Regulation of glucogen metabolism in liver by the autonomic nervous system VI. Possible mechanism of phosphorylase activation by the splanchnic nerve

242

Biochimica et Biophysica Acta, 385 (1975) 242--256 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 27620

R E G U L A T I O N OF GLUCOGEN METABOLISM IN L I V E R BY THE AUTONOMIC N E R V O U S SYSTEM VI. POSSIBLE MECHANISM OF P H O S P H O R Y L A S E ACTIVATION BY THE SPLANCHNIC N E R V E

TAKASHI SHIMAZU and AOI AMAKAWA*

Division of Neurochemistry, Psychiatric Research Institute of Tokyo, Kamikitazawa, Setagaya, Tokyo (Japan), and Department of Anatomy, Osaka University Medical School, Osaka (Japan) (Received September 12th, 1974)

Summary The effects of autonomic-nerve stimulation on the activities of phosphorylase {EC 2.4.1.1), dephospho-phosphorylase kinase (EC 2.7.1.38) and phosphorylase phosphatase (EC 3.1.3.17), and on the concentration of adenosine 3',5'-monophosphate in rabbit liver were investigated. Results were compared with the effects of epinephrine and glucagon on these enzymes. 1. The activity of liver phosphorylase increased rapidly and markedly on electrical stimulation of the splanchnic nerve, or after intraportal administration of epinephrine or glucagon. The activity was n o t affected by vagal stimulation. 2. The activity of dephospho-phosphorylase kinase increased about 2--3-fold 1 min after injections of epinephrine and glucagon, glucagon causing more activation than epinephrine. The enzyme activity was not altered by splanchnic-nerve, or vagal stimulation. 3. Injections of epinephrine and glucagon caused marked elevation of liver adenosine 3',5'-monophosphate within a few minutes. With epinephrine, the nucleotide concentration rose to a maximum after 1 min and amounted to a b o u t 3-fold increase, while with glucagon the maximum increase of approximately 8-fold increase was observed after 2 min. Stimulation of the splanchnic nerve for 10 min did not affect the adenosine 3',5'-monophosphate level in the liver. Vagal stimulation also had no effect on the level. 4. The activity of phosphorylase phosphatase decreased p r o m p t l y (within 30 s) and markedly on splanchnic-nerve stimulation, b u t did not change sig-

* Present address: S o n o d a W o m e n ' s College, Amagasaki, Japan.

243 nificantly on administration of epinephrine or glucagon. A small but insignificant increase in phosphatase activity was observed upon vagal stimulation. 5. The effect of Ca 2÷ on purified dephospho-phosphorylase kinase was studied. The activity was found to depend partially on free Ca 2÷ at low Ca2÷ concentrations (1 • 10-7--1 • 10 -s M). 6. These results suggest that the rise in hepatic phosphorylase content upon splanchnic-nerve stimulation, unlike that induced by epinephrine and glucagon, is not mediated by adenosine 3',5'-monophosphate and subsequent activation of dephospho-phosphorylase kinase, but rather by inactivation of phosphorylase phosphatase. The possible existence of a new factor in this mechanism is discussed.

Introduction The role of the sympathetic nervous system in the glycogenolytic response of liver has been postulated by previous workers to provide an explanation for the hyperglycemic effects of ether anesthesia, asphyxia and puncture of a certain region of the brain. The effects have been attributed to epinephrine and norepinephrine released in response to these stimuli [1--3]. However, direct proof for this postulation was only recently obtained and the processes and mechanisms involved in neuro-metabolic control have yet to be elucidated [4]. Our recent work has shown that electrical stimulation of the splanchnic nerve of rabbits (sympathetic innervation of the liver) causes glycogenolysis in the liver by rapid activation of phosphorylase(a-l,4-glucan:orthophosphate glucosyltransferase, EC 2.4.1.1) and glucose-6-phosphatase (D-glucose 6-phosphate phosphohydrolase, EC 3.1.3.9) [5,6], while stimulation of the vagus nerve (parasympathetic innervation) causes glycogenesis in the liver by activation of glycogen synthetase(UDPG:a-l,4-glucan a-4-glucosyltransferase, EC 2.4.1.11) [7-9]. These enzymic responses provide a satisfactory explanation for the hypergiycemic and glycogenolytic effects of sympathetic-nerve stimulation in intact, adrenalectomized and pancreatectomized animals of various species [10--14], since the effects have also been found to depend upon the integrity of the hepatic innervation [12,15]. It has also been shown that there are, apparently, some differences between the effects of sympathetic-nerve stimulation and catecholamines on giycogenolytic enzymes in the liver, suggesting that there are two distinct mechanisms for controlling phosphorylase activation and glycogenolysis [16]. In contrast to the growing body of data on the mechanism of activation of phosphorylase by catecholamines and glucagon and the implication of adenosine 3',5'-monophosphate (cyclic AMP) in this mechanism [17], little is known about the mechanism of increase in phosphorylase content upon sympatheticnerve stimulation. In an attempt to examine this question, we studied the effects of splanchnic-nerve stimulation on the concentration of cyclic AMP and the activity of dephospho-phosphorylase kinase(ATP:dephospho-phosphorylase phosphotransferase, EC 2.7.1.38) in rabbit liver, and compared the results with the effects of catecholamines and giucagon. The concentration of active phosphorylase in liver represents a balance between inactivation by phosphorylase

