Molecular and Cellular Endocrinology
2 (1975) 221-232. 0 North-Holland
Publ. Comp.
PROSTAGLANDIN Ed-SENSITIVE ADENYLATE CYCLASE RAT LIVER PLASMA MEMBRANES Vittorio
TOMASI
ofGeneral Physiology,
Institute
and Enrica University
OF
FERRETTI
of Ferrara, 44100 Ferrara,
Received 14 October 1974
Italy
Accepted 12 December
1974
Adenylate cyclase of isolated rat liver plasma membranes is stimulated at least four-fold by prostaglandin E, (PGE,) provided that EGTA and CTP are present in the assay system. The GTP-dependency is absolute, since in its absence the PGE, activation of adenylate cyclase is small and inconsistent. PGE, is much less effective while prostaglandins Fra and F,, are ineffective under all conditions tested. The stimulatory effect of PGE, is evident at concentrations around 0.5 uM, optimal stimulation requires 3 uM PC&. Several lines of evidence suggest that PGEr interacts with plasma membrane binding sites separate from those specific for glucagon and epinephrine. First, the effect of combinations of PGEl with glucagon or epinephrine at optimal concentrations are additive, while not additive is the combination PGE,-NaF. Second, variations in the temperature of incubation between 25 and 45 “C influence in a different way glucagon and PGEt-sensitive cyclase. Glucagon stimulation continuously increases with the increase of temperature while PGEr stimulation increases up to 30 “C then it levels off and decreases at 45 ‘C. Third, propranolol which nearly completely blocks epinephrine stimulation is much less effective on PC&sensitive cyclase. 0.1 mM phentolamine has little effect, if any, on hormonal sensitivity. Lubrol at very low doses (1 mg/iOO ml) reduces the effect of epinephrine and glucagon, but increases that of PGE,. Keywords:
PGEr ; adenylate
Prostaglandins
are known
cyclase;
liver plasma membrane;
to be capable
of raising
epinephrine;
cyclic
AMP
glucagon.
levels
in
several cells and tissues (Ramwell and Shaw, 1970) including platelets (Robison et al., 1969), ovary (Kuehl et al., 1970), lung and spleen (Butcher and Baird, 1968). Adenylate cyclase activities sensitive to prostaglandins have been described in membranes isolated from platelets (Butcher et al., 1967) thyroid (Kaneko et al., 1969), ovary (Marsh, 1970), and heart (Sobel and Robison, 1969). In liver a prostaglandin E, (PGE,) stimulation of adenylate cyclase of broken-ceil homogenates has been reported by Bitensky et al. (1972) and more recently by Sweat and Wincek (1973).
V. Tomasi and E. Ferretii
222
In this paper, the stimulation of adenylate cyclase of isolated rat liver plasma membrane by PGE, and the absolute dependency of this activation on GTP are reported. In addition, evidence is presented indicating that PGE, interacts with plasma membrane sites which are separate from those specific for glucagon and epinephrine. While this work was in progress it has been shown (Smigel and Fleisher, 1974) that rat liver plasma membranes possess high affinity binding sites for the E type prostaglandins.
MATERIALS
AND
METHODS
Plasma membranes were isolated from livers of rats weighing less than 100 g by the method of Ray (1970). The use of young rats greatly facilitated the observation of a response of adenylate cyclase to epinephrine. Membranes were used within 2 h after the final preparative step. Adenylate cyclase was assayed in a system containing in a final volume of 0.4 ml: I .O mM ATP, 3.75 mM MgS04, 25 mM Tris-HCI, pH 8.0, 6.2 mM aminophylline and 50-100 c(g of membrane proteins. 2.5 mM phosphoenolpyruvate and 50 ug/ml of pyruvate kinase were generally used as ATP regenerating system. However it was found that in our experimental conditions omission of the regenerating system had negligible eflects on basal and hormone stimulated activities at least up to 5 min of incubation at 37 “C. Unless otherwise stated GTP was 0.5 mM and EGTA 0.1 mM. Incubations were generally carried out for 5 min at 37 “C and the reactions were terminated by immersing the tubes in boiling water for 2 min. The tubes frozen at -20 “C were then thawed and centrifuged at 1000 g for IO min. The supernatants were diluted I : 5 with 0.01 M Tris-HCI, pH 7.5 and cyclic AMP was assayed as described by Brown et al. (1972) with the precautions previously reported (Barnabei et al., 1974). The amount of cyclic AMP formed was linear with the time of incubation at least up to IO min (up to 5 min in the absence of a regenerating system) and with the membrane concentration within 100 ug of proteins. Values are usually averages of triplicate determinations; standard errors are less than 10% for the same membrane preparation. Some variations between different membrane preparations were observed as far as the extent of hormonal stimulation is concerned. Proteins were determined according to Lowry et al. (1951) using bovine serum albumin as a standard. Albino Wistar rats fed ad libitum were used. ATP disodium salt, GTP lithium or sodium salts, phosphoenolpyruvate, pyruvate kinase, epinephrine bitartrate, aminophylline were products of Sigma Chem. Co., St. Louis, U.S.A. Glucagon (crystalline) was a gift of Dr. R. Chance, E. Lilly, Indianapolis, U.S.A. Prosta-
PGE,-stimulated
glandins
adenylate cyclase
were kindly
provided
mazoo, U.S.A. Adenosine Ci/mmole) was a product
223
by Dr. J. Pike, The Upjohn
3’-5’ (7)3H-monophosphate of Radiochemical Centre,
Company,
Kala-
cyclic (spec. act. 27 Amersham, England.
EGTA was a product of Serva, Heidelberg, Germany. Lubrol 12A-9 (formerly known as Lubrol PX) was a gift of Imperial Chemical Industries, England. Phentolamine (Regitin) was a product of Ciba, Basel, Switzerland. Propranolol was a product of Ayerst Laboratories, New York, U.S.A.
