Regulation of glycogen metabolism in liver by the autonomic nervous system II. Neural control of glycogenolytic enzymes

BIOCHIMICA ET BIOPHYSICA ACTA

335

BBA 26023 REGULATION OF GLYCOGEN METABOLISM IN L I V E R BY T H E AUTONOMIC NERVOUS SYSTEM II. N E U R A L CONTROL OF GLYCOGENOLYTIC ENZYMES TAKASHI SHIMAZU A~3 AOI AMAKAWA Departmen~ of Anatomy, Osaka University Medical School, Osaka (Japan) (Received J u n e 25th, 1968)

SUMMARY

I. The effects of electrical stimulation of the autonomic nerves on glycogen phosphorylase (~-I,4-glucan:orthophosphate glucosyltransferase, EC 2.4.1.1) and on glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9) of rabbit liver were investigated. The activities of these two enzymes increased markedly after stimulation of the splanchnic nerve (sympathetic), and the effect was not eliminated b y adrenalectomy or pancreatectomy of the rabbits. 2. The response of the enzymes reached a maximum within 30 sec after the onset of splanchnic-nerve stimulation, and the half-time of the increase was approximately 14 sec. 3. The effect of splanchnic-nerve stimulation was completely counteracted by simultaneous stimulation of the vagus nerve (parasympathetic), although vagal stimulation alone had little effect on the enzymes. 4. The dephosphophosphorylase and dephosphophosphorylase kinase (ATP: dephosphophosphorylase phosphotransferase, EC 2.7.1.38 ) from rabbit liver were partially purified and their properties were studied. 5. Total phosphorylase of the liver was determined by assaying phosphorylase activity after conversion of dephosphophosphorylase to the active form. The total amount of phosphorylase was not altered significantly by splanchnic-nerve stimulation or vagal stimulation. In the resting state, about one fourth of the total phosphorylase in the liver was active. The amount increased to three fourths of the total after stimulation of the splanchnic nerve, but was unchanged during vagal stimulation. 6. Previous treatment of the rabbits with actinomycin S failed to block the increase in phosphorylase activity after splanchnic-nerve stimulation. These results indicate that the increase in phosphorylase activity caused by splanchnic-nerve stimulation can be attributed to enzymic conversion of the dephosphophosphorylase to the active form during stimulation.

INTRODUCTION

The autonomic nervous system originates in the hypothalamus and is made up of the sympathetic and parasympathetic nerves. These nerves are known to Biochim. Biophys. Acta, 165 (1968) 335-348

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T. SHIMAZU, A. AMAKAWA

regulate the activities of the visceral organs continuously and involuntarily, and hence are likely to be important in controlling the metabolism of the organs. The role of the autonomic nervous system in regulating the activities of liver enzymes has recently been recognized1-7. Direct stimulation of the autonomic nervous system affects the activities of liver enzymes which are concerned with amino acid metabolism1, ~ and carbohydrate metabolism 5-7. Of these, glycogen metabolism in liver is of particular interest because of its major role in the rapid provision of energy and in the maintenance of the proper amount of glucose in the blood. Electrical stimulation of the sympathetic nerve of rabbits causes a rapid increase in the activities of glucogen phosphorylase (c¢-I,4-glucan:orthophosphate glucosyltransferase, EC 2.4.1.1 ) and glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase, EC 3-1.3-9) in the liver, followed by an increase in the concentration of glucose in the blood and a pronounced decrease in the content of glycogen in the liverS, s. The stimulation of the parasympathetic nerve, however, causes a marked increase in the activity of glycogen synthetase (UDPglucose:¢c-I,4-glucan *¢-4-glucosyltransferase, EC 2.4.1.11 ) in the liver, followed by a decrease in blood glucose s, 7. Our previous work has now been extended. It was shown that the activities of liver phosphorylase and glucose-6-phosphatase increase almost instantaneously after stimulation of the splanchnic nerve (sympathetic) and that this effect is completely counteracted by a simultaneous stimulation of the vagus nerve (parasympathetic), although vagal stimulation alone has little effect on these enzymes. Total phosphorylase (phosphorylase plus its inactive form, dephosphophosphorylase) in liver can also be determined. Phosphorylase activity rose from less than 30 % to nearly 8o % of the total phosphorylase activity within 30 sec after the onset of splanchnicnerve stimulation. EXPERIMENT