244 phosphatase(phosphorylase phosphohydrolase, EC 3.1.3.17) and reactivation by dephospho-phosphorylase kinase. Therefore, we also studied changes in the activity of phosphorylase phosphatase. These results are reported in this paper and a plausible mechanism for augmentation of the active phosphorylase content of the liver by the sympathetic nerve is presented. Materials and Methods

General procedure Adult male rabbits, weighing 2.3--3.0 kg, were used. The animals were fed ad libitum on laboratory pellet chow and water. The rabbits were lightly anesthetized by intravenous injection of sodium pentobarbital (20 mg/kg). L a p a r o t o m y was done under additional anesthesia with ether. Electrical stimulations of the splanchnic nerve (sympathetic) and vagus nerve (parasympathetic) were carried out as described previously [6,8]. 15--20 min after placing the electrode on the nerve of an anesthetized and laparotomized rabbit, a small portion of the liver (approx. 2--3 g) was removed by ligation and immediately immersed in liquid N2. This was used for measuring resting enzyme levels. The nerve was then stimulated. After a certain period of stimulation, another portion of the liver was quickly removed and similarly frozen in liquid N2 while stimulation was still continuing. Epinephrine bitartrate (76 pg/kg) and glucagon (100 pg/kg) dissolved in 0.7--1.0 ml of water were injected into the portal vein of laparotomized rabbits under anesthesia. Before and after the injection, portions of the liver were quickly removed and immersed in liquid N.. Enzyme assays All enzymes were assayed in extracts prepared from the frozen samples of liver in chilled 60% glycerol solution. Phosphorylase activity was analyzed as described previously [6] except that the supernatant was used after centrifugation of the liver extract at 1000 X g for 5 min. For assay of dephospho-phosphorylase kinase, 1 g of frozen liver was pulverized in a chilled mortar and ground a t - - 2 0 ° C with 2 ml of 60% glycerol solution containing 50 mM sodium ~-glycerophosphate, 250 mM NaF and 2 mM caffeine (pH 8.0). Then 8 m! of cold aqueous solution of the same salts were added. The suspension was centrifuged at 1000 X g for 5 min at 0°C and the resulting supernatant was immediately analyzed for dephospho-phosphorylase kinase activity. The assay mixture contained 3 units of dephosphophosphorylase (inactive phosphorylase) purified from rabbit liver (see below), 2.5 mM ATP, 5 mM MgC12, 50 mM NaF, 2% glycogen, 10 mM Tris/10 mM sodium fl-glycerophosphate buffer (pH 7.4) and a suitable volume of the liver supernatant in a total volume of 0.5 ml. The mixture was incubated at 37°C for 5 min, and the phosphorylase formed was determined by further incubation for 5 min with 0.5 ml of phosphorylase substrate composed of 50 mM Glu-I-P, 10 mM EDTA and 100 mM sodium citrate buffer (pH 6.1). Liberation of Pi was measured as described previously [6]. A control tube containing a mixture of the same composition was run in the second incubation only. The activity of

245 dephospho-phosphorylase kinase is given as milliunits of phosphorylase per mg of protein per min and the results are expressed in terms of the percentage changes in enzyme activity relative to that in the resting state of each animal. In the experiments on the effect of Ca 2. on purified dephospho-phosphorylase kinase, Ca • EGTA(ethylene glycol bis(~-aminoethylether)-tetraacetic acid) buffer was added to the assay mixture for dephospho-phosphorylase kinase. The concentration of Ca • EGTA plus EGTA was fixed at 9 • 10 -s M, and the concentrations of free Ca 2÷ in media with various Ca" E G T A / E G T A ratios were calculated from the binding constant of EGTA for Ca 2÷ [18]. For assay of phosphorylase phosphatase, 1 g of frozen liver was pulverized and ground at --20°C with 2 ml of 60% glycerol containing 5 mM EDTA (adjusted to pH 7.4). Then 8 ml of 10 mM Tris containing 5 mM EDTA (pH 7.4) were added and the suspension was centrifuged at 1000 X g for 5 min at 0°C. The resulting supernatant was immediately assayed for phosphorylase phosphatase. Phosphorylase phosphatase activity was estimated by following the loss of phosphorylase activity in incubation mixture containing a known a m o u n t of phosphorylase. Thus, the assay mixture contained 3 units of phosphorylase (purified from rabbit liver as described), 5 mM caffeine, 5 mM EDTA, 40 mM Tris buffer (pH 7.4) and suitably diluted liver extract in a total volume of 0.5 m!. After incubation at 37°C for 10 min, the reaction was stopped b y adding 4.5 ml of a cold solution of 55.5 mM NaF and 5 mM EDTA. Mixture at zero time was used as a control and this mixture was similarly diluted with cold N a F / E D T A solution. A 0.5-ml aliquot of the diluted reaction mixture was transferred to a new t u b e and the remaining phosphorylase activity was determined b y incubation with 0.5 ml of phosphorylase substrate composed of 50 mM Glu-l-P, 2% glycogen and 100 mM sodium citrate buffer (pH

