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Control by calcium of protein discharge and membrane permeability to potassium in the rat lacrimal gland
Pergamon Press
Life Sciences Vol. 20, pp . 1905-1912, 1977 . Printed in the U.S .A .
CONTROL BY CALCIUM OF PROTEIN DISCHARGE AND MEMBRANE PERMEABILITY TO POTASSIUM IN THE RAT LACRIMAT. GLAND James W . Putney, Jr ., Ralph J . Parod and Susan H . Marier Department of Pharmacology Wayne State University School of Medicine Detroit, Michigan 48201 (Received in final form May 9,
1977)
Summary Discharge of protein from slices of rat exorbital lacrimal gland was stimulated by 10 -5 M carbachol. This response was blocked by -4 M atropine or by the omission of extracellular calcium In 10 . the latter case, secretion could be restored by the reintroduction of calcium ~~ the medium. Carbachol (10-5 M) also stimulated the This release of Rb (a marker for potassium) from the slices . effect was completely blocked by 10 -4 M atropine . The initial transient release of 86Rb was only partially inhibited by Ca removal, but the later sustained phase of release was completely blocked . Ae with protein secretion, this effect of Ca removal It is could be reversed by re-introduction of Ca to the medium . concluded that activation of cholinergic receptors in the lacrimal gland stimulates protein discharge and increases potassium permeability by mechanisms utilizing extracellular calcium ions . In mammals, the flow of tears comprised of water, electrolytes and proteins is controlled (at least in part) by nerve activity (1) . It is generally agreed that lacrimation ie initiated by stimulation of predominantly cholinergic innervation resulting in activation of muscariaic-type receptors within the gland (2-4) . Discharge of macromolecûles into tear fluid occurs _via exocytoais (5) and regulation of water flow is probably mediated by changes is ion transport and permeability (6-10) . Beyond this, little information is available regarding the cellular mechanisms by which cholinergic receptor activation initiates secretioy processes in lacrimal glands . By contrast, knowledge of the cellular mechanisms of salivary secretion has advanced at a considerable rate in the last decade, owing to the judicious employment of is vitro models for study (11-13) . The involvement of Ca ions in exocytoais due to cholinergic agoniste has been aptly demonstrated with slices of parotid gland. Thus, the release of the digestive enzyme, a-amylase, from parotid slices requires the presence of Ca in the bathing medium (13) . Cholinergic agoniste also increase the potassium permeability of parotid cells, and this response is similarly dependent on Activation of cholinergic receptors enhances extracellular Ca (14, 15) . Ca influx which triggers exocytoais se well as an increase in potassium permeability that presumably mediates secretion of water (13-16) . The purpose of this investigation was to examine the processes of protein discharge and ion permeability in the lacrimal gland as affected by the 14D5
Ca and Lacrimal Gland
1906
Vol . 20, No . 11, 1977
cholinergic agoniat carbachol and to determine whether Ca is required for these functions . Methode Slices of the rat exorbital gland were used because of its accessibility, its similarity in structure to the lacrimal gland in man (6), and its viability and ability to respond to cholinergic stimuli _in vitro (5) . Protein discharge with slices of the rat exorbital gland was measured with a method similar to that described previously (17) . Male Wiatar rata (150-250 g) were anesthetized with pentobarbital Na Slices were (50 mg/kg) and the exorbital glands removed and decapsulated . prepared with a Stadie-Rigga microtome and incubated is a physiological salt solution of the following composition (mM) : NaCl, 120 ; KC1, 5 ; CaC12, 3 ; MgC12 , 1 ; ß-hydroxybutyrate-Na, 5 ; tris(hydroxymethyl)aminomethane, 20 ; pH, 7 .40; gas phase, 100X 02 ; temperature, 37°C . The slices (~ 50 mg each, .5 mm thick) were each placed in a small basket of glass and nylon net or polyethylene and nylon net to facilitate transfer to various lutions . The -leucine for twenty slices were incubated in the above medium plus 10 uCi/ml minutes, and then in medium without 3H for 60 min to permit incorporation of Slices were then transferred the label and