RESULTS in a first series of experiments (table 1) we tested the effect of four prostaglandins, glucagon and sodium fluoride on rat liver plasma membrane adenylate cyclase. In comparison to the marked effects of glucagon and fluoride (Cfold stimulation) a slight stimulation of the enzyme by PGE, and PGF,, and no effects with PGE, and PGF,, were observed. However in the light of the observations by Rodbell et al. (1971) on the role of GTP on the glucagonsensitive adenylate cyclase and by Sweat and Wincek (1973) on the PGE, sensitive enzyme, it was considered of importance to test the effect of GTP. In table 1 it is shown that in the presence of 0.5 mM GTP in the assay
Table I The effects of prostaglandins, glucagon and fluoride on adenylate cyclase activity assayed in the absence and presence of GTP. Adenylate cyclase was assayed as described under Materials and Methods in the absence of EGTA. All prostaglandins except PGFZa which was dissolved in water, were dissolved in 95% ethanol and 10 ul of the solutions (8 ug) were added to the assay system. 10 pl of 95% ethanol was found to have no effect on control activity. Glucagon was 1 x 1Od6 M, sodium fluoride 10 mM and GTP 0.5 mM. Data are means I SE, in parenthesis the number of experiments is reported. Adcnylate cyclase activity (pmoles/mg -
Control (4) PGF,, (2) PGF,, (2) PGF, (4) PGE, (2) Glucagon (4) NaF (4)
83 96 143 145 65 337 339
GTP
-1: 7.1
$z 22 :t 20 _rt 24
i
100 115 172 173 78 406 408
109 95 87 600 164 745 618
protein/5 min)
GTP
i_
8.4
i 45 & 51 f_ 60
100 81 80 550 150 683 567
1.3 1.0 0.6 4.1 2.5 2.2 1.8
224
V. Tomasi and E. Ferretfi
system, PGE, was a potent stimulant of adenylate cyclase, while PGE, was much less active and the F type prostaglandins were ineffective. Also glucagon, as expected (Rodbell et al., 1971), had an higher efficacy when GTP was present, while the effect of GTP on fluoride sensitivity is in contrast with the studies of Rodbell et al. (1971).
lOOOr
900
700 _
I
/I 0
"
16
lo-6 "@
Fig.
I 1O-3 M
[GTP]
I. The effect of PGE, on adenylate cyclase activity was assayed at GTP concentrations from 1 Y 10m6 to I >~ 10m3 M. The points represent the means of three experiments.
ranging PGE,
was present
at 4 ug/ml. activity
In these experiments in the absence
of GTP
the effects of PGE,
on adenylate
cyclase
was not significant.
In fig. 1 the GTP concentration was varied between lo-’ and lop3 M. The half-maximal GTP concentration for PGE, stimulation was around 2 x 10e5 M, the nucleotide having practically no effect on basal activity. The most effective of other nucleotides tested in addition to GTP, was UTP. However
PGE,-stimulated
adenyiate cyctase
225
PGE,
1 0
b
’
2
p
*
4
0
’
6
’
’
*
8
’ 10
’
’
’
12 PGE,
’
0
14
16
p9/0,4ml
Fig. 2. The effect of PGE, at different concentrations on adenylate cyclase activity. The insert was drawn on semilog paper. Different solutions of PGE, in 957; ethanol were prepared in order to use not more than 15 ~1 of solution (0.1 Mg/O.4ml is equivalent to 0.7 pM PGE,). The points represent the means of three determinations.
in the presence
of UTP, stimulation
by PGEl
was about
15 % of that observed
with GTP (not shown). Therefore in all successive experiments 0.5 mM GTP was included in the assay system. In fig. 2 the PGE, concentration was varied between 7 x 10e7 and 10T4 M. The stimulatory effect was evident at concentrations as low as 7 x lo-’ M (0.1 pg per tube) and the optimal effect was obtained with 1.4 x lop5 M PGE, (2 pg per tube). At higher concentrations the extent of stimulation declined. In the successive experiments therefore PGE, was added at concentrations ranging from 2 to 4 pg per tube. We observed that addition of EGTA to the incubation system markedly enhanced the effects of hormones used and of sodium fluoride (fig. 3). Of particular interest was the effect of EGTA on epinephrine sensitive cyclase. As previously observed (Marinetti et al., 1969; Pohl et al., 1971) this hormone is slightly effective on the liver plasma membrane cyclase, however in the
V. Tomusi and E. Fermtti
226
0.
NaF O_
0.
Adrenahne PGE,
4yJ
/ 0.
I 0
/
i
I,
0
1O-6
1cr4M
10-s EGTA
Fig. 3. The effect of EGTA
on adenylate cyclase activity. EGTA
concentration was varied
between lo-” and 10m4M. Epinephrine was I > IO-” M, glucagon 1 Y IO--” M and fluoride 10 mM.
presence of 10m4 M EGTA it becomes much more effective (more than d-fold stimulation). Also the sensitivity of adenylate cyclase to glucagon and fluoride and to a lesser extent to PGE1, was enhanced by EGTA. The effect of EGTA seems to be due not simply to a chelation of calcium ion since we observed that addition of Ca2+ does not completely reverse the effect of EGTA (not shown). On the other hand its effect on basal and NaF stimulated activities is probably due to chelation of calcium ions which membranes may bind during the preparation ; as a matter of fact Ray’s method (1970) involves the inclusion of 0.5 mM calcium in the homogenization buffer. It was considered of interest to investigate whether or not the effects of PGE, on adenylate cyclase were mediated by a receptor separate from those of
PGEl-stimulated
227
adenylate cyclase
Table The effect of combinations
of PGE,,
activity.
conditions
For
cyclase
experimental
was assayed
in the presence
glucagon,
2 epinephrine
see Materials
and
of 0.5 mM GTP
and fluoride Methods
and
and 0.1 mM EGTA.
on adenylate table
cyclase
1. Adenylate
In parenthesis
are
in the case of additivity. In experiment 1 membranes were prepared from a rat weighing 90 g, in experiment 2 two rats of 40 g were used. Data are means of
the values
calculated
duplicate Increase
determinations.
of adenylate
cyclase
Exp.
