Synergistic Stimulation of the Ca2+ Influx in Rat Hepatocytes by Glucagon and the Ca2+-linked Hormones Vasopressin and Angiotensin 11”

Glucagon was added to isolated rat hepatocytes, either alone or together with vasopressin or angiotensin 11, and the effects on the initial 45Ca’+ uptake rate were investigated. Addition of glucagon alone which increased cyclic AMP content of the cells slightly in- creased the initial 4aCa’’ uptake rate. When glucagon was added together with vasopressin or angiotensin 11-both of which when added separately increase the initial 4aCa’+ uptake rate but did not affect the cellular content of cyclic AMP-the measured initial 45Ca’+ uptake rate was larger than the sum of that seen with each hormone alone. This indicates that glucagon and Ca’+-linked hormones synergistically enhanced the Ca2+ influx in rat hepatocytes. These effects of glucagon can be mimicked by dibutyryl cyclic AMP or for- skolin, suggesting that cyclic AMP augments both the resting Ca’+ and the vasopressin- or angiotensin II- stimulated influx. Measurement of the intitial 45Ca’+ uptake rate as a function of the extracellular Ca’+ concentration indicated that the increase in the ca’+ influx resulting from single or combined glucagon and vasopressin admin-istration occurred through a homogeneous population of Ca’+ gates. These hormones were found to

Glucagon was added to isolated rat hepatocytes, either alone or together with vasopressin or angiotensin 11, and the effects on the initial 45Ca'+ uptake rate were investigated. Addition of glucagon alone which increased cyclic AMP content of the cells slightly increased the initial 4aCa'' uptake rate. When glucagon was added together with vasopressin or angiotensin 11-both of which when added separately increase the initial 4aCa'+ uptake rate but did not affect the cellular content of cyclic AMP-the measured initial 45Ca'+ uptake rate was larger than the sum of that seen with each hormone alone. This indicates that glucagon and Ca'+-linked hormones synergistically enhanced the Ca2+ influx in rat hepatocytes. These effects of glucagon can be mimicked by dibutyryl cyclic AMP or forskolin, suggesting that cyclic AMP augments both the resting Ca'+ and the vasopressin-or angiotensin IIstimulated influx.
Measurement of the intitial 45Ca'+ uptake rate as a function of the extracellular Ca'+ concentration indicated that the increase in the ca'+ influx resulting from single or combined glucagon and vasopressin administration occurred through a homogeneous population of Ca'+ gates. These hormones were found to raise both the apparent K,,, for external Ca'+ and the apparent V , , of the Ca'+ influx. The maximal increase in these two parameters was observed when the two hormones were added together. This suggests that glucagon and vasopressin synergistically stimulate the same Ca" gating mechanism.
The dose-response curves for the action of glucagon or vasopressin applied in the presence of increasing concentrations of vasopressin or glucagon, respectively, showed that each hormone increases the maximal response to the other without affecting its EDSO. It is proposed that glucagon and the Ca'+-linked hormones control the cellular concentration of two intermediates which are both necessary to allow Ca'+ entry into the cells.
Cyclic AMP and calcium, whose intracellular concentrations are regulated by different receptors, play a major role in the control of the cellular activity. They serve as intracellular messengers in the same types of tissue and both of them *Preliminary results of this work have been presented in the "Proceedings of the 9th European Symposium on Hormones and Cell Regulation," Mt. St. Odile. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. convey to the same type of response (for reviews see: Berridge, 1982Rasmussen and Barrett, 1984). In some systems, such as platelets or smooth muscles, cyclic AMP has an inhibitory effect whereas that of calcium is stimulatory (Rasmussen, 1983). However, in others such as the secretory tissues (Berridge, 1982) or the liver (Exton, 1982), the action of both messengers is stimulatory.
However, the cyclic AMP and calcium messenger systems are to some extent interdependent in mammalian liver. Thus, the intracellular Ca2+ concentration was observed to be increased by glucagon (Charest et al., 1983;and Footnote 1, but see Studer et al., 1984) and the 8-adrenergic agonist isoproterenol.' It has also been reported that glucagon can reverse the net movements of Ca2+ induced by the Ca2+-linked hormones (Assimacopoulos-Jeannet et aZ., 1982;Morgan et al., 1983). Vasopressin and angiotensin I1 also inhibit the accumulation of cyclic AMP in isolated hepatocytes stimulated by glucagon (Crane et Keppens and De Wulf, 1984). Finally, cyclic AMP-dependent and Ca2+-linked hormones have been shown to act synergistically to induce hyperpolarization and effluxes of 3zK+ and 4SCa2+ in guinea pig liver slices or hepatocytes (Jenkinson and Koller, 1977;Cocks et al., 1984).