washout of uniacorporated 3H . through small volumes of media (5 ml) for short periods (5-LO min), and the Ia preliminary experiments, half of each sample released activity determined . was counted for total activity, and the other half made 5X in trichloroacetic acid, centrifuged, and the activity in the supernatant measured (TCA soluble) . With this protocol, virtually all of the label emerging from the slit s has Thus, the extra been incorporated into cellular protein (figure 1) . released is response to carbachol (a cholinergic agoniat) is a measure of In defense of this suppoprotein discharge, presumably by exocytosis (5) . aition, similar patterns can be obtained by measuring the release of peroxidase, an enzyme found almost exclusively in the secretory grannulea (5, 18) . In other experiments, the tissue was solubilized and assayed at the end of the experiments so that the data could be expressed as first-order rate is,ooo ~ ia,oooiz,ooo ~ u 0
m
io,oooe,ooo s,ooo
M
4,000
z,ooo° TrT~ 0 10 20 30 40 Time (minutes) FIG 1
50
60
Release of 3ü from rat exorbital slices . Slices were pulsed with 3F1-leucine for 20 min, washed for 60 min, and then transferred through a series of LO min incubations for 60 additional min . The incubation media were precipitated with TCA and the TCA precipitatee and soluble activities determined . Carbachol (10 -5 M) was present from 20-30 min. ~-i, TCA precipi table ; o o, TCA soluble . These results represent a single experiment ; two others were similar in result .
Vol . 20, No . 11, 1977
Ca and Lacrimal Gland
1907
coefficients (X/min) (14) . All experimental modifications of the media were begun at time zero which represents 60 minutes after the removal of the tissues from the 20 minute H-leucine pulse . Modulation of potassium permeability was investigated with the 86gb technique previously utilized is the parotid gland (14) . As before, 86Rb was used preferentially to 42 K because of the longer half-life (18 .7 days for 86Rb _vs 12 .4 . hr for 42K) . Slices were incubated in media containing the appropriate nuclide for 30 min, and then transferred through a series of 2 min incubations for determination of isotope release . The activity remaining in the tissue at the end of the experiment was also determined so that firstorder rate coefficients could be calculated (X/min) . BÇarbachol (10 -5 M) was added in each experiment where indicated . Efflux of bRb from lacrimal slices increases 1~ 2reapoase to cholinergic stimulating is a manner qualitatively similar to K, indicating the suitability of Rb as a marker for potassium movements is this tissue (figure 2) . 6
ar
N
y 4
ô 2
0
0
10
20
30
40
50
Release of 86Rb (o---o) or 42 K (~ ~) from rat exorbital slices . Slices were loaded with the appropriate nuclide for 30 min, and then transferred through a aeries of 2 min incubations for 40 or 50 min . The released activity during each interval and the activity remaining in the tissue at the end of the experiment ware used to calculate first-order rate coefficients . Carbachol (10 5 M) was added where indicated by the arrows . These results were obtained from a single experiment .
Time (minutes) FIG . 2 Results Figure 3 shows that protein secretion induced by carbachol in lacrimal slices apparently occurs through the same receptor mechanisms operable _in vivo since the response is quantitatively blocked by atropine . Protein dis charge was also dependent oa the presence of Ca ions is the bathing medium . -4 When the incubation medium contained no added Ca and 10 M ethylene glycolbis-(2-amiaoethyl)tetraacetic acid (EGTA), a specific Ca chelating agent, stimulation of secretion failed . This effect was reversible ; returning Ca to the medium after the addition of carbachol induced release of protein (figure 3) . In these experiments, summarized in figure 3 as the means of 5 determinations, variability was high, especially in comparison to the 86Rb data reported below. The average standard errors for each curve are included in the figure legend . However, the increases due to carbachol or due to the reintroduction of Ca were both statistically significant, based on paired c arisons (P . < .05) . Table 1 summarizes the mean paired differences in~ release due
1908
Vol . 20, No . 11, 1977
Ca and Lacrimal Gland
to carbachol and due to the reintroduction of Ca .
