;t~ dose of DALA, with an increase from 75.8 + 5.2 % of maximum adenylate cyclase activity to 94.4& 1.7% (P < 0.05) of forskolin-stimulated production of CAMP. A decrease in the presence of 50 pM DALA to 67.6 -t 6.0% of control activity was also reversed in the presence of 1OY’ M ICI 1 74864 to 87.1 + 6,5 “/o of control adenylate cyclase activity. Similar results were obtained with Met”-enk, and Leu’enk: however, ICI 174864 reversal of the inhibition by the heptapeptide was less effective.

90 -

4. Discussion 80 -

T

70 -

I

60 -

50-

Mets-enk

DALA

Leu’-enk

Met’-enk arg’- phe’

Frc. 5. Keversal of opioid inhibition by ICI 174864. Membranes were incubated for IO min in the presence of 30 ,UM for&Olin, 30 ,IIM (A) or 50 I’M (B) enkephalin and increasing dosesof ICI 174864. Values are the mean f S.E. of the percent of forskolin-stimulated activity from three to five determinations per condition. Statistically significant difference of: (*) P < 0.05 ; (**) P < 0.02 : (***) P < 0.01. (0)

(1 ICI;

(m)

l&‘M

ICI;

(a)

10-“M

ICI;

(a)

lo-“MICI.

50 PM, the octapeptide Met”-enk Arg6-Gly’-Leu8 had no significant effect on forskolin stimulated adenylate cyclase activity. The inhibition by the effective enkephalins was dose-dependent (Fig. 4). The doseresponse curves of inhibition of adenylate cyclase for Met”-enk Leu”- and Met’-enk Argfi-PheF were identical to that obtained with DALA. Significant decreases in forskolin-stimulated adenylate cyclase activity occurred in the presence of 30 ,~AM (P < O-01) of either DALA or the endogenous enkephalins. Although the addition to isolated membranes of the octapeptide. Met”-enk Arg6-Gly’-LeuR decreased stimulation of CAMP production by forskolin. the effect was not statistically significant at any dose tested. In separate experiments. the specificity of inhibition of forskolin-stimulated adenylate cyclase was tested by the addition of increasing doses of ICI 174864, a specific S-receptor antagonist [Figs S(A) and (B)].

This study demonstrates the presence of enkephalins in the extraorbital lacrimal gland of the rat and suggests that the endogenous enkephalins may be important neuropeptide regulators of lacrimal gland function. The enkephalins detected in acid extracts of the rat lacrimal gland include the derivatives of proenkephalin A : Met”-enk : Leu”-enk: the heptapeptide Met”-enk Arg6-Phe’; and the octapeptide Met”enk Arg6-Gly’-Leu*. Recent immunohistochemical work (Lehtosala et al., 1989) studied the occurrence and distribution of the proenkephalin A derivatives in the lacrimal gland of the guinea-pig. Immunoreactive nerve fibers were identified in both the intra- and extraorbital glands with all four enkephalin-like immunoreactivities present and evenly distributed throughout these structures. The proenkephalin A molecule contains four copies of Met”-enkephalin and one copy each of leuj-enk, Met”-enk Argfi-PheT and Met”-enk Arg6-Gly’-Leu’ (Gubler et al., 1982). The levels of these peptides in rat lacrimal gland do not reflect the ratio in the precursor but indicate a relative enrichment of the heptapeptide. Furthermore, the level of the octapeptide was considerably lower than expected. Differential tissue-specific processing of proenkephalin A may account for the non-stoichiometric amounts of the lacrimal enkephalins as occurs in the anterior and intermediate lobes of the pituitary (Panula and Lindberg, 1987). In the pituitary, the heptapeptide is four-fold higher in the anterior lobe than in the intermediate. while the level of Met”-enk is identical in the two lobes. The octapeptide in both lobes is 10% of the predicted value. The specific levels of enkephalins found in lacrimal gland may be due to selective susceptibility to or protection from proteolytic degradation, incomplete cleavage of specific sequences from the precursor or the presence of high molecular