These findings combined with the fact that glucagon, like vasopressin, angiotensin 11, and phenylephrine, has long been known to increase 45Ca2+ uptake in rat hepatocytes (Keppens et al., 1977) prompted us to investigate the effect of glucagon on the resting Ca2+ influx in rat hepatocytes and on the influx stimulated by vasopressin and angiotensin 11.
The results show that by raising the intracellular concentration of cyclic AMP, glucagon acts synergistically with the B. Berthon, unpublished observation.  Ca2+-linked hormones to augment the Ca2+ influx through a homogeneous population of gates located in the plasma membranes.

EXPERIMENTAL PROCEDURES
Cell Isolation-Hepatocytes were isolated from female Wistar rats (200-250 g) as described by Burgess et al. (1981) except that the collagenase concentration was lowered to 0.15 mg/ml and the perfusion of the liver and incubation of the disaggregated liver with the collagenase solution were reduced to 7 and 5 min, respectively. The isolated cells were incubated at a concentration of 3 X lo6 cells/ml in Eagle's medium containing in mM: NaCl, 116; KCl, 5.4; CaC12, 1.8; MgC12, 0.81; NaH2P04, 0.92; NaHC03, 25; 1 g/l of glucose, amino acids, and vitamins, supplemented with 1.5% gelatin (Difco) and gassed with 95% 02, 5% Con. Gelatin was preferred to albumin as it allowed a better preservation of cell viability. Thirty min before the experiments began, the hepatocytes were resuspended in fresh solution of the same composition, supplemented with lmg/ml bacitracin to prevent degradation of the peptide hormones. Under these conditions, the cell viability, as judged by trypan blue exclusion, always exceeded 95% and remained stable until the end of the experiment, i.e. 4-5 h after the isolation.
Measurement of the Znitiul *Ca2' UDtake Rate-The initial %a2+ ~~~~ uptake rate was determined as previously described Poggioli et al., 1985). Briefly, cells were incubated in Eagle's medium with 1 pCi/ml of %az+; 100-p1 aliquots were taken at 15,45, 75, and 105 s after adding the &Ca2+. Each aliquot was diluted with 4 ml of ice-cold washing solution containing 144 mM NaC1,5 mM CaCIZ, and 5 mM Tris-HC1, pH 7.4, filtered through a Whatman GF/C glass fiber filter, and washed 3 times with 4 ml of washing solution. The radioactivity on the filter was counted with a liquid scintillation spectrometer. Unless otherwise indicated, the hormones tested were added 30 s before "Ca2'. 0 0 a 0 Cyclic AMP Content-The concentration of cyclic AMP in hepatocytes was determined by radioimmunoassay after deproteinization and acetylation of the samples.
Materials-Collagenase was obtained from Boehringer. (Arginine) vasopressin, angiotensin 11, glucagon, and Bt2cAMP2 were obtained from Sigma. Forskolin was from Calbiochem. 46Ca2+ was from I.R.E., Fleurus Belgium. The cyclic AMP radioimmunoassay kit was from Institut Pasteur Production, Marnes-La-Coquette, France. Fig. 1, maximal doses of glucagon and vasopressin raised the initial 45Ca2+ uptake rate by isolated liver cells. Vasopressin did so without affecting the intracellular content of cyclic AMP. Glucagon stimulated the initial 45Ca2' uptake rate to a lesser extent than vasopressin and increased the intracellular cyclic AMP concentration about 8-fold. These effects of glucagon lasted for at least 2 min. When the hepatocytes were preincubated with vasopressin plus glucagon for 30 s prior to the addition of 4sCa2+, the initial 45Ca2+ uptake rate rose to a higher level than the sum of the rises observed with each hormone alone. initial 45Ca2+ uptake rate was about the same as that seen when both hormones are added together. However, when vasopressin was added 1 min before glucagon, the initial 45Ca2+ uptake rate was stimulated to a lesser extent and the accumulation of cellular cyclic AMP was smaller. When the cells were preincubated for 2 min with both hormones, the initial 45Ca2+ uptake rate and the cyclic AMP concentration began to decrease slowly. These results indicated that the maximal synergistic effects of the hormone were observed when vasopressin was added after or together with glucagon. Consequently, in the experiments described below, the initial '5Ca2+ uptake rate was measured after the cells had been preincubated for 30 s with vasopressin or angiotensin I1 and glucagon. Since glucagon increases both initial "Ca2+ uptake rate and the cellular concentration of cyclic AMP, we tested the ability of the latter to accelerate the initial '5Ca2+ uptake rate and to potentiate the response of the Caz+-linked hormones. Fig. 2 shows that the permeant analog of cyclic AMP, the Bt2cAMP, was able, like glucagon, to increase the initial '5Ca2+ uptake rate slightly and to potentiate the response of vasopressin and angiotensin 11. Qualitatively similar results were obtained when the &terpene forskolin was used to activate the adenylate cyclase and increase the cellular concentration of cyclic AMP (Fig. 2). Fig. 2 also shows that the stimulating effects of vasopressin and angiotensin I1 on the initial 45Ca2+ uptake rate were not additive. This indicates that the response induced by the two Ca2+-linked hormones was not limited by the number of receptors activated. This might rule out the hypothesis that cyclic AMP potentiates the response to vasopressin and angiotensin I1 by increasing the number of available receptors for these hormones.

