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A relationship between ionic environment and hormonal activation of phosphorylase in an insect
Comp. Biochem. Physiol., 1969, Vol. 29, pp. 755 to 763. Pergamon Press. Printed in Great Britain
A RELATIONSHIP BETWEEN IONIC E N V I R O N M E N T A N D HORMONAL ACTIVATION OF PHOSPHORYLASE IN AN INSECT* j. E. S T E E L E t Research Institute, Research Branch, Canada Department of Agriculture, University Sub Post Office, London, Ontario, Canada (Received 17 September 1968)
A b s t r a c t - - 1 . T h e rate of glycogenolysis in insect fat body tissue is greater in a medium containing Na + than one in which the Na + is replaced by K +. 2. Phosphorylase activity decreases to a lower level when the tissue is preincubated in a medium in which K + is the dominant cation than in one in which Na + is the only cation. 3. The preincubation of fat body tissue in a Na+-deficient medium results in loss of this ion from the tissue. Tissue Na + is restored to a level greater than normal when the tissue is returned to a medium containing physiological concentrations of the ion. 4. It is suggested that the effects of the high-Na + and Na+-deficient media on phosphorylase are the result of changes in the intracellular concentration of Na + and consequent effects on phosphorylase phosphatase. INTRODUCTION
STUDIES on glycogen phosphorylase in a variety of animals have led to the conclusion that it may occur within the cell in two forms, one of which usually shows little or no activity. The quantitative relationship between the two forms appears to depend on the physiological state of the animal. Unfortunately, phosphorylase and the enzymes associated with its conversions have not received much attention in insects. However, it is possible that both active and inactive forms may exist since it has been shown that phosphorylase activity is increased in the presence of adenosine-5'-monophosphate (AMP) (Steele, 1964; Stevenson & Wyatt, 1964; Wiens & Gilbert, 1967). This possibility is further strengthened by the observations of Steele (1963) who demonstrated that there is a considerable increase in phosphorylase activity of cockroach fat body incubated with extracts prepared from the corpora cardiaca of the same insect. Additional evidence for the dual nature of phosphorylase in insects is provided by the work of Wiens & Gilbert (1967), who showed a decrease in active phosphorylase of silkmoth fat body upon incubation * Contribution No. 390, Research Institute, Research Branch, Canada Department of Agriculture, University Sub Post Office, London, Ontario, Canada. Present address: Department of Zoology, University of Western Ontario, London, Ontario, Canada. 755
756
J . E . STEELE
without a decrease in total phosphorylase. Furthermore, they showed an increase in activity of the enzyme attendant u p o n the addition of A T P and M g e+ which is indicative of a kinase-type enzyme. T h e reasons for the current study were twofold. First, to examine the possibility that fat b o d y phosphorylase activity in the intact cell, and its activation b y the carbohydrate mobilizing hormone, is affected b y the ionic composition within the cell since earlier studies b y Cahill et al. (1957) had shown that glycogen metabolism in rat liver slices incubated in vitro was strongly influenced b y the ionic environment. A n d secondly, to determine whether it is possible to regulate at will the level of phosphorylase activity. T h i s objective is of considerable importance to in vitro studies on the m e c h a n i s m of hormonal activation of the enzyme.
MATERIALS AND METHODS The cockroaches (Periplaneta americana L.) used in this study were obtained from a colony maintained in the laboratory for many generations. They were used approximately 2 months after the adult moult. The Ringer's solutions used in the study were of the following eomposition--K + medium: 160raM KCI; 2 r a M CaCll; 2 m M KHCO~; 30raM glucose, 5 mM Trishydroxymethylamino-methane, pH 7"5. Na + medium: 150 mM NaC1; 10 mM KCI; 2 mM CaC12; 2 m M NaHCOs; 30 mM glucose, 5 mM Tris-hydroxymethylamino-methane, pH 7"5. Glycogen determinations were made according to the anthrone method of Carroll et al. (1956). The fat bodies were blotted dry, weighed to the nearest milligram and quickly transferred to 3'0 ml of 3 0 ~ KOH. After boiling for 15 rain the digest was centrifuged at 10,000g for 10 rain. A sample of the clear supernatant was removed and the glycogen precipitated by the addition of 1"2 vol. of 95 % ethanol. For the determination of phosphorylase activity each half fat body (right or left side) was homogenized in 1"0 ml 0.1 M NaF +0-01 M ethylenediaminetetraacetate (EDTA) using a glass tissue grinder with Teflon pestle. The temperature was maintained close to 0°C by immersing the homogenizer in a bath of crushed ice. The homogenate was centrifuged at 27,000 g for 10 rain at 4°C and a sample of the clear supematant taken for assay of phosphorylase activity. The phosphorylase assay reagent and technique employed have been described previously (Steele, 1963). Sodium and potassium were determined using a Beckman DK-1 spectrophotometer with a Beckman 9125 Flame Attachment. Fat bodies were prepared for analysis by the wet ashing technique as described by Ballentine & Burford (1957) with only minor modification. Each half fat body (approximately 50 rag) was digested in a 30-ml Kjeldahl flask containing 9.0 ml of concentrated HNO8 and 1"5 ml of HCIO~-H~O (1 : 1). The final residue was made up to 10"0 ml with deionized water. All values are presented as the mean + the standard error of the mean and the number of determinations indicated by the letter n. The results have been tested for significance using the standard t-test.
