Stimulation of glycine catabolism in isolated perfused rat liver by calcium mobilizing hormones and in isolated rat liver mitochondria by submicromolar concentrations of calcium.

Glucagon stimulates flux through the glycine cleavage system (GCS) in isolated rat hepatocytes (Jois, M., Hall, B., Fewer, K., and Brosnan, J. T. (1989) J. Biol. Chem. 264, 3347-3351. In the present study, flux through GCS was measured in isolated rat liver perfused with 100 nM glucagon, 1 microM epinephrine, 1 microM norepinephrine, 10 microM phenylephrine, or 100 nM vasopressin. These hormones increased flux through GCS in perfused rat liver by 100-200% above the basal rate. The possibility that the stimulation of flux by adrenergic agonists and vasopressin is mediated by increases in cytoplasmic Ca2+ which in turn could regulate mitochondrial glycine catabolism was examined by measuring flux through GCS in isolated mitochondria in the presence of 0.04-2.88 microM free Ca2+. Flux through GCS in isolated mitochondria was exquisitely sensitive to free Ca2+ in the medium; half-maximal stimulation occurred at about 0.4 microM free Ca2+ and maximal stimulation (7-fold) was reached when the free Ca2+ in the medium was 1 microM. The Vmax (nanomoles/mg protein/min) and Km (millimolar) values for the flux through GCS in intact mitochondria were 0.67 +/- 0.16 and 20.66 +/- 4.82 in the presence of 1 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid and 3.28 +/- 0.76 and 10.98 +/- 1.91 in presence of 0.5 microM free Ca2+, respectively. The results show that the flux through GCS is sensitive to concentrations of calcium which would be achieved in the cytoplasm of hepatocytes stimulated by calcium-mobilizing hormones.

These hormones increased flux through GCS in perfused rat liver by lOO-200% above the basal rate. The structure and mechanism of action of GCS have been studied in detail especially by Kikuchi and co-workers (Kikuchi, 1973). However, information is lacking on the physiological regulation of GCS activity. Previously proposed mechanisms of regulation of GCS activity include control by branched-chain cu-keto acids (O'Brien, 1978;Kochi et al., 1986) and by the oxidation-reduction state of mitochondrial pyridine nucleotides (Hampson et al., 1983(Hampson et al., , 1984. We have recently shown that flux through GCS is regulated by glucagon and that the mechanism of regulation is independent of changes in the redox state of mitochondrial pyridine nucleotides (Jois et al., 1989). In the present study we show that epinephrine, norepinephrine, phenylephrine, and vasopressin also stimulate flux through GCS in isolated perfused rat liver. We also show that, in isolated mitochondria, flux through GCS is sensitive to submicromolar concentrations of free Ca'+.

MATERIALS AND METHODS
[1-"C]Glycine was obtained from Du Pont-New England Nuclear. All hormones were obtained from Sigma. All other reagents were of analytical grade. Livers of male Sprague-Dawley rats (150-250 g body weight) were perfused without recirculation as described previously (Sies, 1978 (Eriksson, 1979). The stability constants used were those given by Fabiato and Fabiato (1979). Details of measurement of flux through GCS are given by Jois et al. (1989).

