817

Biochem. J. (1990) 266, 817-822 (Printed in Great Britain)

Inhibition of the calcium pump by human parathyroid hormone(1-34) and human calcitonin in liver plasma membranes Roderick C. McKENZIE,*§ Sophie LOTERSZTAJN,t Catherine PAVOINE,t Francoise PECKER,t Richard M. EPAND* I and Ronald C. ORLOWSKIt¶ *Department of Biochemistry, McMaster University, Health Sciences Centre, Hamilton, Ontario. Canada L8N 3Z5, tUnite INSERM 99, Hopital Henri Mondor, 94010 Creteil, France, and lArmour Pharmaceutical Company, Kankakee, IL 60901, U.S.A.

The effect of human parathyroid hormone-(1-34) (hPTH) and human calcitonin (hCT) on the activity of the Ca2"-extrusion pump in liver plasma membranes was studied. Both hormones were found to be potent inhibitors of Ca2l transport and the related high-affinity (Ca2`-Mg2+)-ATPase activity, causing maximal inhibition of 25-30 % at concentrations of 100 nm. Half-maximal inhibition was observed with 20 nM-hPTH and with 0.5 nM-hCT. By comparison, salmon calcitonin and intact bovine parathyroid hormone-(1-84) were inhibitory only at 10 #uM. The effects of hCT and hPTH on the Ca2" pump activity were not mimicked by cyclic AMP. Also, 10 /uM of either hPTH-(1-34) or hCT did not alter the 45Ca2" influx rate into isolated hepatocytes. We conclude that inhibition of Ca2" efflux, rather than the stimulation of Ca2" influx, may play a functional role in the control of hepatic calcium homeostasis by hPTH-(1-34) and hCT.

INTRODUCTION It is known that certain hormones such as the aadrenergic agonists and vasopressin exert their influence on cellular metabolism in the liver through mobilization of intracellular Ca2". A messenger role is thus conferred on free cytosolic Ca2" [1-3]. Ca2" pumps in plasma membranes, which are coupled to high-affinity (Ca2+-Mg2+)-ATPases, are one of the key systems involved in the control of the level of free cytosolic Ca2" [4]. The plasma membrane Ca2l pump in hepatocytes has been well characterized [5-10] and its hormonal sensitivity has already been noted. Thus, in intact hepatocytes, the Ca2" pump is inhibited by a.-adrenergic agents and vasopressin [11,12]. Also, in isolated rat liver plasma membranes, we have shown that glucagon and the proteolytic fragment of glucagon, glucagon-(l9-29), inhibit the Ca2" pump independently of adenylate cyclase activation [13-16]. Classically, the principal target organs of calcitonin (CT) and parathyroid hormone (PTH) are bone and kidney, where the two hormones exert opposite effects on calcaemia. However, recent studies indicate that CT and PTH may also play a major role in regulating metabolic function of liver cells. Thus, Yamaguchi and co-workers have demonstrated the presence of high-affinity binding sites for eel CT [17] and human CT (hCT) [18] in rat liver. In addition, an increase in intracellular Ca2+ is observed after CT binding [19]. Also, it has been shown that eel CT at nanomolar concentrations inhibits the high-affinity (Ca2+-Mg2+)-ATPase [17]. Liver has long been recognized as a major site of PTH metabolism, and cleavage of intact PTH in the liver produces an active N-terminal fragment, PTH-(1-34).

Moreover, PTH binds to high-affinity sites in rat liver plasma membranes [20], and a recent report demonstrates that hPTH-(1-34) can elevate free intracellular Ca2" in rat hepatocytes [21]. It thus seemed interesting to examine in parallel the effects of PTH and CT on the liver Ca2" pump. We report here that hPTH and hCT inhibit the Ca2" pump in rat liver plasma membrane vesicles independently of cyclic AMP and without affecting the net rate of Ca2" influx into hepatocytes. Our results suggest that the Ca2" pump participates in the control of Ca2' homeostasis in liver by hPTH and hCT. EXPERIMENTAL Materials ATP (sodium salt), EGTA (disodium salt) and Argvasopressin (grade VI) were obtained from Sigma. Porcine glucagon was obtained from Eli Lilly and human apolipoprotein Al was a gift from Dr. H. J. Pownall, Methodist Hospital, Houston, TX, U.S.A. Salmon CT, hCT and hPTH-(1-34) were chemically synthesized by Armour Pharmaceuticals, Kankakee, IL, U.S.A. Glycinamide glucagon was prepared as described previously [22]. Bovine PTH-(1-84) was obtained from the National Hormone and Pituitary Program. Malachite Green (colour index 42000) was obtained from Harleco. 45Ca2+ was from Amersham. All other reagents were Analar grade. When specified, hydrolysis of hPTH-(1-34) was performed in 6 M-HCI for 12 h at 110 °C in a vacuumsealed ampoule. HCI was then removed under vacuum in a desiccator. All peptides were made up as 0.1 mm stock

Abbreviations used: sCT, salmon calcitonin; hCT, human calcitonin; hPTH, human parathyroid hormone; bPTH, bovine parathyroid hormone; glycinamide glucagon, [Cy-glycinamide-Asp9"6'21] glucagonyl-(glycinamide). § Present address: Molecular Virology and Immunology Program, McMaster University, Health Sciences Center. l To whom correspondence should be addressed. 1 Present address: 2624 Belmont Canyon Road, Belmont, CA 94002, U.S.A.