.80 ,
.60C
E
~ .40~
C N
0
0
10
20
30
40
Time (minutes) FIG. 3
50
so
Release of 3H protein from rat exorbital slices . The protocol was similar to that for fig . 1, except that the activity remaining in the tissue at the end of the experiment was determined so that first-order rate coefficients could be calculated . Each curve is the mean of 5 determinations . The protocol and average standard error for each experiment were : control, + .022 ; o-o, 10 -~ M carbachol 20-60 min _+ .131 ; ~-~, 10-~ M atropine 0-60 min, 10 -5 M carbachol 20-60 min, + .090 ; D -~, no added Ca + 10 -4 M EGTA 0-60 min, 10 -5 M carbachol 20-60 min, 3 .1 mM CaC12 40-60 min, + .090 .
Carbachol (10 -5 M) induces a substantial and significant increase in membrane permeability to potassium, as determined with the 86Rb technique (figure 4) . The kinetics of release appear biphasic in that an initial transient increase occurs followed by a lesser, sustained phase of release . Both phases are quantitatively blocked by atropine . However, only the sustained phase of release is eliminated when Ca is omitted from the medium, while the transient phase, at least in part, persists . As with exocytosis, the effect of Ca removal is reversible since the reintroduction of Ca The data in figure 4 increases release of 86Rb in the presence of carbachol . are means of 4 determinations . The standard errors averaged less than lOX of Increases due to carbachol in the the mean values and thus are not included . presence and absence of Ca, the increase due to Ca at 40 min, and the difference between the effect of carbachol in presence and absence of Ca were all statistically significant (P < .05) .
Vol . 20, No . 11, 1977
Ca and Lacrimal Gland
1909
TABLE 1 Paired differences in secretion rates of 3 H-protein due to carbachol and Ca in lacrimal slices . ~ Rate of 3H Release (X/mi n) Time~min)
25
30
35
40
45
50
55
60
10 -5 M Carbachol
.237 + .050
.278 + .067
.345 + .085
.418 + .089
.423 + .076
.410 + .084
.367 + .071
.367 + .074
3 .1 mM Ca
--
---
--
---
.098 + .029
.168 + .048
.228 + .081
.183 + .048
All values are means _+ S .E . and are taken from the same experiments summarized in Figure 3 . Each value is the mean paired difference from the rate obtained at the indicated time of the experiment and the value obtained just prior to the addition of 10-5 M carbachol (first row) or 3 .1 mM Ca (second row) . All values are significantly greater than zero (P < .05) with the exception of 3 .1 mM Ca, t ~ 55 min ( .05 < P < .1) . IOti
8-
2~
0
0
10
20
30
Time (minutes)
Fia . 4
40
50
Release of 86 Rb due to carbachol . The protocol was similar to that employed for figure 3. ~ ~, con trol ; o o, 10-5 M carbachol 18-$0 min ; 1 1, 10 - M atropine 12-50 min, 10 -5 M carbachol 18-50 min ; D ~, no added Ca + 10 -4 M EGTA 0-50 min, 10 -5 M carbachol 18-50 min, 3 .1 mM Ca 38-50 min . Each point is the mean from four separate experiments ; i .e ., four slices from each of four rata were subjected to the four protocols . The standard errors were generally less than lOX of the means .