6.1). The chief enzymes causing interference in the assay of phosphorylase phosphatase in crude liver extract are proteases, because of their actions on phosphorylase [ 1 9 ] . Phosphorylase phosphatase is nearly completely inhibited by NaF, whereas most proteolytic enzymes are not. Thus, in the present study phosphorylase phosphatase was assayed in the absence and presence of 100 mM NaF, and the activity of phosphorylase phosphatase was calculated from the difference in the values. One unit of the enzyme was defined as the amount catalyzing the inactivation of 1 unit of phosphorylase in 1 min under these conditions. Results are given as percentage increases or decreases in enzyme activity relative to the resting level. Protein was determined b y the procedure of L o w r y et al. [20].

Purification of dephospho-phosphoylase (inactive form) and phosphorylase (active form) from rabbit liver The method used previously for purification of dephospho-phosphorylase was slightly modified. All steps up to the second ethanol precipitation were carried o u t as previously [6]. In the second ethanol fraction approx. 0.004 vol. of crystalline pancreatic a-amylase (Boehringer, 10 mg/ml solution) and 1/5 vol. of 100 mM Tris buffer containing 30 mM NaC1 (pH 6.9) were added. The mixture was dialyzed for 15 h at 5°C against 20 mM Tris containing 100 mM NaF and 6 mM NaC1 (pH 6.9). Glycogen is completely digested b y amylase

246 TABLE I PURIFICATION OF DEPHOSPHO-PHOSPHORYLASE FROM RABBIT LIVER See Materials a n d M e t h o d s for details o f t h e p r o c e d u r e . Fraction

Specific activity (munits/mg protein)

Purification factor

9500 X g supernatant 40% a c e t o n e ppt. H e a t e d at 45°C for 5 rain 0.33--0.55 (NH4)2SO 4 25% e t h a n o l p p t . Amylase digestion Sephadex G-IO0 fraction

122.8 450.8 1094.5 2652.7 4752.0 4308.5 9601.7

1.0 3.7 8.9 21.6 38.7 35.0 78.8

during the dialysis. The dialyzed material was centrifuged at 78 000 X g for 60 min and the precipitate was discarded. The supernatant solution was passed through a column (2.5 cm X 30 cm) of Sephadex G-100 equilibrated with 20 mM Tris buffer containing 100 mM NaF (pH 7.0). Fractions of dephosphophosphorylase were collected and concentrated in Visking tubing against powdered polyvinylpyrrolidone (molecular weight, approx. 7 • 10 s ). The resulting clear solution of dephospho-phosphorylase could be stored at --20°C for several months without any loss of enzymic activity. Dephospho-phosphorylase activity was determined after activation with Na2 SO4 [6]. The results of a typical fractionation of dephospho-phosphorylase are summarized in Table I. Phosphorylase was purified from rabbit liver by essentially the same procedures as those used for dephospho-phosphorylase, except that the liver was perfused and homogenzied with 200 mM NaF to inhibit phosphorylase phosphatase. The steps of heat denaturation and ethanol precipitations were carried out at neutral pH in the presence of 1/10 vol. of 20 mM AMP to protect phosphorylase from denaturation. After digestion of glycogen in the second ethanol fraction with a-amylase and centrifugation at 100 000 X g for 60 min, the supernatant was subjected to a third ethanol precipitation in the presence of 20 mM AMP. The precipitated phosphorylase was dissolved in 100 mM glycylglycine (pH 7.4). One unit of the enzymes is defined as the amount liberating I pmol of Pi per min under the assay conditions described previously [6].