Electrical stimulation of autonomic nerves The nerve fibers in the liver are autonomic, consisting of sympathetic fibers from the splanchnic nerve and parasympathetic ones from the vagus nerve. Adult male rabbits, weighing 2.3-2.5 kg, were used. The rabbits were lightly anesthetized b y intravenous injections of pentobarbital sodium (IO mg/kg). Laparotomy was conducted under additional anesthesia with ether. For electrical stimulation of the sympathetic nerve, the left splanchnic nerve was exposed and freed from surrounding adipose tissue just under the diaphragm, and a bipolar platinum electrode, fitted in a tiny plastic plate, was placed on the nerve. For electrical stimulation of the parasympathetic nerve, the left vagus nerve was exposed at the neck, and an electrode was placed on the nerve. The proximal end of the nerve was cut off approximately I cm from the site of the electrode. For simultaneous stimulation of the both nerves, two electrodes were separately placed on the splanchnic nerve and the vagus nerve of the rabbit. Electrical stimuli were supplied by an electronic stimulator. Square pulses of o.5-msec duration, IOO per sec frequency and 5o-V amplitude, were delivered to the splanchnic nerve through an isolation unit. In some cases, pulses of smaller amplitude, e.g. 15 V, but the same duration and frequency were applied to the nerve, with essentiBiochim. Biophys. Acta, 165 (1968) 335-348

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ally similar results. For vagal stimulation, square pulses of o.5-msec duration, ioo per sec frequency and 2o-V amplitude, were delivered to the nerve through an isolation unit. Bilateral adrenalectomy and pancreatectomy of the rabbits were carried out b y laparotomy I5-2o rain before stimulation.

Preparation of the liver 15 - 2o rain 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 nitrogen. This was used for measuring the initial or resting levels of enzyme activities and served as the control for each animal. The nerve was then stimulated. After a certain period of stimulation, another portion of the liver was quickly removed and similarly frozen in liquid nitrogen while the stimulus was still continued.

Fixation and assay of liver phosphorylase The basic methods used were as described previously 5. Dephosphophosphorylase kinase (ATP:dephosphophosphorylase phosphotransferase, EC 2.7.1.38) and phosphorylase phosphatase (phosphorylase phosphohydrolase, EC 3.1.3.17) are inhibited b y EDTA and F-, respectively. The selective actions of these inhibitors in a chilled glycerol-water solution and the preparation of the liver extract at a considerably lower temperature were used for measurement of the actual level of phosphorylase in t h e liver, as suggested by DANFORTH, HELMREICH AND CORIs for living muscle. Homogenization of liver in an ice-cold aqueous solution of these inhibitors gave a very high value for the phosphorylase activity in resting liver and failed to detect the effect of nerve stimulation on enzyme activity. This was mainly because the kinase acted very rapidly under these conditions. Inhibitors were, therefore, added as solutions in glycerol-water and cooled nearly to the freezing point (--2o°). This solvent allowed the inhibitors to penetrate the liver powder at a considerably lower temperature. It also acted as a lubricant to facilitate rapid and smooth mixing of the liver powder with the inactivating solution. In practice the procedure was carried out as follows. A portion (1-2 g) of each frozen liver was promptly pulverized in a chilled mortar and ground at --20 ° with 2 vol. of 60 % glycerol solution containing 50 mM NaF and 5 mM EDTA adjusted to pH 6.1 with NaOH and chilled nearly to the freezing point. After the material was ground, 8 vol. of a cooled aqueous solution of the same salts were added. The suspension was centrifuged at 9ooo × g for 5 min at --5 °, and the supernatant was immediately analyzed for phosphorylase activity 9. The assay mixture contained 5° mM glucose 1-phosphate, 1% glycogen, 25 mM NaF, 2.5 mM EDTA (pH 6.I), 50 mM sodium citrate buffer (pH 6.1) and a suitable volume of centrifuged liver extract in a total volume of I ml. Inorganic phosphate released by phosphorylase during a 5- or Io-min incubation at 37 ° was determined by TAKAHASHI'Smethod lo. In this method, spontaneous hydrolysis of the acid-labile glucose 1-phosphate is kept at a minimum. Thus, the reaction was stopped by adding 2 ml of a cold solution of 1 % ammonium molybdate in o.375 M H2SO 4. Then the tube was immediately transferred to an ice-water bath and 4 ml of isobutanol were added. The mixture was shaken vigorously for about I rain. After centrifugation for several rain, 2 ml of the isobutanol layer Biochim. Biophys. Acta, I65 (I968) 335-348

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T. SHIMAZU, A. AMAKAWA

were transferred to a new tube and 2 ml of 0. 5 % ascorbic acid solution and I ml of ethanol were added. After mixing thoroughly, the tube was incubated for 30 mill at 37 °. Absorbance at 66o m# was measured spectrophotometrically. One unit of the enzyme was defined as that amount which caused the liberation of I/,mole PI in I min, under the conditions of the above assay. Results were given as milll-units per mg of protein, or expressed in terms of the percentage increase or decrease in enzyme activity compared with that before nerve stimulation. Protein was determined by the procedure of LowRY et al. 11.