I :’ 10m5 M
Epinephrine Glucagon PGE, NaF
989
M
239
I x IO-’ M
Epinephrine Glucagon NaF
I41
1 ,. 10eh M
3 x lo-”
347
i- PGE, -
336 1380
PGE,
478
~- PGE,
Epinephrine
I
+ glucagon
+ PGE,
Control
1576 120
(380) (1228) (586) (1369)
activity
(pmoles/mg
protein/5
min)
Exp. 2
255 840 430 485 645 1310 725 1510 135
(685) (1270) (915) (1525)
As a first approach to this problem we tested between PGE, and glucagon or epinephrine. The results reported in table 2 indicate that in two experiments there was additivity in the stimulation of adenylate cyclase by combinations of epinephrine or glucagon with PGE,. In parenthesis the values expected in the case of additivity are reported. Clearly not additive was the combination PGE,-NaF. This kind of evidence in favour of a distinct PGE, receptor was corroborated by other lines of evidence. Bitensky et al. (1972) have studied the effect of temperature on glucagon, epinephrine and PGE ,-sensitive adenylate cyclase of liver. They found breaks in the Arrhenius plots for PGE, and for glucagon but not for epinephrine. Our results reported in fig. 4 indicate that variations in the temperature of incubation have different effects on glucagon and on PGEl sensitive cyclases. glucagon whether
and
epinephrine.
or not there
was additivity
Up to 30 “C both hormones become increasingly effective, afterwards the increase of temperature further enhances glucagon effect without influencing PGE, effect, at least up to 37 “C. At 45 “C glucagon is more effective than at 37 “C, while PGE, is less effective. Further evidence about the distinctness of the binding sites come from experiments with blocking agents. In table 3 it is shown that propranolol (10-5-10p4 M) almost abolished the epinephrine sensitivity of adenylate
V. Tomasi and E. Ferretti
228
1300.
1100 .
,
. , N -i Glucagon
I
x, , 900.
I I
,’ E ll? .700. .E 3 :
i5,,. E ," a s $300. 5 xc : :: 100 .
22
Fig.
4. The
25
effect
27
30
of temperature
on
equilibrated
at the temperatures
membranes.
Up to 30 “C the effect glucagon.
35
PGE,
indicated
Each point
and
of PGE,
and
37
glucagon-sensitive
the reaction was
is the mean
not
cyclase.
was started
significantly
4spc
Temperature
Tubes
were
by the addition
different
from
that
of of
of two determinations.
cyclase, while its effect on the PGE, stimulation was much less pronounced. Propranolol had no effect on the sensitivity of the cyclase towards glucagon. On the other hand phentolamine (lop4 M) does not appear to modify greatly the hormonal sensitivity of adenylate cyclase. In fig. 5 the effect of Lubrol on the hormonal sensitivity is reported. When added to the incubation system at very low concentrations (1 mg/lOO ml) the detergent produced a decrease of epinephrine and glucagon stimulation, but potentiated the effect of PGE,. At higher doses of Lubrol a decrease in the sensitivity of adenylate cyclase to hormones and to a lesser extent to fluoride was observed. At 10 mg Lubrol per 100 ml basal and fluoride stimulated activities were not very different from controls while the effect of hormones was markedly reduced.
PGE,-stimulated
229
adenylate cyclase
Table The effect
of a and
experimental while
a reduction
experiments. isolated
e blocking
conditions
agents
see Methods
of 20%
from
pools
on hormonal
and table
was observed
The effect of propranolol of livers
of
Increase
3
with
sensitivity
2. Phentolamine propranolol.
was evaluated
IO-day
old rats, epinephrine.
over basal
Control
of adenylate
Data
are means
in two experiments. in order
activity
to have
(pmoles/mg
M
Epinephrine Glucagon
900 * 74 1616 * 121
776 i 105 1236 % 110
PGEl
1130 + 33
1136 :t 65
basal i
For
activity
SE of three
Membranes
a greater
protein/5
Phentolamine lo-“
cyclase.
did not affect
were
response
to
min)
Propranolol lo-“
M
10m5 M
5 x 10m5 M
142 1763
140 1525
80 1545
690
670
695
q Control q Eplnephrlne qGlucagon q PGE, q N~F
5
10 [Lubrog
Fig.
5. The effect of Lubrol
membrane Means
adenylate
cyclase.
of two experiments
at different For
mg / lOOmI
concentrations
hormones
are reported.
and
on the hormonal NaF
The ATP
concentrations regenerating
sensitivity see under
system
of plasma table
was omitted.
2.
230
DISCUSSION Rat liver plasma membranes have been extensively used for the study of glucagon and epinephrine sensitive adenylate cyclase (Marinetti et al., 1969; Ray et al., 1970; Tomasi et al., 1970; Pohl et al., I971 ; Rodbell et al., 197 I ; Rethy et al., 1971, 1972); as shown here they can be used also for the study of PGE I sensitive adenylate cyclase. Of particular significance was the role of GTP in eliciting the stimulatory effect of PGE,. In the absence of GTP the activation of adenylate cyclase by PGE i was limited and inconsistent, while yet at IO-” M GTP, PGE, stimulated adenylate cyclase S-fold. This requirement of PCE,-sensitive cyclase of liver plasma membrane for GTP is nearly absolute while in the case of glucagon sensitive cyclase the requirement for GTP is only partial since glucagon became about twice eflective in the presence of the nucleotide. This is in accordance with the studies of Sweat and Wincek (1973). Absolute requirements for GTP of PGE,-sensitive cyclases were reported also for thyroid plasma membranes (Mashiter and Field, 1974) and for platelet membranes (Krishna et al., 1972). It is not clear to which extent GTP is specific in this action. In our hands UTP and CTP could not substitute for GTP, however it has been reported (Sweat and Wincek, 1973) that ITP and XTP can replace GTP in liver and that GDP or GMP are as effective as GTP in thyroid plasma membranes (Wolff and Cook, 1973). Adenylate cyclase of isolated plasma membranes is very sensitive to PCE,, threshold stimulation occurred with 0.5 uM PGE ,, while using particulate preparations it has been found that the threshold concentration was I.4 PM. Optimal stimulation of adenylate cyclase required 3 yM PGE,, which compares with 7 uM PGE, in the case of liver particulate fractions (Sweat and Wincek. 1973), with 2 uM in the case of platelet membranes (Krishna et al., 1972) and with 30 uM PGE, in the case of thyroid (Wolff and Cook, 1973). At concentrations higher than 50 uM PGE, became a less effective stimulant. This disagrees with the findings of Sweat an3 Wincek (1973), however a similar finding was reported by Mashiter and Field (1974) for the PGE,-sensitive adenylate cyclase of the thyroid. Several lines of evidence were presented indicating that PGE, is acting on liver adenylate cyclase via binding sites separate from those of glucagon and epinephrine. First, we observed additivity when combinations of PGEi with glucagon or epinephrine were tested on adenylate cyclase. This finding also excludes a role of PGE, as an intermediate in the action of other hormones in liver. Additive effects of PGE, and TSH have been reported on isolated thyroid plasma membranes (Mashiter and Field, 1974), however Sweat and Wincek