Purification of dephospho-phosphorylase kinase An adult rabbit was killed by intravenous injection of sodium pentobarbital (40 mg/kg). The liver was perfused, in situ, with isotonic saline and then immediately excised and homogenized in an ice-cold Waring blendor with 4 vol. of 50 mM sodium ~-glycerophosphate containing 2 mM caffeine and 100 mM NaF (pH 8.0). Subsequent procedures were carried out at temperatures less than 5°C. The homogenate was centrifuged at 1000 X g for 10 min and the supernatant solution was adjusted to pH 5.5 with 10% (v/v) acetic acid. After standing for a b o u t 1 h, the precipitate was collected by centrifugation at 13 000 X g for 10 min and suspended by homogenization in a volume of the

247 homogenizing medium equal to approx. 1/7 vol. of the supernatant. Dephospho-phosphorylase kinase b o u n d to glycogen was solubilized b y digestion with a-amylase. The turbid suspension was mixed with 0.004 vol. of crystalline pancreatic a-amylase (10 mg/ml solution) and 1/5 vol. of 100 mM Tris containing 30 mM NaC1 (pH 6.9), and dialyzed for 15 h against 20 mM Tris containing 100 mM NaF and 6 mM NaC1 (pH 7.0). It was then centrifuged at 78 000 × g for 60 min and the resulting supernatant was used as purified kinase. The purified enzyme was not stable and the activity was gradually lost on freezing at --20 ° C.

Measurement o f adenosine 3',5'-monophosphate (cyclic AMP) in the liver Cyclic AMP was isolated from liver b y the method of Walton and Garren [ 2 1 ] . A portion (approx. 300 mg) of the liver which had been frozen in liquid N2 was homogenized with 2 ml of 6% trichloroacetic acid. A trace of cyclic [3 H] AMP (20 pl, approx. 5000 cpm) was added to the homogenate to allow quantitative estimation of the overall recovery of cyclic AMP in the extraction procedure. The homogenate was centrifuged, and the trichloroacetic acid in the supernatant fraction was removed b y extraction 6 times with 2 vol. of ethylether and the residual ether was expelled with a stream of air. The extract was adjusted to l~H 7.5 with several drops of 1 M Tris and to a volume of 3 ml with water, and treated with 0.2 ml of 5% ZnSO4 neutralized with 150 mM Ba(OH)2. The mixture was placed in ice for 10 min. The precipitate was removed by centrifugation and 1.5 ml of the supernatant was applied to a column of D o w e x 50 (H ÷ form, 200--400 mesh; 0.8 cm X 7 cm). Elution was performed with water, and fractions containing cyclic AMP (detected by measuring the radioactivity of an aliquot of each 1-ml fraction) were pooled and concentrated to dryness. The residue was taken up in 0.5 ml of'water and the radioactivity of a 0.1-ml aliquot in Bray's solution [22] was determined with a liquid scintillation counter. The recoveries of cyclic AMP were calculated as 40--50%. The concentration of cyclic AMP in the above solution was determined by the protein-binding assay of Gilman [23]. Cyclic AMP-binding protein was partially purified from rat liver. The protein kinase B2 fraction of Kumon et al. [ 2 4 ] , obtained b y D E A E , Sephadex column chromatography, was concentrated by ultrafiltration and used as cyclic AMP-binding protein [25]. Results

Effects o f autonomic-nerve stimulation and glycogenolytic hormones on liver phosphorylase and dephospho-phosphorylase kinase As we showed previously [5,6], the activity of liver phosphorylase increased rapidly and markedly on electrical stimulation of the splanchnic nerve, reaching a maximum value (about 3-fold increase over the resting level) within 30 s (Fig. 1). This maximum phosphorylase level was then maintained for at least 5 rain. The half-time of the increase was approx. 14 s [6]. Intraportal administration of glycogenolytic hormones such as epinephrine and glucagon likewise increased the phosphorylase activity. The activity of phosphorylase was not influenced appreciably b y electrical stimulation of the vagus nerve.

248

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Fig. 1. C h a n g e s in h e p a t i c p h o s p h o r y l a s e c o n t e n t on e l e c t r i c a l s t i m u l a t i o n o f t h e s p l a n c h n i c n e r v e a n d v a g u s n e r v e , a n d a d m i n i s t r a t i o n o f e p i n e p h r i n e a n d g l u c a g o n . S m a l l p o r t i o n s o f t h e liver w e r e r e m o v e d serially, 30 s, 1 m i n a n d 5 m i n a f t e r t h e o n s e t o f n e r v e s t i m u l a t i o n or i n t r a p o r t a l i n j e c t i o n o f h o r m o n e s . P h o s p h o r y l a s e a c t i v i t y w a s a s s a y e d as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . R e s u l t s are m e a n s o f v a l u e s in 1 2 - - 1 8 r a b b i t s a n d are e x p r e s s e d as p e r c e n t a g e c h a n g e s in a c t i v i t y c o m p a r e d w i t h t h a t b e f o r e s t i m u l a t i o n or i n j e c t i o n ( r e s t i n g level). T h e m e a n r e s t i n g level o f t h e p h o s p h o r y l a s e a c t i v i t y a n d its s t a n d a r d e r r o r w a s 1 5 . 3 -+ 1 . 4 m u n i t s / m g p r o t e i n . T h e level d i d n o t c h a n g e s i g n i f i c a n t l y a f t e r serial r e m o v a l o f t h e liver in c o n t r o l a n i m a l s . S t a t i s t i c a l s i g n i f i c a n c e w a s d e t e r m i n e d b y t h e S t u d e n t t-test (*, P < 0 . 0 1 c o m p a r e d t o the resting value).