Determination of total phosphorylase in liver Since liver dephosphophosphorylase (also referred to as "inactive phosphorylase"), unlike muscle phosphorylase b, displays little or no activity even when assayed in the presence of AMP 12,la, the measurement of total phosphorylase (phosphorylase and dephosphophosphorylase combined) in liver is not possible by this method. However, liver dephosphophosphorylase, like the muscle enzyme, is converted into phosphorylase by a specific kinase in the presence of ATP and Mg2+ (see ref. 14). Thus, the total phosphorylase can be determined by assaying phosphorylase activity after conversion of dephosphophosphorylase to the active form. Dephosphophosphorylase kinase from rabbit liver was partially purified as described below, and the kinase was added to the system for the assay of phosphorylase. Later, it was noticed that the addition of the kinase was not necessary if endogenous kinase was fully activated. An attempt was made to measure the total phosphorylase activity, based on the fact that dephosphophosphorylase is activated by high concentrations of NazSO4 (ref. 15). However, this method was inferior to the kinase method, because of the inhibitory effect of Na~SO~ on phosphorylase assay and because dephosphophosphorylase activity is only partly restored even in the presence of a high concentration of Na2SO 4 such as o.5 M (see also ref. 13). The method finally adopted was as follows. Centrifuged liver extract prepared for phosphorylase assay was incubated at 37 ° for 3° min in medium containing 2.5 mM ATP, 5 mM MgSO 4, 5o mM NaF and 20 mM sodium glycerophosphate (pH 7.4) in a total volume of 0.5 ml. The change of activity with the time of incubation is shown in Fig. I. During this period (the first stage) dephosphophosphorylase is efficiently converted to phosphorylase. ATPase (ATP phosphohydrolase, EC 3.6.1.4) and phosphorylase phosphatase are inhibited by NaF. Although cyclic 3',5'-AMP 60 "T, "£

~"~ 40

bE ~-~ O~

10 20 30 Incubotion time(min)

4-0

Fig. I. T i m e c u r v e of a c t i v i t y d u r i n g i n c u b a t i o n for a s s a y of t o t a l p h o s p h o r y l a s e in liver. T h e s u p e r n a t a n t of t h e liver e x t r a c t w a s i n c u b a t e d a t 37 ° for t h e period i n d i c a t e d on t h e abscissa in m e d i u m c o n t a i n i n g 2.5 m M ATP, 5 m M MgSO 4, 50 m M 2~aF a n d 20 m M s o d i u m glycerop h o s p h a t e (pH 7.4). Following i n c u b a t i o n , p h o s p h o r y l a s e a c t i v i t y w a s d e t e r m i n e d as i n d i c a t e d in the text.

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has been thought to accelerate the rate of activation of dephosphophosphorylase by the kinase in the presence of ATP and Mgz+ (see refs. 16-18), it had an insignificant effect at concentrations of lO -3 to lO -8 M in the present system. It was, therefore, not included. At the end of the first stage of incubation, the second stage of incubation (for another IO rain at 37 °) was initiated by adding 5o mM glucose 1-phosphate, 1% glycogen, 5 mM AMP and 5o mM sodium citrate (pH 6.1) in a total volume of I ml. Control tubes containing mixtures of the same composition were not incubated during the second stage. The Pi liberated was determined as described above. The activit y of total phosphorylase is expressed as milli-units of phosphorylase per mg of protein.

