to the steady state occupancy of a high affinity receptor by the hormone (the predominant insulin-binding spe- cies in hepatocytes at 37 "C. For glucagon, ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 256, No. 14, Issue of July 25,pp. 7449-7453, Printed in U.S.A.
1981
Insulin and Glucagon Stimulationof (Na'-K')-ATPase Transport Activity in Isolated Rat Hepatocytes* (Received for publication, October 9, 1980, and in revised form, April 27, 1981)
Max Fehlmann$ and Pierre Freychet From the Groupe de Recherches sur les Hormones Polypeptidiques etla Physiopathologie Endocrinienne, Institut National de la Sante et de la Recherche Medicale and Laboratoire deMedecine Experimentale, Facultk deMedecine, Chemin de Vallombrose, 06034 Nice Cedex, France
The effects of insulin and glucagon on the (Na+-K+)- transports by hormones. ATPase transport activity in freshly isolated rat hepa- Freshly prepared suspensions of isolated rat hepatocytes tocytes were investigated by measuring the ouabain- have beenshown to retain metabolic activity (12, 13), to sensitive, active uptake of "Rb'. The active uptake of possess specificreceptors for insulin (14-16) and glucagon (14, '"Rb+was increased by 18%(p c 0.05) in the presence 16, 17), and to respond to these hormones by an increase in of 100 n~ insulin, and by28%(p< 0.005) in the presence neutral amino acid transport (18). In the present study, we of n~ glucagon. These effects were detected as early as have investigated the effects of insulin and glucagon on the 2 min after hepatocyte exposure to either hormone. liver (Na+-K')-ATPase monovalent cation transport activity Half-maximal stimulationwas observed with about0.5 by measuring the ouabain-sensitive uptake of 86Rbfin freshly n~ insulin and 0.3 n~ glucagon.Thestimulationof isolated rat hepatocytes. These effects were also examined in "Rb+ uptake by insulin occurred in direct proportion relation toNa+ influx and to receptor occupancy by the to the steadystate occupancy of a high affinity receptor by the hormone (the predominant insulin-binding spe- hormone. cies in hepatocytes at 37 "C. For glucagon, half-maxiMATERIALSANDMETHODS mal response was obtained with about5% of the total receptors occupied by the hormone. Amiloride (a spe- Chemicals--86RbC1(specificactivity, 2.6 mCi/mg of rubidium) and "NaCl (specific activity, 100-IO00 mCi/mg of sodium) were purchased cific inhibitorof Na+ influx) abolished the insulin stimfrom the Radiochemical Centre, Amersham, England. Porcine monulation of "Rb' uptake while inhibiting thatof gluca- ocomponent insulin and porcine glucagon were & t sfrom the Novo gon only partially. Accordingly, insulin was found to Research Institute (Copenhagen, Denmark). Amiloride-HC1 wasgenrapidlyenhancetheinitialrate of "Na+ uptake, erously supplied by Merck, Sharp and Dohme (Paris, France). All whereasglucagonhadnodetectableeffecton"Na+ other reagents were of the best grade commercially available. influx. Preparation of Isolated Hepatocytes andIncubation ProceThese results indicate that monovalent cation trans- dures-Hepatocytes were isolated from fed male Wistar rats (120-150 port is influenced by insulin and glucagon in isolated g) by collagenase dissociation of the liver as previously described (13, rat hepatocytes.In contrast to glucagon, which appears18). All experiments were carried out at 37 "C in a Krebs-Ringer to enhance "'Rb+ influx through the (Na+-K+)-ATPase bicarbonate buffer, pH 7.4, containing 10 mg/ml of bovine serum albumin (Fraction V), gentamycin (50pg/ml),and bacitracin (0.8 mg/ withoutaffectingNa+influx,insulinstimulatesNa+ ml), and gassed with a mixture of 5% C02/95% 0 2 . When a sodiumentry which in turn may increase pump the activity by free medium was required, NaCl and NaHCOs were replaced by increasing the availabilityof Na+ ions to internal Na+ choline chloride and choline bicarbonate (referred to as choline metransport sites of the(Na+-K+)-ATPase. dium) to adjust isoosmolarity. For measurements of"Rb' (a K'
Monovalent cation transport is influenced by insulin and glucagon in various tissues. Insulin has been shown to enhance (Na'-K')-ATPase transport activity in skeletal muscle and adipocytes (1, 2), lymphocytes (3), and avian salt gland (4), and also to induce hyperpolarization in skeletal muscle, adipose tissue (for review, see Refs. 5 and 6), and embryonic heart cells (7). In the liver, modification of ion distribution and hyperpolarization have been observed in response to glucagon (8-11). Most of the studies in Liver have been performed with the isolated, perfused organ which represents a highly integrated system, making it difficult to analyze the nature of the mechanisms involved in the regulation of ionic
* This work wassupported in part by Grant 78.5.216.4 from Institut National de la Sante et de la Recherche Medicale (Paris, France), research funds from the University of Nice (France), and Fondation pour la Recherche Medicale (Paris,France). 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. f To whom correspondence should be addressed.