ENDOGENOUS

ENKEPHALINS

IN LACRIMAL

833

GLAND

weight forms of the peptide-containing sequences that do not cross-react with the antisera. Studies on the carboxyl-terminally extended enkephalins, Met6-enk Arg’-PheR and Met”-enk Arg’-GlyXLeug, have suggested that these peptides do not simply represent intermediates in the biosynthetic pathway to Mets-enkephalin but function as neurotransmitters (Iadarola et al.. 1985). Our results demonstrate that the inhibition of adenylate cyclase by the heptapeptide is equivalent to the effect of both Met” and Leu”enkephalins. The potency and efficacy of these three opioids were identical to that of DALA, an enkephalin analogue that inhibits lacrimal protein secretion through attenuation of CAMP production (Cripps and Patchen-Moor, 1989 ; Cripps and Bennett, 1990). These results support a physiological role for the heptapeptide and the pentapeptides as modulators of lacrimal gland function. The octapeptide. Met”-enk Arg’-GlyX-Leug decreased lacrimal adenylate cyclase activity: however, the effect was not statistically significant. The reduced inhibitory effect and lowered measurable amount may be related to instability of the octapeptide. In a comparison of the stability of proenkephalin A derivatives released by rat brain slices (Patey et al., 1983) rapid degradation of the octapeptide but not the heptapeptide. Met”- or Leu”enkephalin, occurs once it is released into the extracellular space. The degradation of the octapeptide persists even in the presence of the peptidase inhibitors thiorphan and bestatin included in our assay. Although measurement of enkephalin receptor binding was not done in this study, reversal of inhibition by the &specific antagonist ICI 174684 (Cotton et al.. 1984; Cohen et al., 1986) suggests that in lacrimal gland Met”-enk, Leu6-enk and MeP-enk Arg;-Phex activate the opioid R-receptor subtype. Keversal of the inhibition of adenylate cyclase by the heptapeptide was not complete which indicates possible interaction of Met6-enk Arg’-Phe*-LeuY with additional binding sites. The receptor involved may be the 1” binding site in as much as the extended metenkephalins reportedly may not distinguish between I( and S binding sites (Paterson, Robson and Kosterlitz, 1983). Definitive identification of the receptors activated by enkephalins in rat lacrimal gland will require additional studies with selective opioid agonists and antagonists. The present study and previous work (Nikkinen et al., 1984: Sibony, 1988; Lehtosalo et aI., 1989. ti’usitalo, Mahrberg and Palkana, 1990 ; Walcott, 1990) provide increasing evidence that a number of peptides are endogenous to the lacrimal gland. These include the enkephalins. VIP, substance P and NPY. The peptides are present in fibers terminating near the secretory acini and ducts. suggesting a role in the regulation of lacrimal secretion. The physiological significance of VIP and enkephalin in the dual regulation of lacrimal peroxidase secretion has been established (Dartt et al., 1984; Cripps and Patchen-

Moor, 1989) and it will be of interest to determine the role of other peptides present in the gland and the interactions that may occur between them.

Acknowledgements This work was supported by LJSPHSGrant EYO7380 from the National Eye Institute.