Partial purification of dephosphophosphorylase kinase from rabbit liver and its kinetic properties The complexity of the assay and the presence of inhibitory or interfering materials sometimes made it difficult to estimate the kinase activity of liver homogenates or crude extracts. Purification of dephosphophosphorylase kinase was undertaken to clarify certain kinetic properties of the kinase. This was done by a modification of the method described for purification of muscle phosphorylase b kinase TM. An adult rabbit was fasted for 2 days and then killed by ether anesthesia. The liver was perfused, in situ, with 5o mM sodium glycerophosphate containing 2 mM caffein (pH 8.0) and then immediately excised and homogenized in an ice-cold Waling blendor with 4 vol. of the same medium. Subsequent procedures were carried out at below 3 °. The homogenate was centrifuged at 15o00 × g for 15 min, and the precipitate was homogenized with I vol. of the same medium and recentrifuged. The resulting supernatant solutions were combined and adjusted to pH 5.5 with IO % acetic acid. After standing at o ° for about I h, the acid precipitate was collected by centrifugation and suspended in a volume of 50 mM sodium glycerophosphate (pH 8.0) equal to approximately 1/7 vol. of the supernatant. Brief homogenization was performed using a glass homogenizer to assure complete mixing and fine dispersion of the material. The suspension was centrifuged at 35o00 × g for 30 min and the precipitate was discarded. The supernatant fluid was centrifuged at 1o5o00 × g for 60 min, and the sedimented protein was taken up by homogenization with 5-1o ml of 5o mM sodium glycerophosphate (pH 8.o) and used as purified kinase. Although this preparation still contained considerable amounts of phosphorylase and the overall purification was only about 2o-fold that of the homogenate on a protein basis (the purification factor could only be calculated roughly since the initial kinase activity of the homogenate or crude extract could not be determined accurately), most of the material interfering with the assay of kinase activity was eliminated. Several kinetic properties of the purified kinase were investigated; the optimum p H for the reaction was around 7.4, and the optimum concentrations of ATP and Mg 2+ were 2.5 mM and 5 mM, respectively (Figs. 2 and 3)Kinase activity was estimated by following the increase in phosphorylase activity of the incubation mixture containing the kinase, dephosphophosphorylase (purified from rabbit liver, see below), ATP and Mg 2+. The reaction mixture conrained 3 units of dephosphophosphorylase, 2.5 mM ATP, 5 mM MgSO4, 50 mM NaF, 20 mM sodium glycerophosphate (pH 7.4) and the kinase in a total volume of 0.5 ml. Before and after IO min of incubation at 37 °, phosphorylase activity was estimated as described above in the presence of 5 mM AMP. Biochim. Biophys. Aaa, I65 (1968) 335-348

340

T. SHIMAZU, A. AMAKAWA ._c

0.4

"o o

0.3

g~ ~o.~ ~E

O1::

~.-[: o.1

#o

,,~ o

_ _

10-4

10-3

10-'2

EATP] (M)

~9

o

10-4

10-3 [Mg S04] (M)

10-2

Fig. 2. Effect of ATP concentration on the activity of dephosphophosphorylase kinase. The activity of dephosphophosphorylase kinase was estimated by following the increase in phosphorylase activity of reaction mixture containing kinase (o.i ml of purified preparation), 3 units of dephosphophosphorylase, 5 mM MgSO4, 5° mM NaF, 20 mM sodium glycerophosphate (pH 7.4), and varying concentrations of ATP. The mixture was incubated for io min at 37 °. Fig. 3. Effect of Mg~+ concentration on the activity of dephosphophosphorylase kinase. Conditions of assay for dephosphophosphorylase kinase were as indicated in Fig. 2, except t h a t varying concentrations of MgSO4 were used in the presence of 2.5 mM ATP.

Purification of dephosphophosphorylase from rabbit liver An adult rabbit was fasted for a day and then killed b y ether anesthesia. The liver was perfused, in situ, with 40 mM Tris containing 2 mM caffein and 5 mM E D T A (pH 8.0) and then immediately homogenized in a Waring blendor with 4 vol. of ice-cold medium of the same composition. After standing at o ° for about 2 h, the phosphorylase activity of the homogenate was measured. Almost all the phosphorylase was transformed to dephosphophosphorylase b y the action of phosphorylase phosphatase during homogenization and subsequent standing of the homogenate at o °. All subsequent steps were carried out at 0-3 ° unless specified otherwise. The homogenate was then centrifuged. This and subsequent centrifugations were carried out at 95o0 × g for IO min. Phosphorylase phosphatase was removed b y adsorption on calcium phosphate gel. An amount of gel equal to about o.I vol. of the homogenate was added to the supernatant solution and the mixture was stirred continuously for 20 rain. The gel was removed by centrifugation. Cold (--20 °) acetone was added slowly with stirring, and after standing for I h the o % to 4 ° % acetone fraction was collected b y centrifugation. The precipitate was homogenized with a volume of o.I M N a F equal to half the volume of the homogenate, and the insoluble proteins were removed b y centrifugation. The supernatant fluid was then used for the heat-denaturation step or, alternatively, it could be stored in the frozen state. It was heated with swirling in a b a t h at 55 ° until the temperature rose to 45 °. This temperature was maintained for 5 rain. The solution was then chilled in an ice-water bath. Denatured proteins were discarded after centrifugation and neutralized (NH4) ~S04 solution saturated at room temperature was added to bring the preparation to o.33 saturation. The precipitate was removed b y centrifugation. The supernatant was brought to 0.55 saturation b y addition of more satd. (NH4)~SO 4 solution, and the precipitate at 0.33-0.55 saturation was collected b y centrifugation. This fraction was dissolved in approx. 0.05 vol. of the original homogenate of o.I M NaF, and dialyzed for several hours against cold o.I M N a F containing 0.5 mM KOH. After dialysis, a small amount of insoluble material was removed b y centrifugation. The preparation was then used for ethanol fractionation or could be kept frozen at Biochim. Biophys. Acta, 165 (1968) 335-348