A possible mechanism for this rapid rise in the phosphorylase content in the liver might be that preexisting dephospho-phosphorylase is phosphorylated under the action of dephospho-phosphorylase kinase to form phosphorylase. If this occurs on splanchnic-nerve stimulation and hormone administration, these stimuli should cause increase in the kinase. Accordingly changes in the activity of dephospho-phosphorylase kinase after these stimuli were studied. As shown in Fig. 2, about 90% increase in kinase activity was observed 1 min after injec-

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Fig. 2. C o m p a r i s o n o f r e s p o n s e s o f liver d e p h o s p h o - p h o s p h o r y l a s e k i n a s e to e p i n e p h r i n e , g l u c a g o n , a n d a u t o n o m i c - n e r v e s t i m u l a t i o n . E x p e r i m e n t a l c o n d i t i o n s w e r e as f o r Fig. 1. V a l u e s are p e r c e n t a g e c h a n g e s in e n z y m e a c t i v i t y c o m p a r e d w i t h t h e r e s t i n g v a l u e ( 2 2 . 0 +- "2.8 m u n i t s / m g p r o t e i n ) . S y m b o l s f o r t h e s t a t i s t i c a l s i g n i f i c a n c e a r e as d e f i n e d f o r Fig. 1.

249 tion of epinephrine. A single injection of glucagon caused more pronounced activation of the kinase after 1 min and more than 200% increase in activity was observed 5 min after its injection. These results on kinase activation indicate a mechanism for increase in the phosphorylase content (Fig. 1) after epinephrine and glucagon. However, the kinase activity did not change, even within the first 30 s, on stimulation of either the splanchnic nerve or vagus nerve for 5 min. Effects on cyclic AMP levels in the liver Next the actions of glycogenolytic hormones and splanchnic-nerve stimulation on hepatic cyclic AMP were examined. Livers were frozen at various times after injection of the hormones or autonomic-nerve stimulation, and their cyclic AMP contents were measured. The time courses of changes in cyclic AMP concentrations are shown in Fig. 3. The resting (or basal} level of the nucleotide was 0.88 + 0.07 pmoles per mg wet weight of liver. Significant accumulation of the nucleotide was detected 30 s after injection of epinephrine into the portal vein, and the concentration rose to a maxim u m after 1 min, representing a b o u t 3-fold increase (2.3 pmol/mg liver) over the basal concentration. The cyclic AMP concentration then gradually decreased to the resting level 10 min after the injection. Roughly in proportion to the extent of the dephospho-phosphorylase kinase response, cyclic AMP increased more after glucagon injection than after epinephrine injection. In fact, the peak concentration of the nucleotide (6.8 pmol/mg liver; approx. 8-fold increase) was achieved 2 min after glucagon injection, and the level decreased more rapidly in 10 min than after epinephrine injection. Glucagon thus appears to increase accumulation of cyclic AMP more than epinephrine. These results are essentially similar to those of others on isolated perfused rat liver [26] and the liver of intact animals [ 2 5 , 2 7 ] .

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Fig. 3. T i m e c o u r s e o f c h a n g e s i n c y c l i c A M P c o n c e n t r a t i o n in t h e liver a f t e r a u t o n o m i c - n e r v e s t i m u l a t i o n and injections of epinephrine and glucagon. At the indicated times after nerve stimulation or intraportal i n j e c t i o n o f h o r m o n e s , s m a l l p o r t i o n s o f liver w e r e q u i c k l y f r o z e n , a n d t h e i r c y c l i c A M P c o n t e n t s w e r e a n a l y z e d . E a c h p o i n t is t h e m e a n o f f o u r o r m o r e d e t e r m i n a t i o n s .

250 In contrast to the marked accumulation of cyclic AMP after glucagon and epinephrine injection, no accumulation of nucleotide in the liver was observed on splanchnic-nerve stimulation for 10 min. Vagal stimulation also had no effect on the cyclic AMP concentration. These findings conform with the above observation that splanchnic-nerve stimulation did not induce any increase in the activity of dephospho-phosphorylase kinase, and indicate that the rapid rise in the phosphorylase content upon splanchnic-nerve stimulation is not mediated by cyclic AMP and subsequent activation of the kinase.