1910
Ca and Lacrimal Gland
Vol . 20, No . 11, 1977
Discussion The data in figure 3 show that activation of cholinergic receptors in the lacrimal gland stimulates protein discharge by a mechanism dependent on extracellular Ca . In this respect, the lacrimal gland may resemble a number of other aecretory tissues in which Ca appears to mediate exocytosis (19) . Preaumably, the cholinergic recaptar whe.e activated perma.ts entry of Ca ions that initiate molecular events leading to membrane fusion and protein disi n support of this contention, data has been presented previously charge . suggesting that cholinergic agonista enhance uptake of 45 Ca by the rat exorbital gland (17, 20) . This same Ca influx may mediate the increased potassium permeability Since potassium is the only ion in the rat exorbital seen in figure 4 . lacrimal gland fluid concentrated relative to plasma (6), it can be argued that the effects of carbachol on potassium permeability reflect events associated with tranaepithelial water flow . A similar mechanism may be operable in the salivary glands (14) . The presence of a Ca-independent transient increase in K permeability also resembles the parotid gland and suggests that the molecular mechanism of control of K permeability is strikingly similar in these two exocrine glands . Recently, evidence has been obtained indicating that the transient phase of R release is mediated by Ca released from the same Ca transport site responsible for the sustained, Cadependent phase of release (21) . The question arises as to whether different cells in the lacrimal gland might serve to mediate the two separate functions of exocytosis and ion transport . Herzog and co-workers found stored aecretory material (peroxidase) in all acinar cells of the exorbital gland (5, 18) and implied that all acini respond equivalently to carbachol in terms of the appearance of exocytosic figures (5) . Further, the magnitude of the turnover of K+ shown in figure 4 suggests that a large fraction of total tissue K+ is involved making it These conunlikely that the response corresponds to a select few cells . siderations suggest (but do not prove) that at least some of the s~cretory cells in the gland perform both functions (protein secretion and K release) . It is intriguing that the secretory cells of the lacrimal gland utilize the same receptor and second messenger (Ca) to mediate two such diverse functions as exocytosis and ion (and presumably water) transport . Such a system would be expected to produce a synchronous flow of water whenever protein secretion occurs - an apparently advantageous consequence . Acknowledgements This study was supported in part by grants from the NIH #DE 04067, EY 01978 and GM 07176 . References 1. 2. 3. 4. 5. 6.
S .Y . BOTELHO, Scientific American , _211 : 78-86 (1964) . M.S . ARENSON and H. WILSON, _J . Phyeiol . London , 217 : 201212 (1971) . S .Y . BOTELHO, M. HISADA and N . FUENMAYOR, Arch . Opthamol ., _76 : 581-588 (1966) . J .M . ELSBY and H . WILSON, Brit . _J . Pharmacol . Chemother ., 29 : 1-7 (1967) . 70 : 692-706 V. HERZOG, H. SIES and F . MILLER, _J . Cell Biol . (1976) . J . ALEXANDER, E.W . VAN LENNEP and J .A . YOUNG, Arch . Ges . Phyeiol ., 337 : 299-310 (1972) .
Vol . 20, No . 11, 1977
7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 .
16 . 17 . 18 . 19 . 20 . 21 .
Ca and Lacrimal Gland
M. HISADA, and S .Y . BOTELHO, Am . J . Physiol ., 214 : 1262-1267 (1968) . T . KIKKAWA, Japan J . Ophthamol., 14 : 25-40 (1970) . H . YOSHIMURA and K.HOSOKAWA, Japaa J . Physiol ., 13 : 303-318 (1963) . S .Y . BOTELHO aad E .V . MARTINEZ, _Am. _J . Physiol., 225 : 606-609 (1973) . M. SCHRAMM and Z . SELINGER, _J . Cyclic Nucleotide _Res ., _1 : 181192 (1975) . Z . SELINGER, in Calcium Transport in Contraction and Secretion ., Drabikowski, and (edit . by Carafoli, E., Clementi, F Margreth, A.), 139-146 (North Holland, Amsterdam, 1975) . B .A . LESLIE, J .W . PUTNEY, JR, and J .M . SHERMAN, _J . Physiol . London , 260 : 351-370 (1976) . J .W . PUTNEY, JR ., _J . Pharmacol . Fx~. Ther ., 198 : 375-384 (1976) . M. SCHRAMM and Z . SELINGER, in Advances in Cytopharmacology , vol . 2 (edit . by Ceccarelli, B ., Clementi, F. and Meldolesi, J .), 29-32 (Raven, New York, 1974) . J .W . PUTNEY, JR ., _J . Pharmacol . Exx~ . Ther ., _199 : 526-537 (1976) . G . KERYER and B . ROSSIGNOL, Am . _J . Physiol ., _230 : 99-104 (1976) . 30 : 235-246 (1972) . V . HERZOG and F . MILLER, Hietochemie , R .P . RUBIN, Calcium and the Secretioy Process (Plenum, New York,
W.
1974) . R .H . DREISBACH, _Am. _J . Physiol., 207 : 1015-1020 (1964) . London , (in the press) . J .W . PUTNEY, JR ., J . Physiol .