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--20 ° . Cold ( - - 2 0 °) ethanol was added slowly to the preparation with stirring to reach a final concentration of 25 %. After standing for 2 h, the precipitate was collected b y centrifugation and taken up in o.I M NaF, adding about half that volume before the ethanol precipitation. Ethanol was again added to a final concentration of 25 %, and the precipitate was collected and dissolved as previously mentioned. The second ethanol fraction was stable in the frozen state for several months and was used as substrate for the assay of dephosphophosphorylase kinase (see above). This enzyme preparation still contained large amounts of glycogen but negligible amounts of phosphorylase, phosphorylase phosphatase and dephosphophosphorylase kinase. The results of a typical fractionation of dephosphophosphorylase are summarized in Table I. TABLE I S U M M A R Y OF T H E

PURIFICATION OF DEPHOSPI-IOPHOSPHORYLASE

Fraction

Specific activity (raunits/mg protein)

Yield (%)

Homogenate C a l c i u m p h o s p h a t e gel s u p e r n a t a n t 40% acetone ppt. H e a t e d a t 45 ° for 5 m i n 0.33-o.55 (NH4)zSO 4 ISt e t h a n o l ppt. 2rid e t h a n o l ppt.

26.9 49.1 235.3 387. 3 1194.5 2138.2 2343.2

lOO 63 52 49 5° 43 41

Dephosphophosphorylase activity was determined after activation with Na2SO4 (ref. 15). For assay, the dephosphophosphorylase was subjected to a final concentration of o.5 M NazSO 4 in the phosphorylase assay system. The reaction mixture was adjusted to p H 6.1 and contained 0.5 M Na2S04, 5o mM glucose iphosphate, i To glycogen, 5 mM AMP, 25 mM NaF, and a suitable amount of dephosphophosphorylase in a total volume of I ml. After a Io-min incubation at 37 °, the P1 liberated was determined as described above. One unit of enzyme was defined as the amount liberating I pmole Pl per rain, under the conditions of the assay.

Assay for glucose-6-phosphatase A portion (I g) of each liver which had been frozen in liquid N= was homogenized in a glass homogenizer with 9 vol. of ice-cold isotonic KC1 solution. The homogenate was centrifuged at 120o0 × g for IO min at o °. The supernatant fraction was analyzed for glucose-6-phosphatase, as previously described 5. Results were given as percentage increase or decrease in enzyme activity compared with that before nerve stimulation.

Injection of actinomycin S Actinomycin S was dissolved in a minimum amount of acetone and diluted with water to make a 0.2 % suspension. The suspension was shielded from light and put in a refrigerator until a clear solution was obtained. Rabbits ware injected with 1.5 mg/kg of actinomycin, 2 h (intraperitoneally) or 20 min (intravenously) before being anesthetized. Control animals received appropriate vehicle. Biochim. Biophys. Acta, 165 (1968) 335-348

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Preliminary experiments were carried out on the amount of actinomycin S required to prevent enzyme induction in rabbit liver. Tyrosine aminotransferase (Ltyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) of rabbit liver was chosen for this purpose. This enzyme has been shown to be induced by the administration of glucocorticoid20, 21. Intraperitoneal injection of rabbits with IO mg/kg of prednisolone caused a great increase in the activity of liver tyrosine aminotransferase with a peak of approx. 3 times the control level about 8 h after the injection. The induction of this enzyme was completely suppressed by treatment of the rabbits with 1-1.5 mg/kg of actinomycin S administered intraperitoneally 2 h before prednisolone injection (Table n). Tyrosine aminotransferase was assayed by the method of LIN et al. 2~. RESULTS

The effects of electrical stimulation of the splanchnic nerve, the vagus nerve, and both nerves simultaneously on the activity of glycogen phosphorylase in rabbit liver is shown in Fig. 4. There were considerable variations in the enzyme activities TABLE II

EFFECTOF VARIOUSDOSES OF ACTINOMYCINS ON THE INDUCTIONOF TYROSINEAMINOTRANSFERASE OF RABBITLIVER W h e n indicated, r a b b i t s were injected intraperitoneally with actinomycin S a t zero time and prednisolone after 2 h, and were killed after i0 h. The activity of tyrosine aminotransferase was m e a s u r e d using the s u p e r n a t a n t of the liver h o m o g e n a t e (12000 × g for i0 min).

