Calium, prostaglandins and the phosphatidylinositol effect in exocrine gland cells

Cell Calcium 2:

561-571,

1981

CACLIUM, PROSTAGLANOINS AND THE PHOSPHATIDYLINOSITOL EFFECT IN EXOCRINE GLAND CELLS James W. Putney, Jr., McKinney

Linda M. Dewitt, Peter C. Hoyle and Jerry S.

Department of Pharmacology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298 USA. (Reprint requests to JWP) ABSTRACT The hypothesis that arachidonic acid metabolism might be involved in Ca-mobilization mechanisms in exocrine gland cells was investigated. Arachidonate (JO-4M) failed to stimulate protein secretion from slices of pancreas, parotid or lacrimal glands and failed to stimulate 86Rb efflux from parotid or lacrimal glands. The stimulation of protein secretion (all three glands) or 86Rb efflux (parotid and lacrimal glands) by appropriate secretagogues was unaffected by lo-6M indomethacin. Eicosatetraynoic acid (2xlO-SM) inhibited 86Rb efflux due to carbachol but not that due to physalaemin or ionomycin. Nordihydroguaiaretic acid inhibited lacrimal and parotid gland responses only at high (JO-4M) concentration. Collectively, these results argue against an obligatory role for arachidonate metabolites in Ca-mediated responses of these exocrine glands. In the exocrine glands activation by neurotransmitters (or analogs) of r ceptors that mobilize cellular Ca also stimulates the incorporation of 35PO4 into phosphatidylinositol (l-3). Michell (4,5) has suggested that in some manner this alteration in phospholipid metabolism may be functionally responsible for the opening of surface membrane Ca gates which presumably precedes the expression of a number of Ca-mediated responses by the exocrine cell. That this reaction probably preceeds Ca mobilization is deduced primarily from two experimental observations. First, receptor activation of phosphatidylinositol turnover is not prevented by Ca omission (6-8). Second, the effect is not mimicked by the divalent cationophore A-23187, while other effects of receptor activation are mimicked by this compound (7-9). There has also been some speculation as to the manner in which altered phosphatidylinositol metabolism might be involved in the Ca-gating mechanism (10-14). One such hypothesis suggests that receptor activation may lead to phosphatidylinositol breakdown which in turn leads to the release of free arachidonate (13,111). As free arachidonate is generally believed to be the rate-limiting substrate for prostaglandin 561

synthesis (15), the resulting prostaglandins might act to mobilize Ca or might act in concert with Ca (13,14). There is evidence for this hypothesis for the mouse pancreas, where exogenous arachidonate and prostaglandins can stimulate amylase release (13). The effects of arachidonate, carbachol, caerulein and pancreozmin were all antagonized by sub-micromolar concentrations of indomethacin (13), a potent cyclooxygenase inhibitor (15). Additionally, recent reports have demonstrated stimulation by acetylcholine of prostaglandin E synthesis in mouse pancreas (16,17). The purpose of this study was to examine the general applicability of this hypothesis by investigating the effects of arachidonate and substances that inhibit prostaglandin formation in two other exocrine tissues that show a prominent phosphatidylinositol turnover - the rat parotid and lacrimal glands. METHODS All experiments were performed with thin slices of parotid gland, exorbital lacrimal gland or pancreas from male Wistar-derived rats weighSng 125-200 g. The rocedures for preparing and incubatin the slices and for measuring ! 6Rb efflux have been described (18,193 . The basic Ringer medium used in these studies had the following composition (mM): NaCl, 120; KCl, 5; CaC12, 3; MgC12, 1; Na-B-hydroxybutyrate, 5; tris-(hydroxymethyl) aminomethane, 20. The media were titrated to pH of 7.40 at 370C and gassed with 100% 02. For proiein secretion studies a "pulse-chase" radiolabe!lIng procedure wfth H-leucine was employed. Statistical reproduciblllty was greatly enhanced if a secretagogue was included during the pulse and during the beginning of the chase period. The procedure employed was as follows. Slices were incubated in Ringer containing 3H-leucine (1.25 &i/ml) and an agonist, the nature of which depended on the tissue. As previously described (20), the tissue was suspended in the medium in a basket of polyethylene and nylon net which facilitated the transfer through various incubation conditions. After the 10 min "pulse" period, a one hour "chase" period was employed, which was comprised of three sequential incubations of 20 min each in Ringer containing 2 mM glutamine and 1% of Gibco 100X concentrates of essential and non-essenttal ammino acids. In order to assure a prompt and reproducible end to the stimulation during the chase period, the agonist was included in the media durin the first 20 min chase period and was deleted thereafter, An approprBate antagonist was included during the second 20 min incubation. For the tissues used in this study, the agonists and antagonists (respectively) were: parotid, isoproterenol (lo-6M) and propranolol (lo-6M); lacrimal, epine hrine (lo-5M) and phentolamine (lo-5M); pancreas, carbachol (lo-6MP and atropine (lo-6M). Following the one hour chase period, the tissues were transferred through a series of 10 min incubations (without amino acids) for 80 mtn. During the last 30 min, a secretagogue was included in the Ringer medium. I’n each case, the secretagogue employed was one known to act through a different receptor than the agents used during the pulse-chase

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After the experiment, the radioactivity released into the procedure. medium during each of the 10 min incubations (found to be > 99% insoluble in 5% trichloroacetic acid) and that remaining in the tissue were determined, and apparent first order rate coefficients were calculated as described previously (21).