analogue) and "Na+ uptake, hepatocytes were incubated in conical microfuge tubes (Eppendorf, 1.5-ml capacity) with the additions indicated in the legends to figures in a total volume of 250 $/tube. For incubations longer than 10 min, tubes were gassed and capped. AU determinations were run in triplicate within each individual experiment. Transport assays were terminated by adding 1.2 ml of chilled saline. Hepatocytes were immediately sedimented by a 5-s centrifugation at 2000 X g, resuspended in chilled saline, and centrifuged again. For 22Na+uptake experiments, one additional washing step was carried out to the procedure described above. Radioactivity present in cell pellets was directly counted for "Na in a gamma spectrometer. For "Rb, cells were resuspended in 100 pl of water; radioactivity was measured on the 32Pchannel of a liquid scintillation spectrometer after the addition of1.5 ml of scintillation liquid (Unisolve, KochLight Laboratories). Binding Studies-Insulin and glucagon were iodinated to specific activities of 200-250 pCi/pg and 90-110 pCi/pg, respectively, using a modification (referred to as the "second modification" in Ref. 19) of the chloramine-T method. Binding assays were performed using the same media and incubation conditions as indicated above for transport experiments. Assays were initiated by adding 150 p1of the cell suspension into microfuge tubes containing 50 pl of '251-hormone(0.3 ng/ml of iodoinsulin or 0.7 n g / d of iodoglucagon) and 50 pl of either hormone-free buffer or varying concentrations of unlabeled hormone. After 30 min at 37 "C (insulin) or 5 min at 37 "C (glucagon),conditions under which binding reached a steady state for the corresponding Iz5I-
7449
Hormonal Regulation
7450
Hepatocytes Fluxes ofinIon
hormone (not shown), incubations were terminated by adding 1.0 ml of chilled buffer to each tube and sedimenting the cells by a 10-s centrifugation at 2000 X g . Cell pellets were then washed (without resuspension) with 1.0 ml of chilled buffer, centrifuged again, and counted for Iz6I radioactivity in a Auto-Gamma spectrometer. The nonspecific component of binding was determined graphically by plotting the total amount of hormone bound against the hormone concentration in the medium (16) and was subtracted from total binding. Maximal binding values were derived from abscissa intercepts of Scatchard plots (16).
TABLEI Effect of insulin and glucagon on active"Rb+ uptake in isolated rut hepatocytes Hepatocytes were incubated without (basal) or with insulin (100 nM) or glucagon (100 nM) and with 0.5 pCi of "Rb+ for 60 min at 37 "C, in the absence or presence of 2 mM ouabain. Active uptake was determined by subtracting the ouabain-independent part of transport from total transport.