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solutions in 50 mM-Hepes buffer. The pH ofeach solution was checked and adjusted to 7.5 if necessary. (Ca2`-Mg2`)-ATPase assay (Ca2`-Mg2`)-ATPase activity was measured in liver plasma membranes prepared according to Neville [23] (up to step 11) as described in [13] with the following modifications. The standard assay mixture contained in a final volume of 250 ,1: 5-8 ,ug of membrane protein, 0.25 mM-NaATP, 50 mM-Hepes, pH 7.5, 0.010% bovine serum albumin, 200 ,tM-EGTA, without Ca2" or with 100-200 /SM-CaCl2. This corresponds to a free Ca2+ content of 15.6 nM-1 .6 /UM. Unless mentioned otherwise, all assays were carried out at a free Ca2+ concentration of 49 nm. The assay tubes were kept on ice until the reaction was started. Incubations were carried out in a water bath at 37 °C with shaking. After a 1 min preincubation at 37 °C, the reaction was started by adding ATP. After an additional 20 min, the tubes were taken from the bath and the reaction was terminated by adding 2.5 ml of Malachite Green reagent [24]. After 1 min, 50 ,ul of 10 % aqueous ascorbic acid was added, in order to stabilize the remaining ATP. The tubes were then allowed to stand for 1 h at room temperature before measuring their absorbances at 650 nm. When assays were carried out in the presence of hormones, the membranes and hormones were incubated together on ice before the start of the assay. Results are the means of quadruplicate determinations. (Ca21-Mg2+)-ATPase activity was determined by subtracting values obtained in the presence of EGTA alone from those obtained in the presence of EGTA plus Ca2+. Ca2+ uptake Ca2+ uptake was assayed in plasma membrane vesicles prepared according to Prpic et al. [12] as described in [14]. Preincubation of plasma membrane vesicles (3080 ,tg of protein) with the hormones for 10 min at 4 °C allowed the peptides to interact with intravesicular sites of inside-out vesicles, because of the leakiness of vesicles. After preincubation of the assay medium for 2 min at 32 °C, the incubation was run in a final volume of 100 ,1u which contained 50 mM-Tris/HCl, pH 8, 0.01 % BSA, 400 /tM-EGTA, 393 1sM-CaCl2 (0.1 /LM free Ca2+), 20 #Ci of 45Ca/ml, 10 mM-MgCl2, 1 ,tg of oligomycin/ml, 10 ftMdigitoxigenin, and with either no NaATP added or 2.5mM-NaATP. After 30s, the samples were filtered under vacuum on Millipore filters (HAWP 0.45 /tM) that had been soaked in 0.25 M-KCI for 1 h. The filters were washed three times with 4 ml of ice-cold washing solution which contained 140 mM-KCl, 20 mM-Tris/HCl, pH 8, 5 mM-LaCl3, 10 mM-CaCl2 and 1 mM-EGTA. The filters were dried and counted for radioactivity in 10 ml of Beckman Ready-Solv. The ATP-dependent uptake was determined from the difference in radioactivity bound to the filter in the presence and absence of ATP. Results were obtained from triplicate determinations. Ca2' determination and control of free Ca2+ concentrations The Ca2+ content in membrane preparations and the reagent solutions were determined using a Perkin-Elmer Model 703 atomic absorption spectrophotometer. The reagents contained 1.63 ,tM-Ca2+ in the presence of EGTA and no added Ca2". When 8 mg of membrane protein was included, Ca2` concentrations rose to 2.8 ,UM. Liver