Effects of Glucagon and Ca2+-linked Hormones on the Initial 45Ca2+ Uptake Rate-As shown in
These findings indicated that cyclic AMP augments the Caz+ influx in rat hepatocytes and potentiates the response of the Ca2+-linked hormones. Do Ca2+-linked Hormones and Cyclic AMP Act on the Same Ca2+ Gating System?-The above findings suggest that vasopressin, angiotensin 11, and cyclic AMP act on the same Ca2+ gating systems. We have previously demonstrated that both the resting Ca2+ influx and that stimulated by noradrenaline, vasopressin, and angiotensin I1 followed Michaelis-Menten kinetics when the initial 45Ca2+ uptake rate was measured as a function of external Ca2+ concentrations . In the same report, it was suggested that the Ca2+linked hormones raised the rate of Ca2+ transport through the plasma membrane by the use of a single Ca2+ gating system. The same methodology was used in the present work to define the kinetic properties of the initial 45Ca2+ uptake rate in cells  Table I.

TABLE 1 Effects of glucagon and vasopressin on the K, and V,, of Ca2+ influx
Values are determined from Hofstee plots as described in Fig. 3. incubated with vasopressin and/or glucagon. Fig. 3 shows the Hofstee plots obtained from the initial 45Ca2' uptake rate measured in the presence of external Ca2+ concentrations ranging from 0.15 to 4.8 mM in control cells or in cells stimulated with 0.3 or 10 nM vasopressin in the presence of 10 nM glucagon or in its absence. For all the conditions tested, the Hofstee plots of the data fitted straight lines. This indicates that in each case ca'+ always entered the cells through a homogeneous population of gates. In addition, it was found that glucagon, like vasopressin, increased both the apparent K, and the apparent Vmax for the Ca'+ influx, determined from Fig. 3, and that these increases were larger when maximal doses of the two hormones were added together ( Table  I). The observation of parallel rises in K , and V,, suggests that the 2 hormones act synergistically to stimulate the Ca2+ influx in rat hepatocytes by raising the rate of Ca'+ transport through the plasma membrane, using the same gating system. Stimulation of the Initial 45Ca2+ Uptake Rate As a Function of the Glucagon and Vasopressin Concentrations-To further characterize the synergistic action of the two hormones, doseresponse curves for the action of each one were performed in the presence of increasing concentrations of the other. The data in Fig. 4A show that at all the vasopressin concentrations tested, glucagon increased the rise in the initial 45Ca2' uptake rate which occurred in responses to vasopressin without affecting its EDSO. Conversely, the dose-response curves for the action of glucagon plotted in the presence of increasing doses of vasopressin also show that vasopressin augments the re- sponse to glucagon without affecting its ED, (Fig. 4B). These data suggest that the two hormones enhance each other's maximal effects. This was more obvious when an alternative plot was used to express the results (Fig. 5, A and  B). In Fig. 5A the initial 45Ca2+ uptake rate measured in the presence of vasopressin plus glucagon is plotted as a function of the rate measured in the presence of vasopressin alone, each point being determined in the presence of one concentration of vasopressin. From the figure, which, for greater clarity, only includes 3 curves, it can be seen that the data fit straight lines whose slope is increased by glucagon. This indicates that glucagon enhances the maximal response (apparent Jmm) induced by vasopressin with stimulating factors being determined by the slopes of the lines in Fig. 5A. The dose-response curve for the stimulating effect of glucagon on the apparent J, . of the vasopressin response is shown in the inset of Fig. 5A. Symmetrical results were observed for the effect of vasopressin on the glucagon response, i.e. vasopressin increases in a dose-dependent manner the apparent Jmar of the glucagon response ( Fig. 5B and inset). Note that each hormone increased the apparent Jmax of the other with an ED, quite similar to that observed for stimulating '5Ca2+ uptake rate.
