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Studies on chemical mechanisms of the action of neurotransmitters and hormones
ARCHIVES
OF
Studies
BIOCHEMISTRY
AND
on Chemical
BIOPHYSICS
124, 280-284 (1968)
Mechanisms
of the Action
of Neurotransmitters
and Hormones II. Increased
Incorporation
of 32P into Phosphatides
Response to Pancreozymin
or Acetylcholine MABEL
Departments
of
Psychiatry
Received
in Pigeon
Pancreas
Slices’
R. HOKIN
and Physiological Chemistry, Madison, Wisconsin 53706
August
as a Second, Adaptive
31, 1967; accepted
University
September
of Wisconsin,
7, 1967
The concentrations of pancreozymin or acetylcholine necessary to give hormoneresponsive amylase extrusion, and those necessary to give hormone-responsive increases in 32P incorporation into phosphatides in pigeon pancreas slices, have been investigated. Low concentrations of pancreozymin or acetylcholine stimulated amylase extrusion with little or no effect on 32P incorporation into phosphatides. High concentrations of pancreozymin gave increased incorporation of 32P into phosphatides without increasing amylase extrusion above that observed with lower concentrations of hormone. Amylase extrusion in response to 10m4or lo+ M acetylcholine (plus eserine) was less than in response to lower concentrations of acetylcholine, but a2P incorporation into phosphatides was much higher with the higher concentrations of acetylcholine. These results provide further evidence that increased synthesis or turnover of phosphatidylinositol, phosphatidic acid, phosphatidylethanolamine, and phosphatidylcholine in response to hormone is not a secondary consequence of zymogen granule extrusion (reverse pinocytosis). In view of the requirement for higher concentrations of hormone than those necessary to elicit amylase extrusion, it is suggested that the increased incorporation of 32P into phosphatides may represent a second, adaptive response to hormone-part of a gearing up of the cell to meet greater demands for the secretion of zgmogens than it had previously been meeting.
tidylinositol, phosphatidic acid, and phosphatidylethanolamine (4-6). The present work compares the concentrations of hormone necessary to elicit enzyme extrusion and those necessary to elicit increased 32P incorporation into individual phosphatides.
Extrusion of amylase and other digestive enzymes in pigeon pancreas slices can be elicited by either pancreozymin or acetylcholine (l-3), and each of these hormones2 has also been shown to give rise to an increased incorporation of 32P into several phosphatides in the tissue; the major increase in both total 32Pincorporation and in net specific activity occurred in phospha-
EXPERIMENTAL
PROCEDURE
Pigeon pancreas slices were prepared and incubated in Krebs-Henseleit bicarbonate medium (7), with added glucose, as described previously (5). At the end of the incubation period, the tissue was removed from the incubation flask and was rapidly frozen in a test tube by plunging the tube into a dry ice-alcohol bath. The tissues were then homogenized from the frozen state in 5% trichloroacetic acid, and lipids were extracted as described elsewhere (5). The lipids were separated by chromatography on silicic-acid impregnated paper (8).
1 This investigation was supported by grants NB-01730 and NB-06745-01 from the National Institutes of Health, U.S. Public Health Service. 2 For the sake of brevity, the word “hormone” is used to refer to both pancreozymin and acetylcholine, although it is realized that acetylcholine is a neurotransmitter, and not a hormone in the classical sense. 280
I’ANCREOZYMIN
AND
281
ACETYLCHOLINE
Amylase activity was determined on aliquots of the incubation medium by a slight modification of the method of Smith and Roe (9). Pancreozymin was a commercial preparation of Cesekin (Vitrum Pharmaceutical Co., Stockholm, Sweden). The activity is stated in units which refer to Ivy dog units as standardized by the supplier. In experiments in which acetylcholine was used, eserine was added with the acetylcholine to inhibit endogenous acetylcholine e&erase activity. RESULTS
E$ect of increasing concentrations of hormone on enzyme extl-usion and 32P incorporation into phosphatides. Lower concentrations of hormone would elicit enzyme extrusion, as measured by amylase activity in the incubation medium, than were necessary to give an effect on V incorporation into phosphatides. Figure 1 shows that amylase extrusion occurred in response to concentrations of
LOG,,
Pz
GONG (mts/mll
FIG. 1. Amylase extrusion and the incorporation of 32P into phosphatidic acid and phosphatidylinositol in response to different concentrations of pancreozymin. Pigeon pancreas slices were incubated in Krebs-Henseleit glcuose-bicarbonate medium with added orthophosphate+P, approximately 50 &/ml, for 40 minutes at 38” with shaking. They were then transferred to fresh medium of the same specific activity without and with hormone and incubated for a further 30 minutes. Amylase was assayed in the transfer medium. The values for the control slices were subtracted from the observed values in slices exposed to hormone, and the differences due to hormone are shown. Radioactivities are corrected to a specific activity of 10,000 cpm/mrmole of P for the orthophosphate of the medium. Values are the averages from triplicate incubations.