Effects on phosphorylase phosphatase of the liver The level of phosphorylase activity in liver is mainly due to a balance between the conversion of dephospho-phosphorylase to phosphorylase by the enzyme dephospho-phosphorylase kinase and the reconversion of phosphorylase to dephospho-phosphorylase by the phosphorylase phosphatase. Thus regulation of phosphorylase phosphatase, the enzyme inactivating phosphorylase, is probably also important in regulation of phosphorylase activity. A preliminary experiment showed that the level of the inactivating enzyme (phosphorylase phosphatase) in liver extracts from normal rabbits is extremely higher than that of the enzyme activating phosphorylase (dephospho-phosphorylase kinase) using the present assay conditions and definitions of the two enzyme activities (369.7 -+ 24 munits/mg protein vs 22.0 + 2.8 munits/mg protein). Therefore, the effects of autonomic-nerve stimulation and the glycogenolytic hormones on phosphorylase phosphatase were investigated (Fig. 4). The activity of the phosphatase promptly decreased on splanchnic-nerve stimulation: a significant decrease (approx. 20%) in its activity was observed after 30 s and the minimum activity (40% decrease) was observed after 5 min. +20 Splanchnic- n e r v e stimulation

Vagus-nerve stimulation

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Fig. 4. E f f e c t s o f a u t o n o m i c - n e r v e s t i m u l a t i o n and i n j e c t i o n s o f e p i n e p h r i n e and g l u c a g o n o n the a c t i v i t y o f h e p a t i c p h o s p h o r y l a s e p h o s p h a t a s e . E x p e r i m e n t a l c o n d i t i o n s w e r e similar t o t h o s e for Figs 1 a n d 2. R e s u l t s are e x p r e s s e d as p e r c e n t a g e c h a n g e s in e n z y m e a c t i v i t y f r o m t h a t b e f o r e t r e a t m e n t . T h e m e a n resting value o f p h o s p h o r y l a s e p h o s p h a t a s e w a s 3 6 9 . 7 -+ 24 m u n i t s / m g p r o t e i n . *, P < 0 . 0 1 ; *% P < 0 . 0 5 c o m p a r e d t o t h e resting value.

251 On the other hand, a small but insignificant increase in phosphatase activity was observed upon vagal stimulation. N o significant change in the activity was observed after injection of epinephrine or glucagon. These results suggest that the rise in hepatic phosphorylase content upon splanchnic-nerve stimulation, unlike that u p o n injection of glycogenolytic hormones, is mainly caused by a rapid suppression of phosphorylase phosphatase activity, probably by inactivation of the enzyme [ 2 8 ] , resulting in decrease in the rate of phosphorylase inactivation.

Effect of free Ca 2÷ on the activity of purified dephospho-phosphorylase kinase Ca ~÷ has been shown to enhance phosphorylase b kinase (EC 2.7.1.38) activity of skeletal muscle and this effect of Ca 2÷ is thought to be involved in phosphorylase activation and glycogenolysis coupled with muscle contraction [ 2 9 - - 3 4 ] . Thus, low concentrations (1 • 1 0 : 6 - - 1 • 10 -7 M) of free Ca 2÷, which roughly correspond to those in living muscle, can stimulate active as well as inactive phosphorylase b kinase, and activation of phosphorylase kinase makes the enzyme more sensitive to low concentrations of the metal. It is u n k n o w n whether Ca 2÷ has a similar effect on liver phosphorylase kinase to that observed in muscle. Accordingly, dephospho-phosphorylase kinase was 'purified from rabbit liver and the effect of Ca 2÷ on it was investi-

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10.5

C o n c e n t r a t i o n of C a 2 + ( M )

E f f e c t o f free Ca 2+ c o n c e n t r a t i o n o n d e p h o s p h o - p h o s p h o r y l a s e k i n a s e a c t i v i t y . D e p h o s p h o - p h o s p h o r y l a s e k i n a s e p u r i f i e d f r o m r a b b i t liver w a s a s s a y e d in t h e p r e s e n c e o f v a r i o u s c o n c e n t r a t i o n s o f free Ca 2+. F o r 0 M Ca 2+ c o n c e n t r a t i o n , 9 • 1 0 - 5 M E G T A w a s a d d e d t o t h e assay m i x t u r e o f t h e k i n a s e ( s e e Materials a n d M e t h o d s ) . B y a d d i n g Ca • E G T A b u f f e r , t h e c o n c e n t r a t i o n o f free Ca2+ w a s i n c r e a s e d a n d its c o n c e n t r a t i o n w a s c a l c u l a t e d f r o m t h e b i n d i n g c o n s t a n t o f E G T A f o r Ca 2+. T h e k i n a s e a c t i v i t i e s at v a r i o u s c o n c e n t r a t i o n s o f Ca 2+ are e x p r e s s e d as p e r c e n t a g e s o f t h e original k i n a s e a c t i v i t y , a s s a y e d in t h e s t a n d a r d reaction mixture. Fig. 5.