Actinomycin S

0. 5 i.o 1. 5 1. 5

Prednisolone

(zo mg/#g)

--

--

--

+

mg/kg mg/kg mg/kg mg/kg

+ + + --

Tyrosine aminotransferase activity (re#moles p-hydvoxyphenylpyruvate/mg protein per rnin) 4.4

13.9 I 1.6 4.7 4.6 4.7

+30o

_

o

+200

5"

,lOC

c

31onchnic-nerve s t i m u l o t ion

o n°

~ i 30 60 300

-100

30 60 300 I "" -

-

VQgus-r~er ve stirnuJation

30 60 300(sec) Splonchnic-nerve and vagus-nerve stimulotion

Fig. 4. Effect of electrical stimulation of the splanchnic nerve, the v a g u s nerve, or b o t h nerves simultaneously on t h e activity of p h o s p h o r y l a s e in r a b b i t liver. I n each animal, portions of the liver were r e m o v e d serially, 3° sec, I rain, and 5 min, after the onset of nerve stimulation. Phosphorylase activity in each liver was assayed as described in the text. Results are the m e a n s of values in 8-1o r a b b i t s and are expressed as percentage change in p h o s p h o r y l a s e activity c o m p a r e d with t h a t before nerve stimulation.

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of different animals before stimulation (resting level), ranging from 5.I to 37-7 miUiunits per mg of protein with an average of 14. 9 + 1.3 (mean and S.E. of 45 rabbits). Accordingly results were shown as percentage increases or decreases in the enzyme activity in each animal after nerve stimulation. The activity was increased approx. 3-fold by electrical stimulation of the splanchnic nerve, attained maximum value within 30 sec after the onset of stimulation, and remained fairly constant until the end of a 5-min period of stimulation. The time course of the increase in phosphorylase activity on stimulation of the splanchnic nerve is shown more precisely in Fig. 5. The response of the enzyme to splanchnic-nerve stimulation could be detected after even Io-sec stimulation, and the magnitude of the response increased linearly to the maximum value which was attained after 3o-sec stimulation. Slight, if any, suppression of phosphorylase activity was observed upon vagal stimulation (Fig. 4). Nevertheless, stimulating the vagus nerve at the same time as the splanchnic nerve almost completely counteracted the stimulatory effect of the latter. Similar changes in enzyme activity of glucose-6-phosphatase in rabbit liver were observed upon stimulation of the autonomic nerves (Fig. 6). Stimulation of the splanchnic nerve caused an increase of approximately 4 ° % in the enzyme activity within 30 sec after the onset of stimulation. This effect was counteracted by simultaneous stimulation of the vagus nerve, although stimulation of the vagus nerve alone either had no effect or caused a slight decrease in activity. "~ +30C _e .g ~: +20C

8

,b *100

2'0 40 6'0 J" 3oh (sec) Time of stimulation Fig. 5. Time course of early increase in the activity of liver phosphorylase after splanchnic-nerve s t i m u l a t i o n . After t h e indicated periods of s p l a n c h n i c - n e r v e s t i m u l a t i o n , p o r t i o n s of a liver were q u i c k l y r e m o v e d , and t h e activities of p h o s p h o r y l a s e were measured. R e s u l t s are expressed as percentage increases 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 a t before s t i m u l a t i o n . Circles represent the averages of 4 or m o r e d e t e r m i n a t i o n s ; vertical bars indicate t h e s t a n d a r d errors of t h e means.

~> .c~ 20 u.~

E :-~ 8E

30 60 300 I I I ''

I I 30 60 B"~'O(sec) Splanchnic-nerve ancl vagus-nerve stimulation stimulation Fig. 6. Effect o n rabbit-liver g l u c o s e - 6 - p h o s p h a t a s e of electrical s t i m u l a t i o n of t h e s p l a n c h n i c nerve, t h e v a g u s nerve, or b o t h nerves s i m u l t a n e o u s l y . E x p e r i m e n t a l c o n d i t i o n s were as described in Fig. 4. •_

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Liver phosphorylase is affected by hormones such as epinephrine released from the adrenal and glucagon released from the pancreas 17, and both the adrenal medulla and pancreas are innervated by the splanchnic nerve. Therefore, the effect of splanchnic-nerve stimulation on liver phosphorylase might be due to hormones. This possibility was tested by stimulating the splanchnic nerve of adrenalectomized or pancreatectomized rabbits. As shown in Fig. 7, the activities of liver phosphorylase and glucose-6-phosphatase were increased markedly as in intact rabbits. It is thus very unlikely that the effect of splanchnic-nerve stimulation on glycogenolytic enzymes in the liver is due solely to hormones such as epinephrine or glucagon. The possibility that the increase in activity of liver phosphorylase was associated with synthesis of enzyme protein was investigated after stimulation of the splanchnic nerve. Table III shows the results of experiments in which rabbits received 1.5 mg/kg of actinomycin S, 2 h (intraperitoneally) or 20 rain (intravenously) before stimulation of the nerve. Preliminary experiments showed that actinomycin S completely inhibits the induction in rabbit liver of tyrosine aminotransferase, an enzyme that is known to be induced by glucocorticoid2°, 21, in a dose of 1.5 mg/kg given intraperitoneally 2 h before administration of glucocorticoid (see Table II). It is evident from the