-6 LOG,0
-5 ACh CONC (M)
FIG. 2. Amylase extrusion and 32P incorporation into phosphatidic acid and phosphatidylinositol in response to different concentrations of ACh (plus eserine). Pigeon pancreas slices were incubated as in Fig. 1 for 70 minutes before transfer and for 20 minutes after transfer to medium containing ACh (plus eserine, lo-’ M). Other conditions were as in Fig. 1.
pancreozymin of 0.05-0.15 unit/ml, with only a very small increase in the incorporation of 32Pinto phosphatidylinositol. In this experiment, a major increase in 3zP incorporation into phosphatidylinositol, and increased radioactivity in phosphatidic acid, were not observed until a concentration of pancreozymin of 1.5 units/ml or higher was used. Figure 2 shows an experiment in which the highest amount of hormone-responsive amylase extrusion was elicited in response to 10-’ M acetylcholine (ACh) (plus eserine), with only a very small increase in phosphatidylinositol and phosphatidic acid radioactivity. With higher concentrations of ACh, enzyme extrusion was actually less than with 1O-7M ACh, but the incorporation of 32Pinto phosphatidylinositol and phosphatidic acid both showed a linear increase over the range 1&6-1@4 M ACh (plus eserine). Table I shows a compiled list of responses to different concentrations of pancreozymin or ACh (plus eserine) and to eserine alone. 111 this Table amylase extrusion and 3zP radioactivity in phosphatidic acid, phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine plus phosphatidylserine are each expressed as a percentage of the value obtained from slices from the same
282
HOKIN TABLE
I
EFFECTOFDIFFERENTCONCENTRATIONSOFPANCREOZYMINORACETYLCHOLINE ONAMYLASEEXTRUSION ACID,PHOSPHATIDYLINOSITOL,PHOSPHATIDYLCHOLINE, AND a2P INCORPORATIONINTOPHOSPHATIDIC AND PHOSPHATIDYLETH.4NOLAMINE PLUS PHOSPHATIDYLSERINE
Pigeon pancreas slices were incubated essentially as in Figs. 1 and 2. In each experiment observed in the presence of hormone was expressed as a percentage of the value for control standard error of the mean is shown for means from more than two experiments.
the value slices. The
-
COWL Additions
Radioactivity
y$-$
(units/ ml)
No.
Eserine Eserine ACh Eserine ACh Eserine ACh Eserine ACh
1W4 M 10m4M ilo-’ M lw4 M + 1O-6 M 10-” M f 1O-6 M 1W4 M ilO’+ M
0.005 0.05 0.15 0.5 1.5 5.0 10.0
-
expts.
PA
_None Pancreozymin
in phosphatides”.b
PC
PI
(100)
(100)
(100)
(l@l)
113 119 f 169 161 206 200 f 181 113 f 176
80 107 95 152 196 607 460 95 128
96 125 118 257 380 735 714 98 144
92 110 110 138 136 135 184 96 140
4
22 18
f
11
f
17
i
163
f
19
f
8
f
43
f
122
f
15
PE/PS (100)
f
6
f
17
f
15
f
3
98 110 100 148 161 291 330 91 176
f
15
f
15
f
80
f
26
2 4 2 4 1 6 1 3 2
164
173
214
121
138
2
123
350
344
144
143
2
128
440
520
198
186
2
-
a Expressed as a percentage of control value without hormone. * PA, Phosphatidic acid; PI, phosphatidylinositol; PC, phosphatidylcholine; ethanolamine plus phosphatidylserine (explained in the text).