1911
Life Sciences Vol. 20, pp . 1905-1912, 1977 . Printed in the U.S .A .
CONTROL BY CALCIUM OF PROTEIN DISCHARGE AND MEMBRANE PERMEABILITY TO POTASSIUM IN THE RAT LACRIMAT. GLAND James W . Putney, Jr ., Ralph J . Parod and Susan H . Marier Department of Pharmacology Wayne State University School of Medicine Detroit, Michigan 48201 (Received in final form May 9,
1977)
Summary Discharge of protein from slices of rat exorbital lacrimal gland was stimulated by 10 -5 M carbachol. This response was blocked by -4 M atropine or by the omission of extracellular calcium In 10 . the latter case, secretion could be restored by the reintroduction of calcium ~~ the medium. Carbachol (10-5 M) also stimulated the This release of Rb (a marker for potassium) from the slices . effect was completely blocked by 10 -4 M atropine . The initial transient release of 86Rb was only partially inhibited by Ca removal, but the later sustained phase of release was completely blocked . Ae with protein secretion, this effect of Ca removal It is could be reversed by re-introduction of Ca to the medium . concluded that activation of cholinergic receptors in the lacrimal gland stimulates protein discharge and increases potassium permeability by mechanisms utilizing extracellular calcium ions . In mammals, the flow of tears comprised of water, electrolytes and proteins is controlled (at least in part) by nerve activity (1) . It is generally agreed that lacrimation ie initiated by stimulation of predominantly cholinergic innervation resulting in activation of muscariaic-type receptors within the gland (2-4) . Discharge of macromolecûles into tear fluid occurs _via exocytoais (5) and regulation of water flow is probably mediated by changes is ion transport and permeability (6-10) . Beyond this, little information is available regarding the cellular mechanisms by which cholinergic receptor activation initiates secretioy processes in lacrimal glands . By contrast, knowledge of the cellular mechanisms of salivary secretion has advanced at a considerable rate in the last decade, owing to the judicious employment of is vitro models for study (11-13) . The involvement of Ca ions in exocytoais due to cholinergic agoniste has been aptly demonstrated with slices of parotid gland. Thus, the release of the digestive enzyme, a-amylase, from parotid slices requires the presence of Ca in the bathing medium (13) . Cholinergic agoniste also increase the potassium permeability of parotid cells, and this response is similarly dependent on Activation of cholinergic receptors enhances extracellular Ca (14, 15) . Ca influx which triggers exocytoais se well as an increase in potassium permeability that presumably mediates secretion of water (13-16) . The purpose of this investigation was to examine the processes of protein discharge and ion permeability in the lacrimal gland as affected by the 14D5
Ca and Lacrimal Gland
1906
Vol . 20, No . 11, 1977
cholinergic agoniat carbachol and to determine whether Ca is required for these functions . Methode Slices of the rat exorbital gland were used because of its accessibility, its similarity in structure to the lacrimal gland in man (6), and its viability and ability to respond to cholinergic stimuli _in vitro (5) . Protein discharge with slices of the rat exorbital gland was measured with a method similar to that described previously (17) . Male Wiatar rata (150-250 g) were anesthetized with pentobarbital Na Slices were (50 mg/kg) and the exorbital glands removed and decapsulated . prepared with a Stadie-Rigga microtome and incubated is a physiological salt solution of the following composition (mM) : NaCl, 120 ; KC1, 5 ; CaC12, 3 ; MgC12 , 1 ; ß-hydroxybutyrate-Na, 5 ; tris(hydroxymethyl)aminomethane, 20 ; pH, 7 .40; gas phase, 100X 02 ; temperature, 37°C . The slices (~ 50 mg each, .5 mm thick) were each placed in a small basket of glass and nylon net or polyethylene and nylon net to facilitate transfer to various lutions . The -leucine for twenty slices were incubated in the above medium plus 10 uCi/ml minutes, and then in medium without 3H for 60 min to permit incorporation of Slices were then transferred the label and washout of uniacorporated 3H . through small volumes of media (5 ml) for short periods (5-LO min), and the Ia preliminary experiments, half of each sample released activity determined . was counted for total activity, and the other half made 5X in trichloroacetic acid, centrifuged, and the activity in the supernatant measured (TCA soluble) . With this protocol, virtually all of the label emerging from the slit s has Thus, the extra been incorporated into cellular protein (figure 1) . released is response to carbachol (a cholinergic agoniat) is a measure of In defense of this suppoprotein discharge, presumably by exocytosis (5) . aition, similar patterns can be obtained by measuring the release of peroxidase, an enzyme found almost exclusively in the secretory grannulea (5, 18) . In other experiments, the tissue was solubilized and assayed at the end of the experiments so that the data could be expressed as first-order rate is,ooo ~ ia,oooiz,ooo ~ u 0
m
io,oooe,ooo s,ooo
M
4,000
z,ooo° TrT~ 0 10 20 30 40 Time (minutes) FIG 1
50
60
Release of 3ü from rat exorbital slices . Slices were pulsed with 3F1-leucine for 20 min, washed for 60 min, and then transferred through a series of LO min incubations for 60 additional min . The incubation media were precipitated with TCA and the TCA precipitatee and soluble activities determined . Carbachol (10 -5 M) was present from 20-30 min. ~-i, TCA precipi table ; o o, TCA soluble . These results represent a single experiment ; two others were similar in result .
Vol . 20, No . 11, 1977
Ca and Lacrimal Gland
1907
coefficients (X/min) (14) . All experimental modifications of the media were begun at time zero which represents 60 minutes after the removal of the tissues from the 20 minute H-leucine pulse . Modulation of potassium permeability was investigated with the 86gb technique previously utilized is the parotid gland (14) . As before, 86Rb was used preferentially to 42 K because of the longer half-life (18 .7 days for 86Rb _vs 12 .4 . hr for 42K) . Slices were incubated in media containing the appropriate nuclide for 30 min, and then transferred through a series of 2 min incubations for determination of isotope release . The activity remaining in the tissue at the end of the experiment was also determined so that firstorder rate coefficients could be calculated (X/min) . BÇarbachol (10 -5 M) was added in each experiment where indicated . Efflux of bRb from lacrimal slices increases 1~ 2reapoase to cholinergic stimulating is a manner qualitatively similar to K, indicating the suitability of Rb as a marker for potassium movements is this tissue (figure 2) . 6
ar
N
y 4
ô 2
0
0
10
20
30
40
50
Release of 86Rb (o---o) or 42 K (~ ~) from rat exorbital slices . Slices were loaded with the appropriate nuclide for 30 min, and then transferred through a aeries of 2 min incubations for 40 or 50 min . The released activity during each interval and the activity remaining in the tissue at the end of the experiment ware used to calculate first-order rate coefficients . Carbachol (10 5 M) was added where indicated by the arrows . These results were obtained from a single experiment .
Time (minutes) FIG . 2 Results Figure 3 shows that protein secretion induced by carbachol in lacrimal slices apparently occurs through the same receptor mechanisms operable _in vivo since the response is quantitatively blocked by atropine . Protein dis charge was also dependent oa the presence of Ca ions is the bathing medium . -4 When the incubation medium contained no added Ca and 10 M ethylene glycolbis-(2-amiaoethyl)tetraacetic acid (EGTA), a specific Ca chelating agent, stimulation of secretion failed . This effect was reversible ; returning Ca to the medium after the addition of carbachol induced release of protein (figure 3) . In these experiments, summarized in figure 3 as the means of 5 determinations, variability was high, especially in comparison to the 86Rb data reported below. The average standard errors for each curve are included in the figure legend . However, the increases due to carbachol or due to the reintroduction of Ca were both statistically significant, based on paired c arisons (P . < .05) . Table 1 summarizes the mean paired differences in~ release due
1908
Vol . 20, No . 11, 1977
Ca and Lacrimal Gland
to carbachol and due to the reintroduction of Ca .