RESULTS
Effect of Insulin and Glucagononthe Time Course of 86Rb' Uptake (Fig. 1)-Total 86Rb+uptake by isolated hepatocytes was linear for about 2 min and reached a maximal value after 60 to 120 min. Insulin (100 nM) and glucagon (100 nM) enhanced both the initial rate and subsequent accumulation of 86Rb+.In the presence of 2 mM ouabain, "Rb+ uptake was reduced by about 85% at all time points tested. Neither insulin nor glucagon affected 86Rbf entry in the presence of ouabain (Fig. 1);this indicates that the stimulatory effect of either hormone results from an increase in (Na+-K')-ATPase activity. After 60 min, active (ouabain-sensitive) "Rb+ uptake was increased by 18% in hepatocytes exposed to insulin and by 28% in cells exposed to glucagon (Table I). Effect of Insulin and Glucagon on86Rb+Efflux-To investigate whether 86Rbefflux was altered by insulin or glucagon, hepatocytes were first preloaded with 86Rb+ for60 min. Cells were then sedimented, washedonce, and resuspended in Krebs-Ringer bicarbonate buffer in the absence or presence of 100 nM insulin or 100 mM glucagon. The efflux of 86Rb+was then measured (Fig. 2). Semilogarithmic plots of the fractional efflux yielded straight lines indicating f i s t order kinetic possesses in all three conditions tested (in the absence or presence of insulin or glucagon). Glucagon enhanced the rate of 86Rb+ exit ( t l I z= 32 min and 40 min for basal and glucagon, respectively). Under the same conditions, insulin had no effect on "Rb+ efflux (Fig. 2). Ouabain did not alter basal and glucagonstimulated 86Rb+ efflux(not shown). These data suggest that in hepatocytes glucagon, but not insulin, slightly enhances membrane permeability to K'. Dose Dependence Relationship for Insulin and Glucagon Stimulation of 86Rb+Uptake-The stimulatory effect of in-
"Rb' uptake"
Increase above basal
nmol/lO' eeHsI6O min
9%
11.13 +- 0.39 13.18*+- 0.67 14.27' 28.2 +- 0.54
18.4
Basal (n = 5) Insulin (n = 5) Glucaeon (n = 4)
a Values are means & S.E. for the number of separate experiments given in parentheses. Significantly different ( p < 0.05) compared with basal value. Significantly different ( p < 0.005) compared with basal value.
TIME,mm
20
40
40
GLUCAGON
FIG. 2. Effect of insulin and glucagon on the time course of "Rb+ efflux. Hepatocytes (IO ml at 2 X IO6 cells/ml) in Krebs-Ringer bicarbonate buffer were incubated for 1 h a t 37 "C with "Rb+ (40 pCi) in a culture flask. Cells were then collected by centrifugation (30 s a t 500 X g), resuspended in 15 ml of warm buffer, centrifuged again, and finally resuspended in 15 ml of the same medium; 200 p1of this cell suspension were added to microfuge conical tubes containing 50 pl of buffer (basal) or 50 pl of hormone (100 nM, final concentration). Tubes were shaken every 4 min, and incubations were terminated at the indicated times. The amount of 86Rb+remaining in cells was determined and has been expressed as the percentage of the amount of"'Rb' present in cells at zero time. Each point is the mean of triplicate determinations within one typical experiment. Similar results were obtained in two other experiments.
sulin and glucagon on "Rb+ uptake was dependent on hormone concentration. Half-maximal stimulation occurred with insulin at about 0.5 nM (Fig. 3) and with glucagon at about 0.3 nM (Fig. 4). Maximal effect wasobserved with insulin at 5 nM and with glucagon in the range of 10 to 50 nM (Figs. 3 and 4). Effect of CAMP Alone and Combined Effects of Insulin and Glucagon on "Rb+ Uptake (Table II)-Cyclic AMP (0.1 mM) enhanced "Rb+ uptake tosimilar a extent (a 27Yo Increase above basal) t o glucagon. When used in combination, maxim d y effective concentrations of insulin and glucagon (100nM each) did not stimulate 86Rb+uptake more than glucagon alone (Table 11). Comparison between Hormone Binding and Stimulation of "Rb+ Uptake-At 37 "C in isolated hepatocytes, insulin binds predominantly to one class of high affinity receptors with an apparent Kd of 0.6 nM (16). When the percentage of maximal insulin effect on "Rb+ uptake was plotted against the percentage of maximal insulin binding to total receptor sites at 37 "C the relationship between binding and effect deviated from linearity only slightly, with 50% of maximal effect occurring at about 30% oftotal receptor occupancy (Fig. 5, left). When the same analysis was made only taking into account insulin binding to the high affinity site, a linear '
1
0
60 TIME, mm
120
FIG. 1. Effect of insulin and glucagon on the time course of "Rb+ uptake. Transport assays were initiated by adding 150 pl of a cell suspension (1.8 X loficeb/ml, final concentration) to microfuge tubes containing 50 p1of 86Rb+(0.5 pCi), 25 pl of hormone (to give a final concentration of 100 nM) or buffer (referred to asbasal), and 25 pJ of ouabain (to give a final concentration of 2 mM) or buffer, d in Krebs-Ringer bicarbonate buffer containing 6 mM K+. Incubations were carried out at 37 "C with intermittent shaking, and reactions were terminated at theindicated time points. Each point is the mean of triplicate determinations within one typical experiment. Similar results were obtained in two other experiments.