animal which were not exposed to hormone. Phosphatidylethanolamine and phosphatidylserine did not separate very well on chromatography in some of the experiments used for this Table, so that the radioactivity in both of these lipids was treated as one value in compiling the Table. In experiments in which these two phosphatides did show good separation, radioactivity in phosphatidylethanolamine was greater than that in Eserine alone gave no phosphatidylserine. significant stimulation of either amylase extrusion or the incorporation of 32Pinto phosphatides under these conditions; in experiments in which slices were exposed to eserine for longer periods of time, some increased amylase extrusion has been observed (10). Effects of different concentrations of pancreozymin or ACh (plus eserine) on amylase extrusion and on 32Pincorporation into phosphatidic acid and phosphatidylinositol in the compiled series were similar to those in the individual experiments discussed above. In
PE/PS,
phosphatidyl-
addition, Table I shows that more than halfmaximal stimulation of amylase extrusion could occur in response to 0.15 unit of pancreozymin per milliliter without any increased incorporation of 32P into phosphatidylcholine or phosphatidylethanolamine plus phosphatidylserine and that, as with 32Pincorporation into phosphatidic acid and phosphatidylinositol, increased incorporation of 32P into phosphatidylcholine and phosphatidylethanolamine plus serine occurred with increasing concentrations of either pancreosymin or ACh (plus eserine) which did not give any further increase in amylase extrusion. 1)ISCUSSION
The extrusion of digestive enzymes from the exocrine cells of the pancreas is believed to occur by a process which involves the coalescence of zymogen granules with the plasma membrane of the apical region of the cell (11). The process appears to be anal-
PANCREOZYMIN
AND
ogous to that of pinocytosis and phagocytosis, but in the reverse direction. Because of the increased synthesis of phosphatides which is observed during active phagocyt80sis in polymorphonuclear leucocytes (12-14)) it has been suggested that t’he breakdown and resynthesis of membrane phospholipids during the process of reverse pinocyt’osis may be responsible for the increased synthesis, or turnover, of phosphatides observed in response to hormones which stimulate enzyme extrusion in the pancreas (15, 16). However, t#he experiments of Hokin (17) on the effect of omission of calcium from the incubation medium have demonstrated that hormoneresponsive synthesis of phosphat’ides could occur in the absence of hormone-responsive enzyme extrusion. The results presented here show that hormone-responsive enzyme extrusion can occur in the absence of any meassynthesis of urable hormone-responsive phosphatides. If amylase extrusion is taken as a measure of zymogen granule ext,rusion, t’hese results appear t,o rule out the possibility that the bulk of the increased incorporat,ion of azPinto phosphatides which occurs in response to hormone is a secondary consequence of zymogen granule extrusion. Some other explanation for this phenomenon must therefore be sought. The fact t’hat maximum phosphatide response is elicited by concentrations of hormones greatly in excess of those necessary to elicit maximum enzyme extrusion suggests that the phenomenon may represent part of second, adaptive response to hormonea gearing up of the cell to meet greater demands than it has previously been meetming for the secretion of digestive enzymes. According t’o this interpretat’ion, t,he hormone carries at least two messages: one which is read at low concentrations and which simply inst’ructs the cell to extrude digestive enzymes, and one which is read at higher concentrations which instructs t’he cell to prepare to secrete greater quant,ites of zymogen granules than it has previously been geared up for. This second message would be logical since it would prepare the cell to cope with a continuing contingency of greater hormonal stimulation associated wiSh higher amounts of hormone. The experiments of Redman and Hokin
233
ACETYLCHOLINE
(18) indicate that the increase in phosphatide radioactivity in response to ACh is found in membranous structures which sediment in the microsomal fract#ion from pancreas cells. Hokin and Huebner (19) have shown with autoradiographic t,echniques that the hormone-responsive phosphatidylinositol synthesis resulted in t,he incorporation of the isotopically labelled phosphatidylinositol int’o both the roughsurfaced endoplaemic reticulum and the smooth-surfaced membranes of t’he Colgi region. Reading of the second message would therefore appear to involve changes in membrane phosphatide metabolism in both the rough-surfaced and t’he smoothsurfaced membranes. Webster and Tyor (20) have observed an increase in the incorporation of n-phenylalanine-14C into pancreatic proteins in response to pancreozymin in the pigeon in viva at 60 minutes or later after administration of hormone, but did not observe any increase at 30 minut.es after hormone administration. The synthesis of complete enzyme is not measurably increased in the presence of concentrations of hormone which give a phosphatide effect over the time interval studied in the present, report (1, 3), whereas the hormone-stimulated increase in 32P incorporation into phosphatides begins within 5 minutes of administration of the hormone (6). AdapGve changes in membrane phosphatides, such as are postulated here, would therefore precede in time any increased enzyme synthesis. One might also predict’ that t#he effects reported here would be gross exaggerat,ions of effects which might normally occur in response to much smaller fluctuations in hormonal stimulation which might occur under more physiological conditions in vivo. BCKNOWLE
DQXIENTS
The technical assistance of Mr. Sadeghian is gratefully acknowledged.