R. C. McKenzie and others

plasma membranes contained 49.3 nmol of Ca2"/mg of protein. Submicromolar Ca2" concentrations were controlled by using the buffering ligand EGTA as described by Pershadsingh & McDonald [25]. An apparent dissociation constant of 1.479 x 10-8 M was used in these calculations. Hepatocyte isolation Hepatocytes were isolated from the livers of male Wistar rats (150-200 g) by a collagenase-perfusion technique described by Sweeney et al. [26]. The cells were incubated at 37 °C in Eagle's medium containing 1.8 mmCaC12 in sealed tubes which were gassed with 02/CO2 (19:1). Measurement of "5Ca2+ influx The influx measurements were made using a technique described by Mauger et al. [27]. A cell suspension (1.5 x 106 cells/500 ,l) was incubated in Eagle's medium containing the hormone for 5 min at 37 °C in a water bath with shaking. At the end of this period, 2 ,uCi of 45CaCl2 was added and 100 ,u aliquots were taken at 0, 15, 45, 75 and 125 s. Each aliquot was immediately diluted with 4 ml of an ice-cold wash solution containing 5 mM-CaCl2/144 mM-NaCl/5 mM-Tris/HCl, pH 7.5. The mixture was filtered through a Whatman GF/C glass-fibre filter and washed with 3 x 4 ml of ice-cold wash solution. The radioactivity associated with the filters was determined by liquid scintillation counting after addition of 10 ml of ACS scintillation fluid (Amersham). Protein determination Protein determinations were made using the Bio-Rad Protein Assay, with bovine serum albumin as a standard. RESULTS Inhibition of the Ca2+ pump in plasma membranes by hPTH-(1-34) and hCT The Ca21 pump in liver plasma membranes can be defined by the ATP-dependent Ca2+ uptake, as well as by the high affinity (Ca21-Mg2+)-ATPase activity [5,7]. Direct addition of 1 1M-hPTH-(l-34) to liver plasma membrane vesicles prepared according to Prpic et al. [12] resulted in a 25 % decrease in the ATP-dependent Ca21 uptake (Fig. 1). The inhibitory effect of hPTH-(1-34) was concentration-dependent, half-maximal inhibition occurring in the presence of 20 nM-hPTH (Fig. 1). We have also examined the effect of hPTH-(1-34) on the (Ca2+-Mg2+)-ATPase activity in liver plasma membranes. Fig. 2 shows that inhibition of (Ca21-Mg2+)ATPase was parallel to that of Ca21 transport. Addition of 20 nM-hPTH-(1-34) induced a 20 % inhibition of (Ca2+-Mg2")-ATPase activity. The action of hPTH(1-34) was specific, since bPTH-(1-84) was found to be 1000-fold less potent. The possibility that a low molecular mass contaminant caused the inhibition of the Ca2+ pump is unlikely, since both hCT and hPTH-(1-34) were purified by gel filtration after synthesis. Also, hydrolysed hPTH-(1-34) had no inhibitory activity (results not shown). Kinetic studies of (Ca2+-Mg2+)-ATPase activity in the presence and absence of hPTH-(1-34) indicated that the peptide did not affect the apparent affinity for the 1990

Hormonal inhibition of the liver Ca2+ pump

819 Table 1. Inhibitory effect of hPTH-(1-34) on (Ca2+-Mg2)ATPase activity at different free Ca2+ concentrations

100* 0)

m

(Ca2+-Mg2+)-ATPase activity was measured as described in the Experimental section. Total Ca2+ concentration was varied between 100 and 200 /tM. Human PTH-(1-34) was added to give a final concentration of 10 riM. Results are

90-

0 C

o

° 80-

D

means + S.E.M.

a0 0

7-

(Ca2+-Mg2+)-ATPase activity (nmol of Pi/ 10 min per mg of protein)

70-

60

Free [Ca2+] 8

9

6 7 -log{[hPTH] (M)}

Inhibition

(nM)

Control

+ hPTH-(1-34)

(%)

15.6 23.5 36.7 64.7

65+19 179+20 230+25 260+21

65+11 74+11 127+29 154+35

0 59 45 41

5

Fig. 1. Effect of hPTH-(1-34) on ATP-dependent Ca2l uptake in liver plasma membrane vesicles ATP-dependent Ca2+ uptake was measured as described in the Experimental section in the presence of 393 glM total CaCl2 (0.1 M free Ca2+). In each experiment, data points were paired with control activity determined in the absence of hormone and normalized to 100 %. Results from five experiments were analysed for statistical significance by the Mann-Whitney test (*P < 0.05). Mean control activity was 1.35 + 0.26 nmol of Ca2+/30 s per mg of protein.

1 00 t/?

substrate ATP (results not shown). Similarly, there was no statistically significant change in inhibition when the free Ca2l concentration was varied between 23.5 and 64.7 nm. The only change appeared to be a decrease in the maximal velocity of the reaction (Table 1). Addition of 1O nM-hCT caused a 30 % inhibition of ATP-dependent Ca2" uptake, half-maximal inhibition occurring at 1 nM-hCT (Fig. 3). sCT was 1000-fold less potent than hCT, and caused only a 120% inhibition of ATP-dependent Ca2" uptake at 1 /,M (Fig. 3). Inhibition of (Ca2`-Mg2`)-ATPase activity by hCT occurred concomitantly with that of Ca2l uptake, since a maximal

90 -

:I

0) 0)

4-

(A

Inhibition of the calcium pump by human parathyroid hormone-(1-34) and human calcitonin in liver plasma membranes.

The effect of human parathyroid hormone-(1-34) (hPTH) and human calcitonin (hCT) on the activity of the Ca2(+)-extrusion pump in liver plasma membrane...
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