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Cotton, R.. Giles. M. G., Miller, L., Shaw. J. S. and Timms, D. (19841 ICI 174864: a highly selective antagonist for the opioid (S-receptor. Eur. 1. PharmncoL 97. 3 3 I-2. Cripps. M. M. and Bennett. D. J. (1990). Peptidergic stimulation and inhibition of lacrimal gland adenylate cyclase. Invest. Ophthalmol. Vis. Sci. 31, 2 145-50. Cripps, M. M. and Patchen-Moor, K. ( 1989) Inhibition of stimulated lacrimal secretion by [u-ala:‘] met-enkephalinamide. Am. ]. Pfrysiol. 257 (Suppl. 201, Gl SIX;1 56. Dandekar. S. and Sabol. S. L. (1982). Cell-free translation and partial characterization of mRNA coding for enkephalin-precursor protein. Proc. IL’~d. .Acad. Sci. U.S.A. 79. 1017-21. Dartt. D. A., Baker, A. K.. Vaillant. C. and Rose. P. E. (1984) Vasoactive intestinal polypeptide stimulation of protein secretion from rat lacrimal gland acini. Am. 1. Physiol. 247 (Suppl. IO). G502-GS09. Frey, W. H.. III, Nelson, J. D.. Frick. M. L. and Elde. R. P. ( 1986). Prolactin immunoreactivity in human tears and lacrimal gland: possible implications for tear production. In The Preocular Tear Film in Health, Disease cmtl Contart Lens Wear. (Eds Holly, F. J.). Pp. 79X-807. Dry Eye Institute: Lubbock, TX. Gubler. U.. Seeburg. P.. Hoffmann, B. 1.. Gage. L. P. and Undenfriend. S. (1982 1. Molecular cloning establishes proenkephalin as precursor of enkephalin-containing peptides. Nature 295. 206-8. Iadarola. M. J.. Panula. P.. Mauane. E. A. and Yank. H. Y. T. ( 198 5 ). The opioid octapeptide Met-enkephalin-Arg’Gly-Leu!‘: characterization and distribution in rat spinal cord. Rrain Res. 330. 12i-34. Kondo, H.. Yamamoto. M., Yanaichara. N. and Nagatsu. I. ( 1988 ). Transient involvement of enkephalins in both the sympathetic and parasympathetic innervations of the submandibular gland of rats: light and electronmicroscopic immuno-cytochemical study. Cc/f Tissue RCS.253. 529-37. Lehtosalo. J.. Lusitala, H.. Mahrberg, T.. Pannula. P. and Palkama. A. ( 1989 1. Nerve fibers showing immunoreactivities for proenkephalin A-derived peptides in the lacrimal glands of the guinea-pig. Grclefe’s Arch. C/in. Esp. Ophthnlmol. 227, 45 5-8. Lindberg, I. and White. L. (1986). Reptilian enkephalins: implications for the evolution of proenkephalin. Arch. Biochem. Biophyls. 245. 1-7. Lowry. 0. H.. Rosebrough. N. J.. Farr. A. L. and Randall, R. J. ( 195 1). Protein measurement with the Folin phenol reagent. 1. Rio/. Chem. 193. 265-j.

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Mircheff. A. K.. Conteas, C. N.. Lu. C. C.. Santiago. G., Gray. M. and Lipson, I,. G. (1983). Basal-lateral and intracellular membrane populations of rat exorbital lacrimal gland. Ant. 1. Ph,ysiol. 254 (SuppI.). GI 3 3-G142. Mocchetti. I., Giorgi, 0.. Schwartz, J. P. and Costa, E. ( 1984). A reduction of the tone of 5-hydroxytryptamine neurons decreases utilization rates of striatal and hypothalamic enkephalins. Eur. J. Phnrms~ol. 106. 42 7-30. Nikkinen. A., Lehtosalo, J. I., Uusitalo. H., Palkama. A. and Panula. P. ( 1984). The lacrimal glands of the rat and guinea-pig are innervated by nerve fibers containing immunoreactivities for substance P and vasoactive intestinal polypeptide. Histochemistry 81, 2 3-7. Panula, P. and Lindberg, I. (1987). Enkephalins in the rat pituitary gland : immunohistochemical and biochemical observations. Endocrinology 121, 48-58. Paterson, S. J., Robson, L. E. and Kosterlitz, H. W. (1 Y 8 3). Classification of opioid receptors. Br. Med. Bull. 39, 31-6.

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Patey. G.. Cupo. A.. Mazarguil. f-1..Morgat. J. I,. and Kossier. J. ( 198 3 ). Kelease of proenkephalin-derived opioid peptides from rat striatum in vitro and their rapid degradation. Netrurosrirnct, 15, 10 3 5-34. Sibony. P. A.. Walcott. B., McKeon. C. and Jakohiec. F. A. (1988). Vasoactive intestinal polypeptide and the innervation of the human lacrimal gland. Arch. Ophthalrnol. 106, 1085-8. Uusitalo. H.. Mahrberg. T. and Palkama, A. (1990). Neuropeptides in the autonomic and sensory nerves of the lacrimal gland : an immunohistochemical study. hrst. Ophthalmd. Vis. Sri. 31 (Suppl.), 44. Walcott, B. (19 90). Leu-enkephalin-like immunoreactivity and the innervation of the rat exorbital lacrimal gland. Invest. Ophtludmol. I/is. Sci. 31 (Suppl.), 44. Yonehara, N.. Tsai, H. Y.. Chen. J. Q. and Inoki. R. (1989). Distribution of substance P and methionine-enkephalin in salivary glands and effect of chronic morphine treatment on levels of these peptides. ]pn 1. Phnrmncol. 50. 503-h.