PGE,-stimulated adenylate cyclase
(1973) failed to detect additive
231
effects by glucagon
and PGE,
using liver crude
particulate fractions. A second point in favour of discrete PGE, binding sites is the effect of temperature on PGE, and glucagon sensitive cyclase. Clearly the two reactions were influenced by variations of the temperature of incubation in a very dissimilar way. A third point was that propranolol which nearly completely blocks epinephrine effect, is much less effective towards PGE, stimulation. The effects of Lubrol on hormonal sensitivity are intriguing but, at least at very low doses, the detergent appears to affect positively PGE, sensitivity and negatively epinephrine and glucagon sensitivity. Some recent studies demonstrate binding sites for labeled prostaglandins in plasma membranes of several tissues and cells including liver (Smigel and Fleischer, 1974) adipose tissue (Gorman and Miller, 1973) kidney (Attallah and Lee, 1973) and thymocytes (Schaumburg, 1973). Unfortunately in none of these studies the binding was correlated to adenylate cyclase activation. It is interesting that rat liver plasma membranes contain low and high affinity binding sites for PGE, (Smigel and Fleisher, 1974) and the same is true for glucagon (Giorgio et al., 1974), insulin (De Meyts et al., 1973) and epinephrine (Dunnick and Marinetti, 1972; Tomasi et al., 1974). For these hormones the Scatchard plots are non-linear which according to De Meyts et al., (1973) suggests negative cooperative effects in the interaction between ligand and receptor. Gorman and Miller (1973) have observed that GTP enhances PGE, binding to rat adipocytes plasma membranes. However considerable binding occurs also in the absence of GTP therefore it is dubious whether the most important role of GTP is in the binding step or in some other step of the mechanism of PGE f activation of adenylate cyclase. The in vitro stinlulation by PGE, of rat liver adenylate cyclase independent from that of hormones which act by stimulating this enzyme establishes the possibility that this compound has a direct role in controlling rat liver metabolism by increasing cyclic AMP levels. Studies on perfused rat liver are in contrast with this possibility (Exton et al., 1971). However it was recently observed that addition of PGE, to isolated rat liver cells results in a marked increase of cyclic AMP and in the activation of glycogenolysis (Barnabei et al., 1974; Tomasi et al., 1975).
232
V. Tomasi and E. Ferretti
REFERENCES Attallah, A. A. and Lee, J. B. (1973) Prostaglandins 4, 703. Barnabei, O., Leghissa, G. and Tomasi, V. (1974) Biochim. Biophys. Acta 362, 316. Butcher, R. W., Pike, J. E. and Sutherland, E. W. (1967) In: Prostaglandins, Eds.: S. Bergstrom and B. Samuelsson (Wiley-Interscience, New York) p. 133. Butcher, R. W. and Baird, C. E. (1968) J. Biol. Chem. 243, 1713. De Meyts, P., Roth, J., Neville, D. M., Gavin, J. R. and Lesmak, M. A. (1973) Biochem. Biophys. Res. Commun. 55, 154. Donnick, J. K. and Marinetti, G. V. (1971) Biochim. Biophys. Acta 249, 122. Exton, J. H., Robison, G. A., Sutherland, E. W. and Park, C. R. (1971) J. Biol. Chem. 246, 6166. Giorgio, N. A., Johnson, C. B. and Blecher, M. (1974) J. Biol. Chem. 249, 428. Gorman, R. R. and Miller, 0. V. (1973) Biochim. Biophys. Acta 323, 560. Kaneko, T., Zor, V. and Field, J. B. (1969) Science 163, 1062. Kreiner, P. W., Keirns, J. J. and Bitensky, M. W. (1973) Proc. Natl. Acad. Sci. U.S. 70, 1785. Krishna, G., Harwood, J. T., Barber, A. J. and Jam&on, G. A. (1972) J. Biol. Chem. 247, 2253. Kuehl, F. A., Hames, J. L., Tarnoff, J., Cirillo, V. J. and Ham, E. A. (1970) Science 169, 883. Marinetti, G. V., Ray, T. K. and Tomasi, V. (1969) Biochem. Biophys. Res. Commun. 36, 185. Marsh, J. M. (1970) FEES Letters 7, 283. Mashitez, K. and Field. J. B. (1974) Fed. Proc. 33, 78. Pohl, S. L., Birnbaumer, L. and Rodbeli, M. (1971) J. Biol. Chem. 246, 1849. Ramwell, P. W. and Shaw, J. E. (1970) Recent Progr. Hormone Res. 26, 139. Ray, T. K. (1970) Biochim. Biophys. Acta 196, 1. Ray, T. K., Tomasi, V. and Marinetti, G. V. (1970) Biochim. Biophys. Acta 21 I, 20. Rethy, A., Tomasi, V. and Trevisani, A. (1971) Arch. Biochem. Biophys. 147, 36. Rethy, A., Tomasi, V., Trevisani, A. and Barnabei, 0. (1972) Biochim. Biophys. Acta 290, 58. Robison, G. A., Arnold, A. and Hartmanil, R. C. (1969) Pharm. Res. tommun. 1, 323, Rodbell, M., Birnbaumer, L., Pohl, S. L. and Krans, H. M. (1971) J. Biol. Chem. 246, 1877. Schaumburg, B. P. (1973) Biochim. Biophys. Acta 326, 127. Smigel, M. and Fleischer, S. (1974) Biochim. Biophys. Acta 332, 358. Sobel, B. E. and Robison, A. (1969) Circulation 40, suppl. III, 189. Sweat, F. W. and Wincek, T. J. (1973) Biochem. Biophys. Res. Commun. 55, 522. Tomasi, V., Koretz, S., Ray, T. K., Dunnick, J. and Marinetti, G. V. (1970) Biochim. Biophys. Acta 21 I, 31. Tomasi, V., Poli, A., Ferretti, M. E. and Barnabei, 0. (1975) Adv. Enzyme Regul. (in press). Tomasi, V., Trevisani, A., Barnabei, 0. and Sereni, F. (1974) In: Proceedings of International Symposium on Perinatal Pharmacology, Eds.: P. Morselli and F. Sereni (Raven Press, New York) (in press). Wolff, J. and Cook, G. H. (1973) J. Biol. Chem. 248, 350.