252 gated, using C a . EGTA buffer to control the free Ca ~÷ concentration. The representative results in Fig. 5 show that removal of Ca 2÷ from the purified kinase with chelating agent (EGTA, 9 • 10 -s M) resulted in about 70% decrease in activity. On increasing the concentration of free Ca 2÷ to 2 • 10 -s M, the kinase activity was partially, though not completely restored. With other preparations of the kinase, chelation of Ca 2÷ and subsequent addition of free Ca 2÷ had less effect on the activity than that seen in Fig. 5. These results suggest that the liver kinase, like that from muscle, is stimulated by a minute a m o u n t (1 • 10-s--1 • 10 -7 M) of Ca 2÷, but that reversible stimulation of the liver kinase b y Ca 2÷ is less than that of the muscle enzyme, which is completely inhibited b y removal of Ca 2÷ and reactivated by its addition [30,31]. Therefore, the role of Ca 2÷ in the regulation of liver phosphorylase kinase cannot be ruled out b u t its physiological importance is still uncertain. Discussion The mechanism of phosphorylase activation b y epinephrine and glucagon appears to be well established. Epinephrine and glucagon stimulate membraneb o u n d adenyl cyclase in certain tissues and cause the accumulation of a second messenger, i.e. cyclic AMP in the cell [17,35,36]. The nucleotide thus formed then activates a protein kinase (EC 2.7.1.37) by binding with an inhibitory subunit (regulatory subunit) of the enzyme causing its dissociation from the catalytic subunit in various tissues, including liver and muscle [24,37--40]. In muscle the activated protein kinase phosphorylates the inactive form of phosphorylase b kinase, thereby activating it [41,42]. The activated phosphorylase b kinase phosphorylates phosphorylase b, converting it to an active form, phosphorylase a. It is quite probable that the activation of phosphorylase in liver by epinephrine and glucagon proceeds b y mechanisms similar to those described for muscle [17,43,44]. In the present study, the series of reactions for phosphorylase activation was consistently found to take place in the liver. However, recent studies on the mechanism of epinephrine have revealed that the intermediation of cyclic AMP is not always necessary. Sherline et al. [45] reported that there are t w o independent mechanisms whereby catecholamines can activate phosphorylase. One is through a ~-receptor-mediated rise in cyclic AMP, and the other is a cyclic AMP-independent mechanism mediated by a-receptors. It has also been suggested that the increase in cyclic AMP accumulation due to epinephrine is apparently unrelated to its stimulation of gluconeogenesis in rat liver [46]. It should be emphasized here that splanchnic-nerve stimulation did not induce accumulation of hepatic cyclic AMP or increase in dephospho-phosphorylase kinase activity, at least during early effects on the liver. Sympathetic nervous activity is mediated b y release of norepinephrine and its effect has often been ascribed to that of norepinephrine. Moreover, the local concentration of norepinephrine in the liver following nerve stimulation is presumably much greater than the level in circulation. Thus it might be expected that hepatic cyclic AMP would increase u p o n splanchnic-nerve stimulation, thereby activating a protein kinase, dephospho-phosphorylase kinase, and finally phos-

253 phorylase. However, this could not demonstrated in the present study. Instead, splanchnic-nerve stimulation was found to cause a rapid increase in the phosphorylase content without affecting the hepatic levels of cyclic AMP and dephospho-phosphorylase kinase. The lack of effect of splanchnic-nerve stimulation on cyclic AMP and dephospho-phosphorylase kinase further supports the suggestion [16] that sympathetic nerves and catecholamines (and glucagon) activate liver phosphorylase by different mechanisms, and that the former mechanism may involve release (or formation) of some factor other than norepinephrine. Recently, more emphasis has been put on regulation of phosphorylase at the level of kinase than at the level of phosphatase. The concentration of phosphorylase phosphatase in liver, however, is sufficient under certain conditions to catalize the inactivation of all the phosphorylase present in a few minutes [47,48]. In fact, the content of phosphorylase phosphatase was approx. 20 times higher than that of dephospho-phosphorylase kinase on a protein basis in crude extracts of rabbit liver (369.7 munits/mg protein vs 22.0 munits/mg protein), although the former enzyme might be overestimated by its assay in the presence of caffeine [48]. Thus, it seems very likely that under certain conditions inactivation of phosphorylase phosphatase may play a crucial role in increasing phosphorylase activity and glycogenolysis in the liver in vivo. This effect was shown in our present study: upon splanchnic-nerve stimulation the activity of phosphorylase phosphatase was promptly and markedly depressed, while the activity of the kinase remained unchanged. Phosphorylase phosphatase, like phosphorylase and its kinase, has been suggested to exist in two forms in dog liver [28], avian skeletal muscle [49] and bovine adrenal cortex [50]. The inactive form of the phosphatase can be converted to the active form, in vitro, by a reaction involving ATP and Mg:÷ [28,49--51], while the active form can be inactivated by incubation with ATP alone [28,49--51] or with NaF [52]. In the light of these studies, it is interesting that, as pointed out by Merlevede et al. [28], three enzymes which are implicated in the phosphorylase system, phosphorylase, phosphorylase kinase and phosphorylase phosphatase can be transformed from an inactive to an active form by a reaction involving ATP and Mg2÷. Hence, it is difficult to imagine how phosphorylase activity could be regulated by the concentrations of ATP and Mg2÷, since these factors would apparently activate two enzymes regulating phosphorylase activity in opposite ways: the kinase as well as the phosphatase. Therefore, other factors must control the kinase and phosphatase activity. One factor is probably cyclic AMP, although its action differs in different tissues. In muscle tissue cyclic AMP has been shown to stimulate both activation of phosphorylase kinase [41,42,53] and inactivation of phosphorylase phosphatase [51], while in adrenal cortex this nucleotide can increase phosphorylase activity by affecting the inactivation of the phosphatase without affecting the kinase [50]. In liver, on the other hand, cyclic AMP can stimulate activation of phosphorylase kinase [44] but does not affect inactivation or activation of the phosphatase [28]. The present finding that splanchnic-nerve stimulation promptly decreases the phosphatase but does not influence the kinase, thus adds another factor which is involved in the regulation of phosphorylase phosphatase in liver; its effect is probably in stimulation of phos-