Khosrow
REFERENCES 1.
L. E., Biochem. J. 48, 320 (1951). 2. SCHUCHICR, It., .uw HOKIN, L. E., J. Biol. Chem. 210, 551 (19.54). 3. HOKIN, L. E., AND HOKIN, 31. It., J. Physiol. (London) 132, 442 (1956). HOKIN,
284
HOKIN
L. E., AND HOKIN, M. R., Biochim. 4. HOKIN, Biophys. Acta 18, 102 (1955). 5. HOKIN, L. E., AND HOIIIN, M. R., J. Biol. Chem. 233, 805 (1958). 6. HOKIN, M. R., Arch. Biochem. Biophys. 124, 271 (1968). 7. KREBS, H. A., AND HENSELEIT, K., 2. Physiol. Chem. 210, 33 (1932). 8. MARINETTI, G. V., ERBLAND, J., AND KOCHEN, J., Federation Proc. 16, 837 (1957). 9. SMITH, B. W., AND ROE, J. II., J. Biol. Chem. 179, 53 (1949). 10. HOKIN, M. R., AND HOKIN, L. E., J. Biol. Chem. 209, 549 (1954). Particles,” pp. 11. PALADE, G. E., “Subcellular 64-80. American Physiological Society, Washington, D.C. (1959).
12. SBARRA, A. J., AND KARNOVSKY, M. L., J. Biol. Chem. 236, 2224 (1960). 13. KARNOVSKY, M. I,., AND WALLACH, D. F. H., J. Biol. Chem. 236, 1895 (1961). 14. SASTRY, P. S., AND HOKIN. L. E.. J. Biol. Chem. 241, 3354 (1966). 15. KARNOVSKY, M. L., Physiol. Rev. 42, 143 (1962). 16. FAWCETT, D. W., Ciirculation 26, 1105 (1962). L. E., Bioeh.im. Biophys. Actn 116. 17. HOKIN, 219 (1966). 18. REDMAN, C. M., AND HOKIN, L. E., J. Bionhvs. Biochem. Cytol. 6, 207 (1959). D., J. Cell Biol. 19. HOKIN, L. E., AND HIJEBNER, 33, 521 (1967). 20. WEBSTER P. D., AND TYOR, M. P., Am. J. Physiol. 221, 157 (1966).
OF
Studies
BIOCHEMISTRY
AND
on Chemical
BIOPHYSICS
124, 280-284 (1968)
Mechanisms
of the Action
of Neurotransmitters
and Hormones II. Increased
Incorporation
of 32P into Phosphatides
Response to Pancreozymin
or Acetylcholine MABEL
Departments
of
Psychiatry
Received
in Pigeon
Pancreas
Slices’
R. HOKIN
and Physiological Chemistry, Madison, Wisconsin 53706
August
as a Second, Adaptive
31, 1967; accepted
University
September
of Wisconsin,
7, 1967
The concentrations of pancreozymin or acetylcholine necessary to give hormoneresponsive amylase extrusion, and those necessary to give hormone-responsive increases in 32P incorporation into phosphatides in pigeon pancreas slices, have been investigated. Low concentrations of pancreozymin or acetylcholine stimulated amylase extrusion with little or no effect on 32P incorporation into phosphatides. High concentrations of pancreozymin gave increased incorporation of 32P into phosphatides without increasing amylase extrusion above that observed with lower concentrations of hormone. Amylase extrusion in response to 10m4or lo+ M acetylcholine (plus eserine) was less than in response to lower concentrations of acetylcholine, but a2P incorporation into phosphatides was much higher with the higher concentrations of acetylcholine. These results provide further evidence that increased synthesis or turnover of phosphatidylinositol, phosphatidic acid, phosphatidylethanolamine, and phosphatidylcholine in response to hormone is not a secondary consequence of zymogen granule extrusion (reverse pinocytosis). In view of the requirement for higher concentrations of hormone than those necessary to elicit amylase extrusion, it is suggested that the increased incorporation of 32P into phosphatides may represent a second, adaptive response to hormone-part of a gearing up of the cell to meet greater demands for the secretion of zgmogens than it had previously been meeting.
tidylinositol, phosphatidic acid, and phosphatidylethanolamine (4-6). The present work compares the concentrations of hormone necessary to elicit enzyme extrusion and those necessary to elicit increased 32P incorporation into individual phosphatides.