Hormonal Regulation
:I
w
in Hepatocytes
Fluxes of Ion
/'
7451
TABLEI1 Effect of CAMP and combined effects of insulin and glucagon on active, ouabain-sensitive "Rb+uptake in isolated rat hepatocytes Hepatocytes were incubated without (basal) or with insulin (100 nM), glucagon (100nM), or cAMP (0.1 RIM), and with 0.5 pCi of "Rb' for 60 min at 37 "C. Each determination was run in triplicate within individual exDeriments. Increase above
~fi~,,+
basal nmol/l@ cells/ 60 min
Ph
%
Basal 10.80 f 0.26 Insulin 12.17 f 0.31 12.6 C0.05 Glucagon 13.75 f 0.86 27.2 ~0.05 1 cAMP 26.9 13.71 f 0.33 ~0.005 9 0 7 Insulin + [INSULIN] , -log M glucagon 29.1 f 1.00' c0.05 FIG. 3. Dose response of insulin stimulationof *'Rb+uptake. 13.95 a Values represent means & S.E. of three separate experiments. Hepatocytes (1.8 X IO6 cells/ml final concentration) were incubated Statistical significance compared with basal value. for 1 h at 37 "C in Krebs-Ringer bicarbonate buffer with varying Not significantly different from the value obtained in the presence concentrations of insulin in the presence of 86Rb+(0.5 pCi/tube). Cells were then collected by centrifugation, washed, and counted for =Rb' of glucagon alone. radioactivity. Each determination was run in triplicate within each experiment. Values represent means & S.E. of four separate experiments.
0
[GLUCAGON] ,-log M
FIG. 4. Dose response of glucagon stimulation of *'Rb+ uptake. Hepatocytes (1.8 X lo6 cells/ml final concentration) were incubated for 1 h at 37 "C in Krebs-Ringer bicarbonate buffer with varying concentrations of glucagon in the presence of *'jRb+(0.5 pCi/ tube). Cells were then collected by centrifugation, washed, and counted for H6Rb+radioactivity. Each determination was run in triplicate within each experiment. Values represent means & S.E. of six separate experiments.
relationship was observed (Fig. 5, left), suggesting that the stimulation of &Rb+ transport by insulin is directly proportional to theoccupancy of the high affinity binding sites. A t 37 "C in isolated hepatocytes, glucagon binds to one class of receptors with an apparentKd of about 5 nM (16).The stimulation of "Rb+ uptake by glucagonwas not directly proportional to receptor occupancy, since 50%of the maximal stimulation was observed when about 5%of the totalreceptors were occupied (Fig. 5, right).
50 PERCENT o l UXIYAL BINDING
100
FIG. 5. Comparison between insulin and glucagon stimulation of *'Rb+ uptake and steady state receptor occupancy by the hormone. Insulin binding was measured a t varying hormone concentrations under steady state conditions (30 min at 37 "C). Specific insulin binding, total receptor occupancy (O---O) and occupancy of the high affinity receptor (M were ) determined as indicated under "Materials and Methods" and described in detail elsewhere (16). The uptake ofRb' was determined after 30 min of hepatocyte exposure to "Rb+ in the presence of varying insulin concentrations, under the same experimental conditions as used for the measurement of insulin binding. Similarly, glucagon binding and glucagon stimulation of 86Rb+uptake were measured in identical experimental conditions, after 5 min of hepatocyte exposure to glucagon (a condition under which a steady state of glucagon binding is obtained a t 37 "C (16)). For binding, thedata are expressed as percentage of maximal binding values derived from abscissa intercepts of Scatchard plots. For "Rb+ uptake, the data are expressed as percentage of the maximal response to insulin (as shown in Fig. 3) or to glucagon (as shown in Fig. 4).