RESULTS Exogenously applied arachidonic acid, at lo-4M or 10'5M, did not stimulate efflux of 86Rb from parotid or lacrimal gland slices (Fig. 1). The arachidonate was added (at 20 min) in the absence of Ca, and 6 min later, 3.0 mM Ca was added to the Ringer. No significant response was seen on addition of arachidonate or on addition of Ca, despite the fact that the experimental protocol provides fresh arachidonic acid every 2 min. In the continued presence of arachidonic acid and Ca, a normal response to carbachol could be obtained (at 30 min, Fig. 1).

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Fig. 1. Failure of exogeneous arachidonic acid to affect 86Rb efflux from rat lacrimal (left) or parotid (right) gland slices. Initially the media contained no added Ca and lo-4M EGTA. Arachidonate (0, 10e5M o, lo-4M) was present from 20 min (1) to 40 min, 3.0 mM Ca was added from 26 min (2) to 40 min, and lo-5M carbachol was added from 30 min (3) to 40 min. The figure summarizes the mean of four experiments with each tissue and with each arachidonate concentration. S.E.'s average 10% of the means.

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The 86Rb efflux response of lacrimal and parotid gland slices to carbachol is shown in Fig. 2. Carbachol (lo-6M) was added to the Ringer from 20-40 min. Ca was omitted from O-30 min and subsequently restored to 3.0 mM from 30-40 min. This protocol allows for separate analysis of the two phases of the 86Rb efflux response, the first being due to Ca release and the second to Ca influx (18,22). Also shown in Fig. 2 are the results of simultaneous paired experiments for which the Ringer contained lo-6M indomethacin. For both the parotid and lacrimal glands, no significant effect of indomethacin was detected. In other experiments (not shown) a higher concentration of indomethacin (lo-4M) caused a slight (ca. 30%), but significant inhibition of both phases.

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Fi . 2. Release of 86Rb from slices of rat lacrimal (left) or parotid right) glands, in the presence and absence of lo-5M indomethacin. Initially, the media contained no added Ca and lo-4M EGTA. Carbachol was added for 20-40_min and CaC12 (3.0 mM) was added from 30(10-W 40 min l,control; o, + lo-SM indomethacin. The figure summarizes four S.E.'s experiments with and without indomethacin for each tissue. averaged 10% of the means.

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The effects of indomethacin (10D5M) and exogenous arachidonate (lo-4M) on protein secretion from the lacrimal and parotid gland were also determined. The results of these experiments are shown in Fig. 3. Indomethacin did not significantly affect the rate of protein release due to carbachol. The addition of exogeneous arachidonic acid did not stimulate release of protein.

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Fig. 3. Effects of exogenous arachidonate and indomethacin on 3H-protein release from slices of rat lacrimal (left) and parotid (right) glands. Tissue proteins were pulse-chase labelled with 3H-leucine as described in Methods. A, lo-4M arachidonate, 50-80 min. l , 10-5 carbachol, 50-80 min; o, lo-5M indomethacin, O-80 min, lo-5M carbachol, 50-80 min. The figure summarizes four experiments with each tissue and under each experimental condition. S.E.'s averaged 20% of the means.

The effects on86 Rb efflux of 5,8,11,14-eicosatetraynoic acid (ETYA), an analog of arachidonic acid that inhibits both the lipoxygenase and cycle-oxygenase enzymes, were also examined (Fig. 4). In the lacrimal gland a partSa (slightly greater than 50%) inhibition of the second phase of the response was observed. For the parotid gland, a slight (less than 50%) inhibition that was statistically significant was obtaqned (Fig. 4). However, ETYA had no significant effects agal'nstphysalaemin or the Ca-ionophore, ionomycin, or against carbachol when the concentration was increased to lo-4M (Fig. 5).

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Fig. 4. Effect of ETYA on release of 86Rb from rat lacrimal (left) and parotid (right) gland slices. The protocol was as for Fig. 2. l , con+ trol; o, + 2x10-5M ETYA.

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Fig. 5. Effect of ETYA on release of 86Rb from rat parotid gland slices. The protocol was as for Fig. 4 (right) except that the agonists employed were: left, 10'4M carbachol; center IO-7H physalaemin; right 2.67 x lo-CM ‘ionomycin; e, control, o; + 2x10"5M ETYA.