2 (1975) 221-232. 0 North-Holland
Publ. Comp.
PROSTAGLANDIN Ed-SENSITIVE ADENYLATE CYCLASE RAT LIVER PLASMA MEMBRANES Vittorio
TOMASI
ofGeneral Physiology,
Institute
and Enrica University
OF
FERRETTI
of Ferrara, 44100 Ferrara,
Received 14 October 1974
Italy
Accepted 12 December
1974
Adenylate cyclase of isolated rat liver plasma membranes is stimulated at least four-fold by prostaglandin E, (PGE,) provided that EGTA and CTP are present in the assay system. The GTP-dependency is absolute, since in its absence the PGE, activation of adenylate cyclase is small and inconsistent. PGE, is much less effective while prostaglandins Fra and F,, are ineffective under all conditions tested. The stimulatory effect of PGE, is evident at concentrations around 0.5 uM, optimal stimulation requires 3 uM PC&. Several lines of evidence suggest that PGEr interacts with plasma membrane binding sites separate from those specific for glucagon and epinephrine. First, the effect of combinations of PGEl with glucagon or epinephrine at optimal concentrations are additive, while not additive is the combination PGE,-NaF. Second, variations in the temperature of incubation between 25 and 45 “C influence in a different way glucagon and PGEt-sensitive cyclase. Glucagon stimulation continuously increases with the increase of temperature while PGEr stimulation increases up to 30 “C then it levels off and decreases at 45 ‘C. Third, propranolol which nearly completely blocks epinephrine stimulation is much less effective on PC&sensitive cyclase. 0.1 mM phentolamine has little effect, if any, on hormonal sensitivity. Lubrol at very low doses (1 mg/iOO ml) reduces the effect of epinephrine and glucagon, but increases that of PGE,. Keywords:
PGEr ; adenylate
Prostaglandins
are known
cyclase;
liver plasma membrane;
to be capable
of raising
epinephrine;
cyclic
AMP
glucagon.
levels
in
several cells and tissues (Ramwell and Shaw, 1970) including platelets (Robison et al., 1969), ovary (Kuehl et al., 1970), lung and spleen (Butcher and Baird, 1968). Adenylate cyclase activities sensitive to prostaglandins have been described in membranes isolated from platelets (Butcher et al., 1967) thyroid (Kaneko et al., 1969), ovary (Marsh, 1970), and heart (Sobel and Robison, 1969). In liver a prostaglandin E, (PGE,) stimulation of adenylate cyclase of broken-ceil homogenates has been reported by Bitensky et al. (1972) and more recently by Sweat and Wincek (1973).
V. Tomasi and E. Ferretii
222
In this paper, the stimulation of adenylate cyclase of isolated rat liver plasma membrane by PGE, and the absolute dependency of this activation on GTP are reported. In addition, evidence is presented indicating that PGE, interacts with plasma membrane sites which are separate from those specific for glucagon and epinephrine. While this work was in progress it has been shown (Smigel and Fleisher, 1974) that rat liver plasma membranes possess high affinity binding sites for the E type prostaglandins.
MATERIALS
AND
METHODS
Plasma membranes were isolated from livers of rats weighing less than 100 g by the method of Ray (1970). The use of young rats greatly facilitated the observation of a response of adenylate cyclase to epinephrine. Membranes were used within 2 h after the final preparative step. Adenylate cyclase was assayed in a system containing in a final volume of 0.4 ml: I .O mM ATP, 3.75 mM MgS04, 25 mM Tris-HCI, pH 8.0, 6.2 mM aminophylline and 50-100 c(g of membrane proteins. 2.5 mM phosphoenolpyruvate and 50 ug/ml of pyruvate kinase were generally used as ATP regenerating system. However it was found that in our experimental conditions omission of the regenerating system had negligible eflects on basal and hormone stimulated activities at least up to 5 min of incubation at 37 “C. Unless otherwise stated GTP was 0.5 mM and EGTA 0.1 mM. Incubations were generally carried out for 5 min at 37 “C and the reactions were terminated by immersing the tubes in boiling water for 2 min. The tubes frozen at -20 “C were then thawed and centrifuged at 1000 g for IO min. The supernatants were diluted I : 5 with 0.01 M Tris-HCI, pH 7.5 and cyclic AMP was assayed as described by Brown et al. (1972) with the precautions previously reported (Barnabei et al., 1974). The amount of cyclic AMP formed was linear with the time of incubation at least up to IO min (up to 5 min in the absence of a regenerating system) and with the membrane concentration within 100 ug of proteins. Values are usually averages of triplicate determinations; standard errors are less than 10% for the same membrane preparation. Some variations between different membrane preparations were observed as far as the extent of hormonal stimulation is concerned. Proteins were determined according to Lowry et al. (1951) using bovine serum albumin as a standard. Albino Wistar rats fed ad libitum were used. ATP disodium salt, GTP lithium or sodium salts, phosphoenolpyruvate, pyruvate kinase, epinephrine bitartrate, aminophylline were products of Sigma Chem. Co., St. Louis, U.S.A. Glucagon (crystalline) was a gift of Dr. R. Chance, E. Lilly, Indianapolis, U.S.A. Prosta-
PGE,-stimulated
glandins
adenylate cyclase
were kindly
provided
mazoo, U.S.A. Adenosine Ci/mmole) was a product
223
by Dr. J. Pike, The Upjohn
3’-5’ (7)3H-monophosphate of Radiochemical Centre,
Company,
Kala-
cyclic (spec. act. 27 Amersham, England.
EGTA was a product of Serva, Heidelberg, Germany. Lubrol 12A-9 (formerly known as Lubrol PX) was a gift of Imperial Chemical Industries, England. Phentolamine (Regitin) was a product of Ciba, Basel, Switzerland. Propranolol was a product of Ayerst Laboratories, New York, U.S.A.