Extrusion of amylase and other digestive enzymes in pigeon pancreas slices can be elicited by either pancreozymin or acetylcholine (l-3), and each of these hormones2 has also been shown to give rise to an increased incorporation of 32P into several phosphatides in the tissue; the major increase in both total 32Pincorporation and in net specific activity occurred in phospha-
EXPERIMENTAL
PROCEDURE
Pigeon pancreas slices were prepared and incubated in Krebs-Henseleit bicarbonate medium (7), with added glucose, as described previously (5). At the end of the incubation period, the tissue was removed from the incubation flask and was rapidly frozen in a test tube by plunging the tube into a dry ice-alcohol bath. The tissues were then homogenized from the frozen state in 5% trichloroacetic acid, and lipids were extracted as described elsewhere (5). The lipids were separated by chromatography on silicic-acid impregnated paper (8).
1 This investigation was supported by grants NB-01730 and NB-06745-01 from the National Institutes of Health, U.S. Public Health Service. 2 For the sake of brevity, the word “hormone” is used to refer to both pancreozymin and acetylcholine, although it is realized that acetylcholine is a neurotransmitter, and not a hormone in the classical sense. 280
I’ANCREOZYMIN
AND
281
ACETYLCHOLINE
Amylase activity was determined on aliquots of the incubation medium by a slight modification of the method of Smith and Roe (9). Pancreozymin was a commercial preparation of Cesekin (Vitrum Pharmaceutical Co., Stockholm, Sweden). The activity is stated in units which refer to Ivy dog units as standardized by the supplier. In experiments in which acetylcholine was used, eserine was added with the acetylcholine to inhibit endogenous acetylcholine e&erase activity. RESULTS
E$ect of increasing concentrations of hormone on enzyme extl-usion and 32P incorporation into phosphatides. Lower concentrations of hormone would elicit enzyme extrusion, as measured by amylase activity in the incubation medium, than were necessary to give an effect on V incorporation into phosphatides. Figure 1 shows that amylase extrusion occurred in response to concentrations of
LOG,,
Pz
GONG (mts/mll
FIG. 1. Amylase extrusion and the incorporation of 32P into phosphatidic acid and phosphatidylinositol in response to different concentrations of pancreozymin. Pigeon pancreas slices were incubated in Krebs-Henseleit glcuose-bicarbonate medium with added orthophosphate+P, approximately 50 &/ml, for 40 minutes at 38” with shaking. They were then transferred to fresh medium of the same specific activity without and with hormone and incubated for a further 30 minutes. Amylase was assayed in the transfer medium. The values for the control slices were subtracted from the observed values in slices exposed to hormone, and the differences due to hormone are shown. Radioactivities are corrected to a specific activity of 10,000 cpm/mrmole of P for the orthophosphate of the medium. Values are the averages from triplicate incubations.
-6 LOG,0
-5 ACh CONC (M)
FIG. 2. Amylase extrusion and 32P incorporation into phosphatidic acid and phosphatidylinositol in response to different concentrations of ACh (plus eserine). Pigeon pancreas slices were incubated as in Fig. 1 for 70 minutes before transfer and for 20 minutes after transfer to medium containing ACh (plus eserine, lo-’ M). Other conditions were as in Fig. 1.