present study c o n f i i s this observation since monensin, a Na+ ionophore, stimulates the ouabain-sensitive =Rb+ uptake in hepatocytes by about 50%(Table 111). Effect of Insulin and Glucagon on "Na+ Influx-To investigate whether insulin or glucagon could affect Na+ permeability in isolated hepatocytes, the uptake of "Na+ by cells Sodium Dependence of Basal and Hormone-stimulated previously exposed to ouabain (to inhibit active Na' efflux "Rb+ Uptake (Table IIO-In the presence of amiloride, a through the (Na+-K+)pump) was measured in the absence or drug shown to inhibit Na+ influx in epithelial cells (20, 21), presence of either hormone. Fig. 6 shows that the uptake of primary cultured hepatocytes (22), and freshly isolated hepa- "Na+ increased linearly for at least 1 min (Fig. 6, inset);after tocytes (23), insulin failed to significantly alter 86Rb+uptake. 2 to 5 min, the slope of the curve decreased to give rise to a By contrast, the glucagon stimulation of "jRb+ uptake was new near-linear rate of entry up to 20 min. When added at 0 only partially inhibited. These observations suggest that in- time, insulin enhanced "Na+ entry within 1 min; this effect sulin stimulation of Rb+ uptake through activation of (Na+- was also observed at later time points. By contrast, glucagon K+)-ATPase activity (Fig. 1) is largely dependent on Na+ had no detectable effect on "Na+ uptake at any time point entry, whereas the glucagon effect is not. Stimulation of the tested (Fig. 6). In the absence of ouabain, "Na+ tracer equili(Na+-K+)-ATPasetransport activity by an increased Na+ bration was extremely rapid and no effect of insulin could be influx has been reported in various cell types (24-27). The measured (data not shown).
-
7452
Hormonal Regulation
of
TABLEI11 Effect of amiloride and monensin on basal and hormonestimulated active, ouabain-sensitive=Rb' uptake in isolated rat hepatocytes Hepatocytes were incubated without (basal) or with insulin (100 nM) or glucagon (100 nM), and with 0.5 pCi of 86Rb+ for 60 min at 37 "C. 1 =Rb+ uptake"
Ion Fluxes Hepatocytes in
tive, high affinity sites (16). Comparison between insulinstimulated Rb+ entry andreceptor occupancy, measured under identical experimental conditions, revealed that the activation of the (Na+-K+)-ATPase transport activity closely paralleled insulin binding to the high affinity receptor sites. In aggregates of embryonic heart cells, insulinhas recently been reported to induce an ouabain-resistant hyperpolarization, and this effect was also found to correspond closely to the occupancy of a high affinity receptor ( 7 ) . The physiological importance of rapid alteration ofion Control (n = 9) distribution by insulin in mediating its action on metabolic processes is still poorly understood. In amphibian liver, insulin (n = 7) activation of glycogen synthase is blocked by ouabain (29). However, we were unable to detect any ouabain effect on insulin stimulation of amino acid transport, a relatively late Values are means f S.E. for the number of separate experiments insulin effect in isolated rat hepatocytes (18). Along with other given in parentheses. observations (reviewed in Ref. 6), this makes it unlikely that 'Significantly different (p < 0.025) compared with basal value. all metabolic effects of insulin are mediated by a modification e Significantly different ( p < 0.001) compared with basal value. of (Na+-K+)-ATPase transport activity. Not significantly different from basal value. e Significantly different (p < 0.01) compared with basal value. Our data suggest that theactivation of the (Na+-K+)-ATP'Significantly different (p < 0.005) compared with control. ase transport activity by insulin in isolated hepatocytes is secondary to a rapid and transient increase in Na' influx. A similar mechanism has been proposed for serum stimulation of the (Na+-K+) pump in quiescent fibroblasts (26) and for monensin-induced hyperpolarization of neuroblastomaglioma hydrid cells (27). The fact that monensin, a Na+ 0 ionophore, increased active 86Rb+uptake in hepatocytes (Table 111) indicates that the transport activity of the (Na'-K')ATPase in these cells issensitive to anincrease in Na+ influx. Insulin may thus primarily increase Na' entry into hepatocytes, which in turn may enhance the pump activity by increasing the availability of Na+ ions to internal Na+ transport sites of the (Na+-K')-ATPase (26). However, it cannot be decided from the present experiments whether or not, under physiological conditions ( i.e. in the absence of ouabain), 20 0 10 the overall effect of insulin is to alter intracellular Na' conTIME, mln centration. Insulin is known to promote cell growth and proFIG. 