c ~*200

Intact

Adrenalectomized Pancrea tectornlzed

Fig. 7. Response of liver phosphorylase and glucose-6-phosphatase to splanchnic-nerve stimulation in adrenalectomized and pancreatectomized rabbits. Experimental conditions were similar to those described in Figs. 4 and 6. Bilateral adrenalectomy and pancreatectomy of rabbits were carried out by laparotomy 15-2o rain before splanchnic-nerve stimulation. TABLE III EFFECT

OF ACTINOMYCIN

S ON THE RESPONSE

OF L I V E R P H O S P H O R Y L A S E

TO

SPLANCHNIC-NERVE

STIMULATION Rabbits were given 1.5 mg/kg of actinomycin S intraperitoneally (i.p.) 2 h before, or intravenously (i.v.) 2o rain before stimulation of the splanchnic nerve. Control rabbits received appropriate vehicle.

Phosphorylase activity (munits/mg protein)

Control Actinomycin S (i.p.) Actinomycin S (i.v.)

Before stimulation

After stimulation 3o sec

I rain

5 min

lO.9 I 1.5 14.6

30.3 39.o 32.o

28.3 35.5 44.3

33.4 30.0 41.4

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data t h a t actinomycin S had no inhibitory effect on the response of phosphorylase to splanchnic-nerve stimulation. Essentially the same extent of increase in enzyme activity was seen in rabbits treated with actinomycin as in the control animals which received appropriate sham treatment. This result suggests that new formation of enzyme protein is unnecessary for the rapid increase in phosphorylase activity observed on splanchnic-nerve stimulation. Liver phosphorylase exists in two forms. One is active, and the other is inactive phosphorylase (dephosphophosphorylase). These two forms of phosphorylase have been shown to be interconvertible b y a specific phosphatase and kinaseX2,14. The conversion of the inactive to the active form takes place in the presence of ATP and Mg 2+. In contrast to the muscle enzyme, inactive liver phosphorylase displays little or no catalytic activity even when assayed in the presence of AMP TM,13. Therefore, prior to phosphorylase assay, the inactive phosphorylase must be converted to the active form to determine the total phosphorylase in the liver. This was done with a liver extract fortified with optimum concentrations of ATP and Mg z+. Preliminary experiments described in the experimental section revealed that addition of excess kinase was unnecessary for measurement of total phosphorylase if endogenous kinase was fully activated under proper conditions of incubation. ~ (A/T) 024 0,65 0.74 0.74

3

o.~

Splanchnic-n ePve stimulation

0.28 0.24 Q24 (328

0 30 60 Vagu5-nerve stimulation

300(sec)

Fig. 8. Level of total phosphorylase and the ratio of active phosphorylase to total phosphorylase in rabbit liver after stimulation of the splanchnic nerve or the vagus nerve. Total phosphorylase was determined by assaying phosphorylase activity after conversion of dephosphophosphorylase to the active form (see text). The enzyme activity is expressed as milli-units per mg of protein. Solid parts of columns represent the amount of active phosphorylase, and A/T denotes the ratio of the active to the total phosphorylase. Representative results of experiments are shown in Fig. 8, in which the level of total phosphorylase in the liver was measured after stimulation of the splanchnic nerve or the vagus nerve. The amount of total phosphorylase was not changed significantly b y stimulation of either nerve, although the level of active phosphorylase was markedly increased after splanchnic-nerve stimulation and slightly changed after vagal stimulation. Thus, about one fourth of the total phosphorylase was active in the resting state in the liver; whereas, three fourths of the total phosphorylase became active after stimulation of the splanchnic nerve. By contrast, approximately one fourth of the total phosphorylase was active and this remained fairly constant before and throughout vagal stimulation. DISCUSSION Determination of fiver phosphorylase b y the usual procedure involving homogenization of the liver in an ice-cold glass homogenizer, results in high and variable Biochim. Biophys. Aaa, z65 (z968) 335-348