pancreozymin of 0.05-0.15 unit/ml, with only a very small increase in the incorporation of 32Pinto phosphatidylinositol. In this experiment, a major increase in 3zP incorporation into phosphatidylinositol, and increased radioactivity in phosphatidic acid, were not observed until a concentration of pancreozymin of 1.5 units/ml or higher was used. Figure 2 shows an experiment in which the highest amount of hormone-responsive amylase extrusion was elicited in response to 10-’ M acetylcholine (ACh) (plus eserine), with only a very small increase in phosphatidylinositol and phosphatidic acid radioactivity. With higher concentrations of ACh, enzyme extrusion was actually less than with 1O-7M ACh, but the incorporation of 32Pinto phosphatidylinositol and phosphatidic acid both showed a linear increase over the range 1&6-1@4 M ACh (plus eserine). Table I shows a compiled list of responses to different concentrations of pancreozymin or ACh (plus eserine) and to eserine alone. 111 this Table amylase extrusion and 3zP radioactivity in phosphatidic acid, phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine plus phosphatidylserine are each expressed as a percentage of the value obtained from slices from the same
282
HOKIN TABLE
I
EFFECTOFDIFFERENTCONCENTRATIONSOFPANCREOZYMINORACETYLCHOLINE ONAMYLASEEXTRUSION ACID,PHOSPHATIDYLINOSITOL,PHOSPHATIDYLCHOLINE, AND a2P INCORPORATIONINTOPHOSPHATIDIC AND PHOSPHATIDYLETH.4NOLAMINE PLUS PHOSPHATIDYLSERINE
Pigeon pancreas slices were incubated essentially as in Figs. 1 and 2. In each experiment observed in the presence of hormone was expressed as a percentage of the value for control standard error of the mean is shown for means from more than two experiments.
the value slices. The
-
COWL Additions
Radioactivity
y$-$
(units/ ml)
No.
Eserine Eserine ACh Eserine ACh Eserine ACh Eserine ACh
1W4 M 10m4M ilo-’ M lw4 M + 1O-6 M 10-” M f 1O-6 M 1W4 M ilO’+ M
0.005 0.05 0.15 0.5 1.5 5.0 10.0
-
expts.
PA
_None Pancreozymin
in phosphatides”.b
PC
PI
(100)
(100)
(100)
(l@l)
113 119 f 169 161 206 200 f 181 113 f 176
80 107 95 152 196 607 460 95 128
96 125 118 257 380 735 714 98 144
92 110 110 138 136 135 184 96 140
4
22 18
f
11
f
17
i
163
f
19
f
8
f
43
f
122
f
15
PE/PS (100)
f
6
f
17
f
15
f
3
98 110 100 148 161 291 330 91 176
f
15
f
15
f
80
f
26
2 4 2 4 1 6 1 3 2
164
173
214
121
138
2
123
350
344
144
143
2
128
440
520
198
186
2
-
a Expressed as a percentage of control value without hormone. * PA, Phosphatidic acid; PI, phosphatidylinositol; PC, phosphatidylcholine; ethanolamine plus phosphatidylserine (explained in the text).
animal which were not exposed to hormone. Phosphatidylethanolamine and phosphatidylserine did not separate very well on chromatography in some of the experiments used for this Table, so that the radioactivity in both of these lipids was treated as one value in compiling the Table. In experiments in which these two phosphatides did show good separation, radioactivity in phosphatidylethanolamine was greater than that in Eserine alone gave no phosphatidylserine. significant stimulation of either amylase extrusion or the incorporation of 32Pinto phosphatides under these conditions; in experiments in which slices were exposed to eserine for longer periods of time, some increased amylase extrusion has been observed (10). Effects of different concentrations of pancreozymin or ACh (plus eserine) on amylase extrusion and on 32Pincorporation into phosphatidic acid and phosphatidylinositol in the compiled series were similar to those in the individual experiments discussed above. In
PE/PS,
phosphatidyl-
addition, Table I shows that more than halfmaximal stimulation of amylase extrusion could occur in response to 0.15 unit of pancreozymin per milliliter without any increased incorporation of 32P into phosphatidylcholine or phosphatidylethanolamine plus phosphatidylserine and that, as with 32Pincorporation into phosphatidic acid and phosphatidylinositol, increased incorporation of 32P into phosphatidylcholine and phosphatidylethanolamine plus serine occurred with increasing concentrations of either pancreosymin or ACh (plus eserine) which did not give any further increase in amylase extrusion. 1)ISCUSSION
The extrusion of digestive enzymes from the exocrine cells of the pancreas is believed to occur by a process which involves the coalescence of zymogen granules with the plasma membrane of the apical region of the cell (11). The process appears to be anal-
PANCREOZYMIN
AND