6. Effect of insulin and glucagon onthe time course of liferation of a variety of cultured cells. Since an increase in '%a+ influx. Hepatocytes (3 X lo6cells/ml final concentration) were NaCinflux appears to be implicated in initiation of cell prolifpreincubated in Krebs-Ringer bicarbonate buffer for10 min at 37 "C eration in primary cultures of adult rat hepatocytes (22, 30) in the presence of 2 mM ouabain. Sodium uptake assays were then initiated by adding 150 pl of the cell suspension to prewarmed micro- and in cultured mouse fibroblasts (26), it is possible that the fuge conical tubes containing 50 pl of "NaCl(1 pCi/tube) and 50 pl of stimulatory effect of insulin on Na+ influx may represent an buffer (basal) or hormone (to give a fiial concentration of 100 nM). early signal wherebythe hormone triggers its mitogenic effects Reactions were terminatedat the indicated time points by adding 1.2 in animal cells. The subsequent activation of the (Na+-K+) ml of chilled saline and immediately collecting the cells by centrifu- pump is also in agreement with the general observation that gation (5 s at 2000 X g).Cell pellets were washed twiceby resuspension in chilled saline and centrifugation anc! counted for "Na radio- an enhanced pump activity is associated with cell proliferation activity. Each point is the mean of triplicate determinations within (reviewed in Ref. 31). In the perfused liver, hyperpolarization is observed in reone typical experiment. sponse to glucagon but not to insulin (10). In isolated hepatocytes, our data suggest that glucagon enhances K' efflux, possibly through an increase in K+ permeability, and also DISCUSSION exerts a stimulatory effect on the (Na+-K+)-ATPase transport It has long been recognized that insulin modulates the distribution of Na' and K' across the plasma membrane of activity. The former observation is in agreement with a previous study (28),and both phenomena are consistent with the target cells. This was interpreted to result from eithera decreased permeability for both Na+ and K+ (5) or a stimu- glucagon-induced hyperpolarization observed in the perfused lation of the (Na+-K+)-ATPase transportactivity (1-4, 7). In liver (8, 10). The ion redistribution evoked by glucagon apisolated rat liver cells,insulin has previously been reported to pears to be closely related to thestimulation of gluconeogenesis ( 10). Moreover, hyperpolarization (32) and increased Rb+ increase K+ influx (28). In this study, we have extended this uptake (33) are associated with the prereplicative period that observation showing that insulin rapidly enhances Rb' entry follows partial hepatectomy. This period is also characterized by stimulating the (Na'-K+)-ATPase transport activity. Insulin stimulation of amino acid transport in isolated hepato- by a transient elevation of CAMPlevels (34) and by hyperglucytes has been shown to require a long term exposure of the cagonemia (35).It is thus possible that the effects of glucagon cells to hormone (2 to 3 h) and to involve protein synthesis. In on monovalent cation fluxes are implicated in the expression of a variety of actions of this hormone in liver. contrast, activation of the (Na'-K+) pump by insulin is a rapid effect occurring after only a few minutes of hepatocyte expoAcknowledgments-We thank A. Kowalski for technical assistance sure to the hormone. We have recently shown that at 37 "C and illustration work and J. Duch for secretarial assistance. We are insulin binds predominantly to a single class of noncoopera- grateful to Dr. R. Mengual for providingus with monensin. 0
c
2oool
Hormonal Regulation
of Ion Fluxes Hepatocytes in
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