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Figure 6 summarizes experiments on the effects of arachidonate and indomethaain on protein secretion from rat pancreas slices. Arachidonate (lo-4M) did not induce protein release, and the response to lo-7M caerulein was apparently not affected by lo-5M indomethacin.

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Fig. 6. Effects of exogenous arachidonate and indomethacin on 3H-protein release from slices of rat pancreas. The protocol was as for Fig. 3. A, lo-4M arachidonate, 50-80 min (n=6); l , lo-7M caerulein, 50-80 min (n=Z); o, lo-5M indomethacin, O-80 min, 10m7M caerulein, 50-80 min (n=Z).

In experiments not shown but similar to those shown in Fig. 2-4 the effects of nordihydroguaiaretic acid (NDGA; a lipoxygenase inhibitor) were ascertained. A concentration of lo-4M NDGA (but not lo-5M) caused a partial inhibition of responses of both parotid and lacrimal slices to both carbachol and A-23187, a calcium ionophore. By using a protocol similar to that shown in Fig. 1, it was shown that in the parotid gland prostaglandins E2, 12, F2 and D (at 10e5M) did not stimulate 46 Rb efflux in the presence ov absent g of Ca. In the lacrimal, only prostaglandins E2 and 12 were tested, again with negative results.

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DISCUSSION The results of these experiments do not support any direct role of arachidonate metabolites in Ca mobilization or in other aspects of stimulus-secretion coupling or stimulus-permeability coupling in the rat parotid gland. It seems unlikely that such products are important for the lacrimal gland as well. The strongest evidence is the failure of rather high concentrations of arachidonate to mimic the effects of receptor stimulation. In most systems studied thus far, addition of this ratelimiting substrate results in prostaglandin formation and the expression of appropriate tissue responses, if such exist (15,23,24).

In the parotid gland, consistent with the lack of biological activity of exogenous arachidonate was the failure of indomethacin, ETYA or NDGA (when applied in appropriate concentrations) to affect Ca gating mechanisms. NDGA was found to be inhibitory in the lacrimal and parotid at 'IO-4M, but not at lo-5M. As the concentration required for lipoxygenase inhibition is in the uM range (25), these effects are more likely due to other less specific actions of this reactive antioxidant. The effects of ETYA on Ca-gating in the lacrimal gland may also be due to actions other than inhibition of arachidonate metabolism. Otherwise, it is difficult to rationalize the failure of indomethacin, a cyclo-oxygenase inhibitor, or NDGA (in appropriate concentrations), a lipoxygenase inhibitor, to produce similar effects. Additionally, high concentrations of arachidonate failed to induce either 86Rb efflux or protein secretion. Since in the parotid gland, the inhibitory effects of ETYA were specific for the muscarinic agonist, carbachol, and since the inhibition could be surmonted by increasing the concentration of carbachol, it is quite possible that ETYA has some receptor blocking activity. While the actions of ETYA and NDGA in the pancreas were not examined, an attempt was made to reproduce at least the reported results of Marshall, Dixon and Hokin (13). These investigators observed almost complete (ca. 80%) inhibition of agonist stimulated amylase secretion by lo-5M indomethacin; in our system this concentration produced no effect. They (13) also reported a doubling of amylase secretion by lo-4M arachidonate; in this study no response was obtained. The data obtained here are similar to those reported previously by Chauvelot et al (26). Recently, Marshall, Dixon and Hokin (personal communication) have also observed that slices of rat pancreas do not show any apparent involvement of prostaglandins. The difference appears due to the fact that for the earlier studies, the whole mouse pancreas (rather than slices) was employed. This and other experimental results lead them to conclude that prostaglandins formed in the acinar cells may act on the ductal cells to enhance the transport of enzyme through the ducts. Such an hvoothesis can readily explain the apparent functional involvement of prbstaglandins in the-exocrine pancreas. In addition to the exocrine pancreas, however, the phosphatidylinositol turnover is a response ubiquitous for Ca-mob ilizing receptors (includ ing those in the parotid and

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lacrimal glands) regardless of the end response of the tissue (4,5). Therefore, that the fundamental function of this reaction may be to effect Camobilization (4,5) remains a tenable hypothesis. Since we observed no apparent involvement of arachidonate metabolites in the Ca-mediated responses of our preparations, we conclude that an obligatory role for arachidonate metabolites in this primary Ca-mobilizing mechanism seems unlikely.

ACKNOWLEDGEMENTS

This study was supported by grants from NIH # DE-05764 and EY-03533. We thank B. Leslie, Drs. J. Poggioli, S. Weiss, R. Rubin and S. Laychock for helpful criticisms and suggestions.

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20.

(1976). a-AdreLeslie, B.A., Putney, J.W.,Jr. and Sherman, J.M. nergic, B-adrenergic and cholinergic mechanisms for amylase secretion by rat parotid gland in vitro. J. Physiol. (Lond.) 260, 351370.

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