RESULTS in a first series of experiments (table 1) we tested the effect of four prostaglandins, glucagon and sodium fluoride on rat liver plasma membrane adenylate cyclase. In comparison to the marked effects of glucagon and fluoride (Cfold stimulation) a slight stimulation of the enzyme by PGE, and PGF,, and no effects with PGE, and PGF,, were observed. However in the light of the observations by Rodbell et al. (1971) on the role of GTP on the glucagonsensitive adenylate cyclase and by Sweat and Wincek (1973) on the PGE, sensitive enzyme, it was considered of importance to test the effect of GTP. In table 1 it is shown that in the presence of 0.5 mM GTP in the assay
Table I The effects of prostaglandins, glucagon and fluoride on adenylate cyclase activity assayed in the absence and presence of GTP. Adenylate cyclase was assayed as described under Materials and Methods in the absence of EGTA. All prostaglandins except PGFZa which was dissolved in water, were dissolved in 95% ethanol and 10 ul of the solutions (8 ug) were added to the assay system. 10 pl of 95% ethanol was found to have no effect on control activity. Glucagon was 1 x 1Od6 M, sodium fluoride 10 mM and GTP 0.5 mM. Data are means I SE, in parenthesis the number of experiments is reported. Adcnylate cyclase activity (pmoles/mg -
Control (4) PGF,, (2) PGF,, (2) PGF, (4) PGE, (2) Glucagon (4) NaF (4)
83 96 143 145 65 337 339
GTP
-1: 7.1
$z 22 :t 20 _rt 24
i
100 115 172 173 78 406 408
109 95 87 600 164 745 618
protein/5 min)
GTP
i_
8.4
i 45 & 51 f_ 60
100 81 80 550 150 683 567
1.3 1.0 0.6 4.1 2.5 2.2 1.8
224
V. Tomasi and E. Ferretfi
system, PGE, was a potent stimulant of adenylate cyclase, while PGE, was much less active and the F type prostaglandins were ineffective. Also glucagon, as expected (Rodbell et al., 1971), had an higher efficacy when GTP was present, while the effect of GTP on fluoride sensitivity is in contrast with the studies of Rodbell et al. (1971).
lOOOr
900
700 _
I
/I 0
"
16
lo-6 "@
Fig.
I 1O-3 M
[GTP]
I. The effect of PGE, on adenylate cyclase activity was assayed at GTP concentrations from 1 Y 10m6 to I >~ 10m3 M. The points represent the means of three experiments.
ranging PGE,
was present
at 4 ug/ml. activity
In these experiments in the absence
of GTP
the effects of PGE,
on adenylate
cyclase
was not significant.
In fig. 1 the GTP concentration was varied between lo-’ and lop3 M. The half-maximal GTP concentration for PGE, stimulation was around 2 x 10e5 M, the nucleotide having practically no effect on basal activity. The most effective of other nucleotides tested in addition to GTP, was UTP. However
PGE,-stimulated
adenyiate cyctase
225
PGE,
1 0
b
’
2
p
*
4
0
’
6
’
’
*
8
’ 10
’
’
’
12 PGE,
’
0
14
16
p9/0,4ml
Fig. 2. The effect of PGE, at different concentrations on adenylate cyclase activity. The insert was drawn on semilog paper. Different solutions of PGE, in 957; ethanol were prepared in order to use not more than 15 ~1 of solution (0.1 Mg/O.4ml is equivalent to 0.7 pM PGE,). The points represent the means of three determinations.
in the presence
of UTP, stimulation
by PGEl
was about
15 % of that observed
with GTP (not shown). Therefore in all successive experiments 0.5 mM GTP was included in the assay system. In fig. 2 the PGE, concentration was varied between 7 x 10e7 and 10T4 M. The stimulatory effect was evident at concentrations as low as 7 x lo-’ M (0.1 pg per tube) and the optimal effect was obtained with 1.4 x lop5 M PGE, (2 pg per tube). At higher concentrations the extent of stimulation declined. In the successive experiments therefore PGE, was added at concentrations ranging from 2 to 4 pg per tube. We observed that addition of EGTA to the incubation system markedly enhanced the effects of hormones used and of sodium fluoride (fig. 3). Of particular interest was the effect of EGTA on epinephrine sensitive cyclase. As previously observed (Marinetti et al., 1969; Pohl et al., 1971) this hormone is slightly effective on the liver plasma membrane cyclase, however in the
V. Tomusi and E. Fermtti
226
0.
NaF O_
0.
Adrenahne PGE,
4yJ
/ 0.
I 0
/
i
I,
0
1O-6
1cr4M
10-s EGTA
Fig. 3. The effect of EGTA
on adenylate cyclase activity. EGTA
concentration was varied
between lo-” and 10m4M. Epinephrine was I > IO-” M, glucagon 1 Y IO--” M and fluoride 10 mM.
presence of 10m4 M EGTA it becomes much more effective (more than d-fold stimulation). Also the sensitivity of adenylate cyclase to glucagon and fluoride and to a lesser extent to PGE1, was enhanced by EGTA. The effect of EGTA seems to be due not simply to a chelation of calcium ion since we observed that addition of Ca2+ does not completely reverse the effect of EGTA (not shown). On the other hand its effect on basal and NaF stimulated activities is probably due to chelation of calcium ions which membranes may bind during the preparation ; as a matter of fact Ray’s method (1970) involves the inclusion of 0.5 mM calcium in the homogenization buffer. It was considered of interest to investigate whether or not the effects of PGE, on adenylate cyclase were mediated by a receptor separate from those of
PGEl-stimulated
227
adenylate cyclase
Table The effect of combinations
of PGE,,
activity.
conditions
For
cyclase
experimental
was assayed
in the presence
glucagon,
2 epinephrine
see Materials
and
of 0.5 mM GTP
and fluoride Methods
and
and 0.1 mM EGTA.
on adenylate table
cyclase
1. Adenylate
In parenthesis
are
in the case of additivity. In experiment 1 membranes were prepared from a rat weighing 90 g, in experiment 2 two rats of 40 g were used. Data are means of
the values
calculated
duplicate Increase
determinations.
of adenylate
cyclase
Exp.