Correlation between the Initial 45Ca2+ Uptake Rate and the Cellular Level of Cyclic AMP-The results shown in Figs. 1  and 2 suggest that the effects of glucagon on the Ca2+ influx were mediated by cellular cyclic AMP. The data shown in Fig.  6, obtained in the same series of experiments as those described in Figs. 4 and 5, give the initial 45Ca2+ uptake rate as a function of the cellular concentration of cyclic AMP measured in the presence of increasing doses of glucagon or in its absence and in the presence of 0.3 or 10 nM vasopressin or in its absence. Under the 3 conditions, initial 45Ca2+ uptake rates increased with the concentrations of cyclic AMP, but reached a plateau at the highest concentrations. Vasopressin increased the maximal effect obtained in the presence of high cyclic AMP concentrations. 45Ca2+ uptake rates increased directly as the concentrations of cyclic AMP were raised above the basal level by the addition of glucagon. This might mean that the basal level of cyclic AMP is sufficient to allow the resting Ca2+ influx, as well as the stimulating effects of vasopressin and angiotensin 11.

DISCUSSION
Two types of receptor lead to an increase in the glycogen breakdown in mammalian liver by stimulating cyclic AMPor Ca2+-dependent pathways, which converge to activate phosphorylase kinase. The results of the present work indicate that the two pathways are not independent. We have shown ( Figs. 1 and 2) that in the rat hepatocytes glucagon acts synergistically with vasopressin and angiotensin I1 to increase the Ca2+ influx. This effect of glucagon can be mimicked by Bt2cAMP or forskolin, which suggests that glucagon stimulates the Ca2+ influx by raising the cellular cyclic AMP concentration. The dose-response curves in Fig. 4 indicated that the enhancement of the Ca2+ influx by glucagon and vasopressin even occurs at concentrations as low as 0.1 nM. This concentration is in the range of the concentrations of glucagon and vasopressin found under physiological conditions in the plasma. Consequently, cyclic AMP-dependent and Ca2+-linked hormones may be involved in regulating cellular Ca2+ in the mammalian liver in uiuo.
The rise in the Ca2+ influx induced by the synergistic effect of glucagon and Ca2+-linked hormones might explain the observation that the combination of the two types of hormones induces the accumulation of calcium in the hepatocytes, whereas a net loss of calcium occurs if these hormones are added separately (Assimacopoulos-Jeannet et al., 1982;Morgan et al., 1983). A larger Ca2+ influx could counterbalance the increase in the unidirectional Ca2+ efflux resulting from the release of Ca2+ from internal stores induced by the hormones.
A synergistic effect has also been observed in guinea pig liver cells where simultaneous treatment with a cyclic AMPdependent and a Ca2+-linked hormone caused a much larger loss of K+ than the sum of the loss observed with each hormone alone (Cocks et al., 1984). Assuming that the loss of K+ is induced by a rise in the intracellular Ca2+ concentration (Cocks et al., 1984), the exerting of a synergistic effect by the two types of hormones on the Ca2+ influx might help to increase this loss.
The question arises as to the stage at which the cyclic AMP and the Ca2+-dependent pathways give rise to a stimulated Ca2+ influx. Measurements of the initial 46Ca2+ uptake rate as a function of the extracellular Ca2+ concentration (Fig. 3) lead to the conclusion that glucagon and vasopressin activate the same Ca2+ entry mechanism via the same Ca2+ gating system. We previously proposed that the Ca2+-linked hormones increase the rate constant of the reaction or reactions by which the Ca2+ ion is transported into the cell after being bound on its recognition site (Mauger et al., 1984).