.80 ,
.60C
E
~ .40~
C N
0
0
10
20
30
40
Time (minutes) FIG. 3
50
so
Release of 3H protein from rat exorbital slices . The protocol was similar to that for fig . 1, except that the activity remaining in the tissue at the end of the experiment was determined so that first-order rate coefficients could be calculated . Each curve is the mean of 5 determinations . The protocol and average standard error for each experiment were : control, + .022 ; o-o, 10 -~ M carbachol 20-60 min _+ .131 ; ~-~, 10-~ M atropine 0-60 min, 10 -5 M carbachol 20-60 min, + .090 ; D -~, no added Ca + 10 -4 M EGTA 0-60 min, 10 -5 M carbachol 20-60 min, 3 .1 mM CaC12 40-60 min, + .090 .
Carbachol (10 -5 M) induces a substantial and significant increase in membrane permeability to potassium, as determined with the 86Rb technique (figure 4) . The kinetics of release appear biphasic in that an initial transient increase occurs followed by a lesser, sustained phase of release . Both phases are quantitatively blocked by atropine . However, only the sustained phase of release is eliminated when Ca is omitted from the medium, while the transient phase, at least in part, persists . As with exocytosis, the effect of Ca removal is reversible since the reintroduction of Ca The data in figure 4 increases release of 86Rb in the presence of carbachol . are means of 4 determinations . The standard errors averaged less than lOX of Increases due to carbachol in the the mean values and thus are not included . presence and absence of Ca, the increase due to Ca at 40 min, and the difference between the effect of carbachol in presence and absence of Ca were all statistically significant (P < .05) .
Vol . 20, No . 11, 1977
Ca and Lacrimal Gland
1909
TABLE 1 Paired differences in secretion rates of 3 H-protein due to carbachol and Ca in lacrimal slices . ~ Rate of 3H Release (X/mi n) Time~min)
25
30
35
40
45
50
55
60
10 -5 M Carbachol
.237 + .050
.278 + .067
.345 + .085
.418 + .089
.423 + .076
.410 + .084
.367 + .071
.367 + .074
3 .1 mM Ca
--
---
--
---
.098 + .029
.168 + .048
.228 + .081
.183 + .048
All values are means _+ S .E . and are taken from the same experiments summarized in Figure 3 . Each value is the mean paired difference from the rate obtained at the indicated time of the experiment and the value obtained just prior to the addition of 10-5 M carbachol (first row) or 3 .1 mM Ca (second row) . All values are significantly greater than zero (P < .05) with the exception of 3 .1 mM Ca, t ~ 55 min ( .05 < P < .1) . IOti
8-
2~
0
0
10
20
30
Time (minutes)
Fia . 4
40
50
Release of 86 Rb due to carbachol . The protocol was similar to that employed for figure 3. ~ ~, con trol ; o o, 10-5 M carbachol 18-$0 min ; 1 1, 10 - M atropine 12-50 min, 10 -5 M carbachol 18-50 min ; D ~, no added Ca + 10 -4 M EGTA 0-50 min, 10 -5 M carbachol 18-50 min, 3 .1 mM Ca 38-50 min . Each point is the mean from four separate experiments ; i .e ., four slices from each of four rata were subjected to the four protocols . The standard errors were generally less than lOX of the means .