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T. SHIMAZU, A. AMAKAWA

values which are thought to be artifacts. Application of techniques to arrest enzymic reactions, consisting mainly of fixation of the liver by rapid freezing and extraction of the enzyme at a low temperature with glycerol-fluoride-EDTA solution, makes it possible to study the time course of the increase in phosphorylase content in the liver during and following autonomic-nerve stimulation in rabbits. The complication of individual variation of phosphorylase activity in different animals can also be overcome by rapid removal of one lobe before stimulation and then comparing activity in this with that of another lobe of the same liver after stimulation of the nerve. This technique permits the use of each rabbit as its own control in determining responses to different periods of nerve stimulation in the same animal. It may be stated that the function of the sympathetic nerve is to prepare the animals for emergency action. This was proved to be true on the enzymic level. Stimulation of the sympathetic nerve caused almost instantaneous activation of phosphorylase and glucose-6-phosphatase in the liver; the half-time of the increase in phosphorylase was 14 sec. This rapid response of enzymes permits a rapid supply of glucose to the blood in an emergency. Our previous work has shown that sympathetic stimulation of the hypothalamus (ventromedial hypothalamic nucleus) causes a great and rapid increase in blood glucosee. It is known that the sympathetic and parasympathetic nerves act antagonistically in regulation of the activity of the viscera. Balance of the activities of these two autonomic nerves takes part in homeostasis in higher animals. The antagonistic actions of the autonomic nerves have been shown in glycogen metabolism in liverS-L Stimulation of the sympathetic nerve causes glycogenolysis in the liver by stimulating the activation of phosphorylase and glucose-6-phosphatase, while stimulation of the parasympathetic nerve causes glycogenesis in the liver by increasing the activity of glycogen synthetase. The antagonistic actions of the sympathetic and parasympathetic nerves were also shown on individual enzymes implicated in glycogen metabolism. Although the parasympathetic nerve had little effect on phosphorylase and glucose6-phosphatase, this nerve completely suppressed the effect of the sympathetic nerve on these enzymes. A similar antagonism has already been shown on glycogen synthetase of rabbit liver; the effect of the vagus nerve on glycogen synthetase is suppressed by simultaneous stimulation of the splanchnic nerve 7. The response of phosphorylase and glucose-6-phosphatase in rabbit liver to splanchnic-nerve stimulation was not abolished by adrenalectomy or pancreatectomy. This shows that the effect of the sympathetic nerve is not solely due to epinephrine or glucagon which are known to activate liver phosphorylase 17. Several differences were found between the effect of sympathetic-nerve stimulation and that of epinephrine administration on glycogenolytic enzymes. The results are presented and discussed in the succeeding paper 23. Attempts to measure total phosphorylase in crude liver extracts have so far been unsuccessful since liver dephosphophosphorylase, unlike mucle phosphorylase b, is not activated by AMP 12. In the present studies, dephosphophosphorylase and dephosphophosphorylase kinase were partially purified from rabbit liver and their kinetic properties were investigated. APPLEMAN, KREBS AND FISCHER13 have already purified dephosphophosphorylase from pig and rabbit liver. Dephosphophosphorylase displays about o.2 % of the catalytic activity of active enzyme, in the presence of AMP, and 20 %, in the presence of AMP and high concentrations (o.7 M) of NazSO4. Biochim. Biophys. Acta, I65 (I968) 335-348

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Active phosphorylase, however, was strongly inhibited by high concentrations of NazSO 4. Therefore, it was impossible to measure total phosphorylase activity in the presence of this salt. Accordingly, dephosphophosphorylase was converted to the active form by the kinase and then total phosphorylase was determined by assaying phosphorylase activity. Insignificant change in the total phosphorylase activity was observed on splanchnic-nerve stimulation or vagal stimulation. In agreement with this result, previous treatment of the rabbits with a dose of actinomycin S sufficient to inhibit protein synthesis failed to block the increase in phosphorylase activity after splanchnic-nerve stimulation. These results indicate that increase in phosphorylase activity caused by splanchnic-nerve stimulation can be attributed to the enzymic conversion of dephosphophosphorylase to the active form during the stimulation, and not to synthesis de novo of the enzyme. An attempt to detect an effect of splanchnic-nerve stimulation on dephosphophosphorylase kinase of the liver was not successful, probably because of the presence in the crude extract of materials inferfering with the assay of the kinase. The mechanism of increase in liver glucose-5-phosphatase after splanchnicnerve stimulation is not fully understood. This enzyme is localized in the microsomal fraction of the liver homogenate. Therefore, it might be possible that certain changes in the structure of the endoplasmic reticulum are involved. An apparent hypertrophy of the agranular reticulum of the liver cell has been observed under the electron microscope after sympathetic stimulation of rabbit hypothalamus (M. SAKAGAMI, T. SHIMAZU,Y. SHIOTANIAND T. BAN, unpublished results). ACKNOWLEDGEMENTS

We wish to thank Professor T. BAN,Department of Anatomy, Osaka University Medical School, and Professor M. SUDA,Protein Research Institute, Osaka University, for their helpful suggestions and discussions throughout this work. We also wish to thank Professor M. SUDA for a generous gift Of actinomycin S.

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BiocMm. Biophys. Acta, I65 (1968) 335-348