AND DISCUSSION
We have previously shown that glucagon stimulates flux through GCS in isolated rat hepatocytes (Jois et al., 1989). Dibutyryl-CAMP was also equally effective in stimulating the flux suggesting that the effects of glucagon may be mediated by changes in hepatic CAMP levels. However, as many of the metabolic effects of glucagon can be mimicked by ai-adrenergic agonists via CAMP-independent mechanisms, the present experiments were carried out to examine the effects of aladrenergic agonists on flux through GCS in rat liver. We employed the isolated perfused rat liver so as to avoid any receptor damage caused by proteases used in the preparation of hepatocytes. Fig. 1 shows that 0.1 pM glucagon, 1 I.LM epinephrine, 1 pM norepinephrine, 10 KM phenylephrine, and 0.1 pM vasopressin all increased flux through GCS by lOO-200% above basal rate in isolated perfused rat liver. In general the stimulatory effects were evident 10 min after hormone infusion and maximal stimulation was evident after about 20 min. cY1-Adrenergic agonists and vasopressin are known to exert their effects in the liver by increasing the concentration of free Ca*+ in the cytoplasm (Williamson et al., 1985;Exton, 1985). In isolated hepatocytes, these hormones increase cytosolic free Ca2+ concentration from resting values of 0.1-0.2 pM to about 0.6 pM (Murphy et al., 1980;Blackmore et al., 1982;Charest et al., 1983;Thomas et al., 1984). Thus, the present experiments along with our previous work (Jois et al., 1989) suggest that both CAMP and Ca*+ may be important signals in hormonal stimulation of glycine catabolism in rat liver. However, the possibility that the effects of glucagon are also mediated by increase in cytoplasmic Ca*+ cannot be ruled out as both glucagon and CAMP have been shown to elevate cytoplasmic Ca2+ levels (Charest et al., 1983;Sistare et al., 1985). Fig. 2 shows data from experiments in which rat liver mitochondria were incubated in the presence of varying concentrations of free Ca*+. Flux through GCS in rat liver mitochondria was exquisitely sensitive to concentrations of free Ca*+ which would be expected to occur in the cytosol of hormonally stimulated hepatocytes. Half-maximal stimulation occurred around 0.4 pM whereas maximal stimulation (7fold) occurred around 1 pM free Ca*+. The stimulation of flux through GCS by Ca2+ was also observed when mitochondria were incubated under state 4 conditions (in absence of ADP in the medium): the values for flux in absence and presence of 0.5 PM free Ca*+ were 0.05 f 0.01 and 0.90 + 0.26 nmol/mg protein/min, respectively (mean + S.E., n = 8). The stimulation of flux by Ca2+ was dependent on entry of Ca2+ into mitochondria as it was abolished by 1 pg/ml ruthenium red, an inhibitor of Ca*+ uptake by mitochondria (Fig. 3). Cysteamine (1 mM), a known inhibitor of GCS (Yudkoff et al., 1981) abolished i4C02 production from [1-'*C]glycine both in presence and absence of Ca*+ (data not shown). The basal flux through GCS measured in the present study is significantly lower than values previously reported by us (Jois et al., 1989) as well as others (Yoshida and Kikuchi, 1970;Hampson et al., 1983). This discrepancy is due to failure to use EGTA to maintain low Ca2+ concentrations during mitochondrial isolation and incubation in the previous studies. In the absence of a Ca2+ chelating agent or specific precautions to prevent contamination, the incubation medium is likely to have a large contamination of free Ca*+, up to 20-30 pM (Campbell and Siddle, 1976). The results from the present experiment draw attention to the necessity of controlling the Isolated rat liver was perfused with Krebs-Henseleit buffer containing 0.3 mM [l-'4C]glycine, 2.1 mM lactate, 0.3 mM pynivam, and various hormones as shown. The hormones were introduced into the perfusate entering the portal vein to give the following final concentrations: glucagon, 0.1 pM; epinephrine, 1 pM; norepinephrine, 1 pM; phenylephrine, 10 pM; and vasopressin, 0.1 FM. Details of measurements of 14C0, released and calculation of flux through GCS are given under "Materials and Methods." Results are expressed as the mean + SE. of four separate experiments. *, p < 0.05, compared to mean of values before infusion of hormone by paired t test.
free Ca*+ in media when measuring flux through GCS.
The release of i4C02 as a function of glycine concentration was examined in the presence (0.5 pM) and absence of free Ca*+ (Fig. 4). The V,.. and K,,, were 0.67 f 0.16 and 20.66 f 4.82 in absence of free Ca2+ (1 mM EGTA present) and 3.28 + 0.76 and 10.98 f 1.92 in presence of 0.5 pM free Ca*+, respectively (mean + S.E., n = 6). Thus a submicromolar concentration of free Ca*+ increased the V,., by 5-fold and decreased the K,,, by 50%.
That glycine catabolism is stimulated by glucagon, catacholamines, and vasopressin is probably related to the fact that glycine is a glucogenic amino acid (Hetenyi et al., 1988) and that the gluconeogenic pathway from glycine involves the combined action of GCS and serine hydroxymethyl transferase such that two molecules of glycine produce one molecule each of serine and CO,. glucose. Glucagon, catacholamines, and vasopressin have all been shown to increase hepatic glucose output by stimulating glycogenolysis and gluconeogenesis. Thus the physiological importance of the stimulation of flux through GCS by these three different classes of hormones probably relates to their stimulation of gluconeogenesis.
A number of mechanisms have been suggested whereby calcium acts as an intramitochondrial mediator of hormonal effects (Williamson et al., 1981;Denton and McCormack, 1985;and Halestrap, 1989). These include stimulation of calcium-sensitive enzymes, stimulation of respiration and an increase in matrix volume which, in turn, has been linked to stimulation of pyruvate carboxylation, citrulline synthesis, glutaminase, activity, etc. Further work is required to establish the precise mechanism by which Ca2+ stimulates flux through GCS in isolated liver mitochondria. However, the experiments reported in this paper clearly demonstrate the regulation of this enzyme in the perfused liver by calciummobilizing hormones. The experiments in isolated mitochondria clearly demonstrate that calcium itself is capable of mediating effects of these hormones.