1910
Ca and Lacrimal Gland
Vol . 20, No . 11, 1977
Discussion The data in figure 3 show that activation of cholinergic receptors in the lacrimal gland stimulates protein discharge by a mechanism dependent on extracellular Ca . In this respect, the lacrimal gland may resemble a number of other aecretory tissues in which Ca appears to mediate exocytosis (19) . Preaumably, the cholinergic recaptar whe.e activated perma.ts entry of Ca ions that initiate molecular events leading to membrane fusion and protein disi n support of this contention, data has been presented previously charge . suggesting that cholinergic agonista enhance uptake of 45 Ca by the rat exorbital gland (17, 20) . This same Ca influx may mediate the increased potassium permeability Since potassium is the only ion in the rat exorbital seen in figure 4 . lacrimal gland fluid concentrated relative to plasma (6), it can be argued that the effects of carbachol on potassium permeability reflect events associated with tranaepithelial water flow . A similar mechanism may be operable in the salivary glands (14) . The presence of a Ca-independent transient increase in K permeability also resembles the parotid gland and suggests that the molecular mechanism of control of K permeability is strikingly similar in these two exocrine glands . Recently, evidence has been obtained indicating that the transient phase of R release is mediated by Ca released from the same Ca transport site responsible for the sustained, Cadependent phase of release (21) . The question arises as to whether different cells in the lacrimal gland might serve to mediate the two separate functions of exocytosis and ion transport . Herzog and co-workers found stored aecretory material (peroxidase) in all acinar cells of the exorbital gland (5, 18) and implied that all acini respond equivalently to carbachol in terms of the appearance of exocytosic figures (5) . Further, the magnitude of the turnover of K+ shown in figure 4 suggests that a large fraction of total tissue K+ is involved making it These conunlikely that the response corresponds to a select few cells . siderations suggest (but do not prove) that at least some of the s~cretory cells in the gland perform both functions (protein secretion and K release) . It is intriguing that the secretory cells of the lacrimal gland utilize the same receptor and second messenger (Ca) to mediate two such diverse functions as exocytosis and ion (and presumably water) transport . Such a system would be expected to produce a synchronous flow of water whenever protein secretion occurs - an apparently advantageous consequence . Acknowledgements This study was supported in part by grants from the NIH #DE 04067, EY 01978 and GM 07176 . References 1. 2. 3. 4. 5. 6.
S .Y . BOTELHO, Scientific American , _211 : 78-86 (1964) . M.S . ARENSON and H. WILSON, _J . Phyeiol . London , 217 : 201212 (1971) . S .Y . BOTELHO, M. HISADA and N . FUENMAYOR, Arch . Opthamol ., _76 : 581-588 (1966) . J .M . ELSBY and H . WILSON, Brit . _J . Pharmacol . Chemother ., 29 : 1-7 (1967) . 70 : 692-706 V. HERZOG, H. SIES and F . MILLER, _J . Cell Biol . (1976) . J . ALEXANDER, E.W . VAN LENNEP and J .A . YOUNG, Arch . Ges . Phyeiol ., 337 : 299-310 (1972) .
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7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 .
16 . 17 . 18 . 19 . 20 . 21 .
Ca and Lacrimal Gland
M. HISADA, and S .Y . BOTELHO, Am . J . Physiol ., 214 : 1262-1267 (1968) . T . KIKKAWA, Japan J . Ophthamol., 14 : 25-40 (1970) . H . YOSHIMURA and K.HOSOKAWA, Japaa J . Physiol ., 13 : 303-318 (1963) . S .Y . BOTELHO aad E .V . MARTINEZ, _Am. _J . Physiol., 225 : 606-609 (1973) . M. SCHRAMM and Z . SELINGER, _J . Cyclic Nucleotide _Res ., _1 : 181192 (1975) . Z . SELINGER, in Calcium Transport in Contraction and Secretion ., Drabikowski, and (edit . by Carafoli, E., Clementi, F Margreth, A.), 139-146 (North Holland, Amsterdam, 1975) . B .A . LESLIE, J .W . PUTNEY, JR, and J .M . SHERMAN, _J . Physiol . London , 260 : 351-370 (1976) . J .W . PUTNEY, JR ., _J . Pharmacol . Fx~. Ther ., 198 : 375-384 (1976) . M. SCHRAMM and Z . SELINGER, in Advances in Cytopharmacology , vol . 2 (edit . by Ceccarelli, B ., Clementi, F. and Meldolesi, J .), 29-32 (Raven, New York, 1974) . J .W . PUTNEY, JR ., _J . Pharmacol . Exx~ . Ther ., _199 : 526-537 (1976) . G . KERYER and B . ROSSIGNOL, Am . _J . Physiol ., _230 : 99-104 (1976) . 30 : 235-246 (1972) . V . HERZOG and F . MILLER, Hietochemie , R .P . RUBIN, Calcium and the Secretioy Process (Plenum, New York,
W.
1974) . R .H . DREISBACH, _Am. _J . Physiol., 207 : 1015-1020 (1964) . London , (in the press) . J .W . PUTNEY, JR ., J . Physiol .
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