As regards the respective intermediates involved in the coupling between the receptor and the Ca2+ gate, we showed here that glucagon acted by raising the intracellular cyclic AMP concentration, which probably in turn stimulated cyclic AMP-dependent protein kinase activity. It has been suggested that cyclic AMP can modulate the al-adrenergic receptor properties in rat liver cells (Morgan et al., 1984). However, this mechanism does not seem to be involved in stimulation of the Ca2+ influx. This conclusion is based on the observation that the responses to vasopressin and angiotensin I1 are not additive, indicating that the number of receptors activated is not the limiting step in the response to individual hormone. As regards the response to combined glucagon and Ca2+-linked hormone administration, it seems probable that cyclic AMP acts either on the coupling mechanism leading from the Ca2+linked hormone receptor to the increased Caz+ influx or on the Ca2+ gate itself. In agreement with this hypothesis are the observations that, in several cell types, protein phosphorylation regulates ion channels such as those of K+ and Ca2+ (see, for example, Levitan et al., 1983). Injection into neurones or cardiac myocytes of cyclic AMP or of the catalytic subunit of the cyclic AMP-dependent protein kinase has been shown to induce Ca2+ currents (see, for example, Reuter, 1983).
Our knowledge of the mechanisms involved in the action of the Ca2+-linked hormones has recently progressed (see Nishisuka, 1984;Berridge and Irvine, 1984). In liver, as in other tissues, these hormones trigger the degradation of membrane polyphosphoinositides (Rhodes et al., 1983;Creba et al., 1983;Litosch et al., 1983;Seyfred and Wells, 1984). This leads to: 1) diminution of the concentrations of the phosphatidyli-nositol4-phosphate and the phosphatidylinositol 4,5-biphosphate; 2) the production of inositol triphosphate, which releases intracellular Ca2+ (Burgess et al., 1984;Thomas et al., 1984;and Joseph et al., 1984); and 3) the production of diacylglycerol which helps to activate protein kinase C (Hughes et al., 1984;Nishisuka, 1984). Although the breakdown or turnover of the phosphoinositides has been suggested to control Ca2+ gating (Michell, 1975(Michell, , 1982 we do not know which of the above three features of this complex messenger system leads to the increased Ca2+ influx. The nature of the interaction between cyclic AMP and the polyphosphoinositides metabolism is poorly documented. However, it has been shown that glucagon does not affect the phosphoinositides turnover whether administered alone or concomitantly with a Ca2+-linked hormone (Prpic et al., Kaibuchi et al., 1982) and does not lead to the disappearance of polyphosphoinositides (Creba et al., 1983). Consequently, cyclic AMP does not seem to potentiate the response of the Ca2+-linked hormones by increasing hydrolysis of phosphatidylinositol 4,5-bisphosphate, but more probably works with one of the intermediates produced by the degradation of phosphatidylinositol 4,5-bisphosphate to trigger the Ca2+ influx.
The analysis of Figs. 4 and 5 may provide information about the mode of the interaction between cyclic AMP and the inositol lipid-dependent messengers. The results in these figures show that each hormone increases, in a dose-dependent manner, the maximal response to the other without affecting its EDb0. If G is the intermediate involved in the glucagon response (possibly cyclic AMP) and V, the intermediate involved in the vasopressin response, each intermediate can influence the maximal response induced by the other according to the following equations: For the glucagon response: and for the vasopressin response: Where Jmax is the maximal response obtained in the presence of infinite G and V concentrations: J,,,, and J,,., are the maximal responses obtained with infinite concentrations of G and V in the presence of given concentrations of V and G, respectively, and K v and K C are the apparent dissociation constants for V and G, respectively. The Ca2+ influx (Jic.) will then follow the equation: This equation (3) is typical of reactions involving two substrates which form complexes with an enzyme. This model, which accounts for the data in Figs. 4 and 5 implies that there is a Ca2+ influx, provided that minimal concentration of both G and V are present in the cytosol. The data in Fig. 6 suggest that the basal cyclic AMP concentration, which is about 2 pmol/mg dry weight or 1 ~L M is sufficient to allow basal and vasopressin-stimulated Ca2+ influxes. This also implies that even in the absence of a Ca2+-linked hormone, there is a polyphosphoinositides turnover that gives rise to an intracellular concentration of the intermediate called V in our model sufficient to account for both the resting and cyclic AMPstimulated Ca2+ influxes.
In conclusion, the present results indicate that in rat hepatocytes, cyclic AMP-and Ca2+-linked hormones increase synergistically the Ca2+ influx by activating the same Ca2+ gating system. It is suggested that both the intermediates, respectively, arising from the increase in cellular cyclic AMP content and degradation of the polyphosphoinositides are substrates necessary to allow resting and stimulated Ca2+ entry into the cells.