I :’ 10m5 M
Epinephrine Glucagon PGE, NaF
989
M
239
I x IO-’ M
Epinephrine Glucagon NaF
I41
1 ,. 10eh M
3 x lo-”
347
i- PGE, -
336 1380
PGE,
478
~- PGE,
Epinephrine
I
+ glucagon
+ PGE,
Control
1576 120
(380) (1228) (586) (1369)
activity
(pmoles/mg
protein/5
min)
Exp. 2
255 840 430 485 645 1310 725 1510 135
(685) (1270) (915) (1525)
As a first approach to this problem we tested between PGE, and glucagon or epinephrine. The results reported in table 2 indicate that in two experiments there was additivity in the stimulation of adenylate cyclase by combinations of epinephrine or glucagon with PGE,. In parenthesis the values expected in the case of additivity are reported. Clearly not additive was the combination PGE,-NaF. This kind of evidence in favour of a distinct PGE, receptor was corroborated by other lines of evidence. Bitensky et al. (1972) have studied the effect of temperature on glucagon, epinephrine and PGE ,-sensitive adenylate cyclase of liver. They found breaks in the Arrhenius plots for PGE, and for glucagon but not for epinephrine. Our results reported in fig. 4 indicate that variations in the temperature of incubation have different effects on glucagon and on PGEl sensitive cyclases. glucagon whether
and
epinephrine.
or not there
was additivity
Up to 30 “C both hormones become increasingly effective, afterwards the increase of temperature further enhances glucagon effect without influencing PGE, effect, at least up to 37 “C. At 45 “C glucagon is more effective than at 37 “C, while PGE, is less effective. Further evidence about the distinctness of the binding sites come from experiments with blocking agents. In table 3 it is shown that propranolol (10-5-10p4 M) almost abolished the epinephrine sensitivity of adenylate
V. Tomasi and E. Ferretti
228
1300.
1100 .
,
. , N -i Glucagon
I
x, , 900.
I I
,’ E ll? .700. .E 3 :
i5,,. E ," a s $300. 5 xc : :: 100 .
22
Fig.
4. The
25
effect
27
30
of temperature
on
equilibrated
at the temperatures
membranes.
Up to 30 “C the effect glucagon.
35
PGE,
indicated
Each point
and
of PGE,
and
37
glucagon-sensitive
the reaction was
is the mean
not
cyclase.
was started
significantly
4spc
Temperature
Tubes
were
by the addition
different
from
that
of of
of two determinations.
cyclase, while its effect on the PGE, stimulation was much less pronounced. Propranolol had no effect on the sensitivity of the cyclase towards glucagon. On the other hand phentolamine (lop4 M) does not appear to modify greatly the hormonal sensitivity of adenylate cyclase. In fig. 5 the effect of Lubrol on the hormonal sensitivity is reported. When added to the incubation system at very low concentrations (1 mg/lOO ml) the detergent produced a decrease of epinephrine and glucagon stimulation, but potentiated the effect of PGE,. At higher doses of Lubrol a decrease in the sensitivity of adenylate cyclase to hormones and to a lesser extent to fluoride was observed. At 10 mg Lubrol per 100 ml basal and fluoride stimulated activities were not very different from controls while the effect of hormones was markedly reduced.
PGE,-stimulated
229
adenylate cyclase
Table The effect
of a and
experimental while
a reduction
experiments. isolated
e blocking
conditions
agents
see Methods
of 20%
from
pools
on hormonal
and table
was observed
The effect of propranolol of livers
of
Increase
3
with
sensitivity
2. Phentolamine propranolol.
was evaluated
IO-day
old rats, epinephrine.
over basal
Control
of adenylate
Data
are means
in two experiments. in order
activity
to have
(pmoles/mg
M
Epinephrine Glucagon
900 * 74 1616 * 121
776 i 105 1236 % 110
PGEl
1130 + 33
1136 :t 65
basal i
For
activity
SE of three
Membranes
a greater
protein/5
Phentolamine lo-“
cyclase.
did not affect
were
response
to
min)
Propranolol lo-“
M
10m5 M
5 x 10m5 M
142 1763
140 1525
80 1545
690
670
695
q Control q Eplnephrlne qGlucagon q PGE, q N~F
5
10 [Lubrog
Fig.
5. The effect of Lubrol
membrane Means
adenylate
cyclase.
of two experiments
at different For
mg / lOOmI
concentrations
hormones
are reported.
and
on the hormonal NaF
The ATP
concentrations regenerating
sensitivity see under
system
of plasma table
was omitted.
2.
230
DISCUSSION Rat liver plasma membranes have been extensively used for the study of glucagon and epinephrine sensitive adenylate cyclase (Marinetti et al., 1969; Ray et al., 1970; Tomasi et al., 1970; Pohl et al., I971 ; Rodbell et al., 197 I ; Rethy et al., 1971, 1972); as shown here they can be used also for the study of PGE I sensitive adenylate cyclase. Of particular significance was the role of GTP in eliciting the stimulatory effect of PGE,. In the absence of GTP the activation of adenylate cyclase by PGE i was limited and inconsistent, while yet at IO-” M GTP, PGE, stimulated adenylate cyclase S-fold. This requirement of PCE,-sensitive cyclase of liver plasma membrane for GTP is nearly absolute while in the case of glucagon sensitive cyclase the requirement for GTP is only partial since glucagon became about twice eflective in the presence of the nucleotide. This is in accordance with the studies of Sweat and Wincek (1973). Absolute requirements for GTP of PGE,-sensitive cyclases were reported also for thyroid plasma membranes (Mashiter and Field, 1974) and for platelet membranes (Krishna et al., 1972). It is not clear to which extent GTP is specific in this action. In our hands UTP and CTP could not substitute for GTP, however it has been reported (Sweat and Wincek, 1973) that ITP and XTP can replace GTP in liver and that GDP or GMP are as effective as GTP in thyroid plasma membranes (Wolff and Cook, 1973). Adenylate cyclase of isolated plasma membranes is very sensitive to PCE,, threshold stimulation occurred with 0.5 uM PGE ,, while using particulate preparations it has been found that the threshold concentration was I.4 PM. Optimal stimulation of adenylate cyclase required 3 yM PGE,, which compares with 7 uM PGE, in the case of liver particulate fractions (Sweat and Wincek. 1973), with 2 uM in the case of platelet membranes (Krishna et al., 1972) and with 30 uM PGE, in the case of thyroid (Wolff and Cook, 1973). At concentrations higher than 50 uM PGE, became a less effective stimulant. This disagrees with the findings of Sweat an3 Wincek (1973), however a similar finding was reported by Mashiter and Field (1974) for the PGE,-sensitive adenylate cyclase of the thyroid. Several lines of evidence were presented indicating that PGE, is acting on liver adenylate cyclase via binding sites separate from those of glucagon and epinephrine. First, we observed additivity when combinations of PGEi with glucagon or epinephrine were tested on adenylate cyclase. This finding also excludes a role of PGE, as an intermediate in the action of other hormones in liver. Additive effects of PGE, and TSH have been reported on isolated thyroid plasma membranes (Mashiter and Field, 1974), however Sweat and Wincek