ogous to that of pinocytosis and phagocytosis, but in the reverse direction. Because of the increased synthesis of phosphatides which is observed during active phagocyt80sis in polymorphonuclear leucocytes (12-14)) it has been suggested that t’he breakdown and resynthesis of membrane phospholipids during the process of reverse pinocyt’osis may be responsible for the increased synthesis, or turnover, of phosphatides observed in response to hormones which stimulate enzyme extrusion in the pancreas (15, 16). However, t#he experiments of Hokin (17) on the effect of omission of calcium from the incubation medium have demonstrated that hormoneresponsive synthesis of phosphat’ides could occur in the absence of hormone-responsive enzyme extrusion. The results presented here show that hormone-responsive enzyme extrusion can occur in the absence of any meassynthesis of urable hormone-responsive phosphatides. If amylase extrusion is taken as a measure of zymogen granule ext,rusion, t’hese results appear t,o rule out the possibility that the bulk of the increased incorporat,ion of azPinto phosphatides which occurs in response to hormone is a secondary consequence of zymogen granule extrusion. Some other explanation for this phenomenon must therefore be sought. The fact t’hat maximum phosphatide response is elicited by concentrations of hormones greatly in excess of those necessary to elicit maximum enzyme extrusion suggests that the phenomenon may represent part of second, adaptive response to hormonea gearing up of the cell to meet greater demands than it has previously been meetming for the secretion of digestive enzymes. According t’o this interpretat’ion, t,he hormone carries at least two messages: one which is read at low concentrations and which simply inst’ructs the cell to extrude digestive enzymes, and one which is read at higher concentrations which instructs t’he cell to prepare to secrete greater quant,ites of zymogen granules than it has previously been geared up for. This second message would be logical since it would prepare the cell to cope with a continuing contingency of greater hormonal stimulation associated wiSh higher amounts of hormone. The experiments of Redman and Hokin
233
ACETYLCHOLINE
(18) indicate that the increase in phosphatide radioactivity in response to ACh is found in membranous structures which sediment in the microsomal fract#ion from pancreas cells. Hokin and Huebner (19) have shown with autoradiographic t,echniques that the hormone-responsive phosphatidylinositol synthesis resulted in t,he incorporation of the isotopically labelled phosphatidylinositol int’o both the roughsurfaced endoplaemic reticulum and the smooth-surfaced membranes of t’he Colgi region. Reading of the second message would therefore appear to involve changes in membrane phosphatide metabolism in both the rough-surfaced and t’he smoothsurfaced membranes. Webster and Tyor (20) have observed an increase in the incorporation of n-phenylalanine-14C into pancreatic proteins in response to pancreozymin in the pigeon in viva at 60 minutes or later after administration of hormone, but did not observe any increase at 30 minut.es after hormone administration. The synthesis of complete enzyme is not measurably increased in the presence of concentrations of hormone which give a phosphatide effect over the time interval studied in the present, report (1, 3), whereas the hormone-stimulated increase in 32P incorporation into phosphatides begins within 5 minutes of administration of the hormone (6). AdapGve changes in membrane phosphatides, such as are postulated here, would therefore precede in time any increased enzyme synthesis. One might also predict’ that t#he effects reported here would be gross exaggerat,ions of effects which might normally occur in response to much smaller fluctuations in hormonal stimulation which might occur under more physiological conditions in vivo. BCKNOWLE
DQXIENTS
The technical assistance of Mr. Sadeghian is gratefully acknowledged.
Khosrow
REFERENCES 1.
L. E., Biochem. J. 48, 320 (1951). 2. SCHUCHICR, It., .uw HOKIN, L. E., J. Biol. Chem. 210, 551 (19.54). 3. HOKIN, L. E., AND HOKIN, 31. It., J. Physiol. (London) 132, 442 (1956). HOKIN,
284
HOKIN
L. E., AND HOKIN, M. R., Biochim. 4. HOKIN, Biophys. Acta 18, 102 (1955). 5. HOKIN, L. E., AND HOIIIN, M. R., J. Biol. Chem. 233, 805 (1958). 6. HOKIN, M. R., Arch. Biochem. Biophys. 124, 271 (1968). 7. KREBS, H. A., AND HENSELEIT, K., 2. Physiol. Chem. 210, 33 (1932). 8. MARINETTI, G. V., ERBLAND, J., AND KOCHEN, J., Federation Proc. 16, 837 (1957). 9. SMITH, B. W., AND ROE, J. II., J. Biol. Chem. 179, 53 (1949). 10. HOKIN, M. R., AND HOKIN, L. E., J. Biol. Chem. 209, 549 (1954). Particles,” pp. 11. PALADE, G. E., “Subcellular 64-80. American Physiological Society, Washington, D.C. (1959).
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