254 phatase inactivation or inhibition of the activation process, which otherwise proceeds rapidly in liver in vivo. However, the possibility cannot be ruled out that splanchnic-nerve stimulation may inhibit the phosphatase directly. Fig. 4 shows that there was a slight but insignificant increase in the phosphatase activity upon vagal stimulation. This increase may have been related to activation of the glycogen synthetase system in the liver, since hepatic glycogen synthesis can be enhanced by stimulation of this nerve, probably due to activation of glycogen synthetase phosphatase [8,9]. It has sometimes been suggested that a single enzyme (phospho-protein phosphatase) might be responsible for both the inactivation of phosphorylase (phosphorylase phosphatase reaction) and the activation of glycogen synthetase (glycogen synthetase phosphatase reaction). Recent studies on the substrate specificity of purified muscle glycogen synthetase phosphatase have revealed that this enzyme can also dephosphorylate phosphorylated histone [54,55], active phosphorylase b kinase [55,56], and phosphorylase a [55]. However, further studies are required on this hypothesis. It now seems apparent that the increase in hepatic phosphorylase content after splanchnic-nerve stimulation, unlike that after injection of epinephrine or glucagon, is not mediated by cyclic AMP and the subsequent activation of Epinephrine

I Sympathetic nerves

oN

Glucagon ATP ca2+

Adenyl cyclase

X (?)

Inactive phosphorylase kinase \

cychc

AMP

P r"o t e i n " Kinase

""N

.... J /

/

,s

Active #'* phosphoPylase kinase

Dephospho- phosphorylase

PhosphoPylase

Active pllosphorylase phosphatase

Inactive phosprlorylase plTosphatase

Fig. 6. Possible m e c h a n i s m s for p h o s p h o r y l a s e a c c u m u l a t i o n in liver b y s y m p a t h e t i c nerves and g l y c o genolytic hormones.

255

phosphorylase kinase, but mainly by inactivation of phosphorylase phosphatase (Fig. 6). This effect of sympathetic-nerve stimulation, therefore, may be mediated by release (or formation at the nerve terminals on liver cells) of some factor (Factor X in Fig. 6) other than norepinephrine. The existence of this factor was first suggested several years ago [16] but its nature is still unknown. However, it seems relevant in this connection to note that the release of catecholamines in response to nerve stimulation from the catecholamine-containing vesicles (or granules) in the adrenal medulla as well as sympathetic neurones occurs by a process of exocytosis in which the entire soluble contents of the granule are secreted into the extracellular spaces. In fact, the catecholaminestorage vesicles (chromaffin granules) of the adrenal medulla contain a large proportion of specific soluble proteins called chromogranins and these proteins are secreted together with ATP and catecholamines [ 5 7 - 6 0 ] . Furthermore, evidence has been presented that a protein having immunological properties identical with those of purified chromogranin A, a major component of the chromogranins, is present in the amine-storing granules of the sympathetic neurones and ganglia, and that this protein is secreted when the nerves are stimulated [ 5 7 , 6 1 - 6 3 ]. Acknowledgements The authors are deeply indebted to Drs M. Takeda and Y. Ohga of the Department of Biochemistry, Kobe University School of Medicine, for determination of cyclic AMP by protein-binding assay. Thanks are also due to Dr Y. Nishizuka of Kobe University School of Medicine, for his valuable suggestion. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

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