PGE,-stimulated adenylate cyclase
(1973) failed to detect additive
231
effects by glucagon
and PGE,
using liver crude
particulate fractions. A second point in favour of discrete PGE, binding sites is the effect of temperature on PGE, and glucagon sensitive cyclase. Clearly the two reactions were influenced by variations of the temperature of incubation in a very dissimilar way. A third point was that propranolol which nearly completely blocks epinephrine effect, is much less effective towards PGE, stimulation. The effects of Lubrol on hormonal sensitivity are intriguing but, at least at very low doses, the detergent appears to affect positively PGE, sensitivity and negatively epinephrine and glucagon sensitivity. Some recent studies demonstrate binding sites for labeled prostaglandins in plasma membranes of several tissues and cells including liver (Smigel and Fleischer, 1974) adipose tissue (Gorman and Miller, 1973) kidney (Attallah and Lee, 1973) and thymocytes (Schaumburg, 1973). Unfortunately in none of these studies the binding was correlated to adenylate cyclase activation. It is interesting that rat liver plasma membranes contain low and high affinity binding sites for PGE, (Smigel and Fleisher, 1974) and the same is true for glucagon (Giorgio et al., 1974), insulin (De Meyts et al., 1973) and epinephrine (Dunnick and Marinetti, 1972; Tomasi et al., 1974). For these hormones the Scatchard plots are non-linear which according to De Meyts et al., (1973) suggests negative cooperative effects in the interaction between ligand and receptor. Gorman and Miller (1973) have observed that GTP enhances PGE, binding to rat adipocytes plasma membranes. However considerable binding occurs also in the absence of GTP therefore it is dubious whether the most important role of GTP is in the binding step or in some other step of the mechanism of PGE f activation of adenylate cyclase. The in vitro stinlulation by PGE, of rat liver adenylate cyclase independent from that of hormones which act by stimulating this enzyme establishes the possibility that this compound has a direct role in controlling rat liver metabolism by increasing cyclic AMP levels. Studies on perfused rat liver are in contrast with this possibility (Exton et al., 1971). However it was recently observed that addition of PGE, to isolated rat liver cells results in a marked increase of cyclic AMP and in the activation of glycogenolysis (Barnabei et al., 1974; Tomasi et al., 1975).
232
V. Tomasi and E. Ferretti
REFERENCES Attallah, A. A. and Lee, J. B. (1973) Prostaglandins 4, 703. Barnabei, O., Leghissa, G. and Tomasi, V. (1974) Biochim. Biophys. Acta 362, 316. Butcher, R. W., Pike, J. E. and Sutherland, E. W. (1967) In: Prostaglandins, Eds.: S. Bergstrom and B. Samuelsson (Wiley-Interscience, New York) p. 133. Butcher, R. W. and Baird, C. E. (1968) J. Biol. Chem. 243, 1713. De Meyts, P., Roth, J., Neville, D. M., Gavin, J. R. and Lesmak, M. A. (1973) Biochem. Biophys. Res. Commun. 55, 154. Donnick, J. K. and Marinetti, G. V. (1971) Biochim. Biophys. Acta 249, 122. Exton, J. H., Robison, G. A., Sutherland, E. W. and Park, C. R. (1971) J. Biol. Chem. 246, 6166. Giorgio, N. A., Johnson, C. B. and Blecher, M. (1974) J. Biol. Chem. 249, 428. Gorman, R. R. and Miller, 0. V. (1973) Biochim. Biophys. Acta 323, 560. Kaneko, T., Zor, V. and Field, J. B. (1969) Science 163, 1062. Kreiner, P. W., Keirns, J. J. and Bitensky, M. W. (1973) Proc. Natl. Acad. Sci. U.S. 70, 1785. Krishna, G., Harwood, J. T., Barber, A. J. and Jam&on, G. A. (1972) J. Biol. Chem. 247, 2253. Kuehl, F. A., Hames, J. L., Tarnoff, J., Cirillo, V. J. and Ham, E. A. (1970) Science 169, 883. Marinetti, G. V., Ray, T. K. and Tomasi, V. (1969) Biochem. Biophys. Res. Commun. 36, 185. Marsh, J. M. (1970) FEES Letters 7, 283. Mashitez, K. and Field. J. B. (1974) Fed. Proc. 33, 78. Pohl, S. L., Birnbaumer, L. and Rodbeli, M. (1971) J. Biol. Chem. 246, 1849. Ramwell, P. W. and Shaw, J. E. (1970) Recent Progr. Hormone Res. 26, 139. Ray, T. K. (1970) Biochim. Biophys. Acta 196, 1. Ray, T. K., Tomasi, V. and Marinetti, G. V. (1970) Biochim. Biophys. Acta 21 I, 20. Rethy, A., Tomasi, V. and Trevisani, A. (1971) Arch. Biochem. Biophys. 147, 36. Rethy, A., Tomasi, V., Trevisani, A. and Barnabei, 0. (1972) Biochim. Biophys. Acta 290, 58. Robison, G. A., Arnold, A. and Hartmanil, R. C. (1969) Pharm. Res. tommun. 1, 323, Rodbell, M., Birnbaumer, L., Pohl, S. L. and Krans, H. M. (1971) J. Biol. Chem. 246, 1877. Schaumburg, B. P. (1973) Biochim. Biophys. Acta 326, 127. Smigel, M. and Fleischer, S. (1974) Biochim. Biophys. Acta 332, 358. Sobel, B. E. and Robison, A. (1969) Circulation 40, suppl. III, 189. Sweat, F. W. and Wincek, T. J. (1973) Biochem. Biophys. Res. Commun. 55, 522. Tomasi, V., Koretz, S., Ray, T. K., Dunnick, J. and Marinetti, G. V. (1970) Biochim. Biophys. Acta 21 I, 31. Tomasi, V., Poli, A., Ferretti, M. E. and Barnabei, 0. (1975) Adv. Enzyme Regul. (in press). Tomasi, V., Trevisani, A., Barnabei, 0. and Sereni, F. (1974) In: Proceedings of International Symposium on Perinatal Pharmacology, Eds.: P. Morselli and F. Sereni (Raven Press, New York) (in press). Wolff, J. and Cook, G. H. (1973) J. Biol. Chem. 248, 350.