RESULTS Glycogen has long been recognized as the principal carbohydrate reserve in animals. Both the position occupied by glycogen with respect to the glycolytic pathway and the relationship between its utilization and the physiologicalstate o~ the animal indicate the prime importance of this substrate as a source of energy. The ability of hormones to regulate the rate at which glycogen is degraded in
IONIC ENVIRONMENT AND ACTIVATION OF PHOSPHORYLASE IN AN INSECT
757
vertebrate animals is well known. A comparable situation has now been shown to exist in insects (Bowers & Friedman, 1963; Steele, 1963; Ralph & McCarthy, 1964). Cahill et al. (1957) have succeeded in demonstrating that the rate of glycogenolysis, in liver slices incubated in vitro, may be modified by using a Ringer's solution in which the usually dominant Na + ion is replaced by K +. Under these conditions the rate of glycogenolysis was found to be appreciably reduced. We have repeated some of these experiments using insects, not only to determine whether glycogenolysis in the insect fat body is affected by the ionic environment but also to determine whether there is any effect on hormone-mediated glycogenolysis by certain of the ions present. The tissues were removed and divided into left and right halves, one half being placed in 2.0 ml of the Na + medium and the other in 2.0 ml of the K + medium. Glucose was omitted from both media. Incubations were carried out at 30°C with gentle shaking. Following the period of incubation the tissues were blotted and the glycogen content of the tissue determined. The concentration of glycogen in fat body incubated in the Na + medium was found to be 17.56 + 4.43 mg/g wet wt. as compared with 23.28 + 6-43 mg/g wet wt. (n = 4) for tissue incubated in the K + medium. The results suggest more rapid glycogenolysis in the Na + medium since tissue incubated in this medium contains only 75 per cent as much glycogen as that incubated in the K+ medium ( P < 0.01). The higher rate of glycogenolysis in the medium in which Na + is the predominant cation is in complete agreement with the findings of Cahill et al. (1957) for liver slices. In the preliminary studies of the effects of hyperglycaemic hormone on fat body phosphorylase the tissues were first preincubated in a buffered NaC1 medium on the assumption that phosphorylase would be converted to the inactive form. The tissue was then transferred to fresh medium to which had been added hyperglycaemic hormone or control extracts. The following experiment was designed to show that preincubation results in decreased phosphorylase activity. The left half of the fat body was homogenized in the N a F + E D T A mixture immediately upon removal and its phosphorylase activity represents the initial value. The right half was incubated for 30 min at 30°C in 0.5 ml of the Na + medium before determination of phosphorylase activity of the unincubated tissue which was 267.75 + 30.00/xmoles Pi/g per 15 min whereas that of the tissue incubated in the Na + medium was 140"75 + 30.23 /xmoles Pi/g per 15 rain (n = 8). This represents a decline in activity of 47 per cent
(P< 0.01). The inactivation of phosphorylase in liver requires the participation of a phosphorylase phosphatase. Wosilait & Sutherland (1956) have shown that the latter enzyme is inhibited 78 per cent by 0-15 M NaC1 while the studies of Cahill et al. (1957) indicate that the inhibition is due to the Na + ion. The studies of Cahill et al. (1957) showed that liver slices incubated in a medium high in K + and lacking Na + exhibited lower phosphorylase activity than that of liver slices incubated in a medium in which Na + was predominant and K + absent. One
758
J.E. STEELE
explanation may be that phosphorylase activity is reduced as a result of enhanced phosphorylase phosphatase activity due to loss of Na + from the tissue. Should insect fat body contain a phosphorylase phosphatase similar to that found in mammalian liver, inhibition of this enzyme by Na ÷ ion would be expected. Furthermore, incubation of the tissue in a medium lacking this ion ought to result in greater conversion of phosphorylase to the inactive form. Because of the higher rate of glycogenolysis shown to occur in the Na + medium it was of considerable importance to determine the relative abilities of Na + and K + to reduce the level of phosphorylase activity in isolated fat body. The fat bodies were removed and separated into left and right halves. The left half was rinsed briefly in the Na ÷ medium, blotted dry and transferred to 2.0 ml of the same medium. The right half was treated in a similar manner using the K+ medium. The tissues were incubated for 30 min at 30°C in a shaker bath, following which phosphorylase activities were determined. Enzyme activity of the tissue which had been incubated in the Na ÷ medium was equivalent to 194.25 _+16.08 /~moles Pi/g per 15 min while that of tissue incubated in the K÷ medium was 152.25 _+10.46/~moles Pi/g per 15 min (n = 4). The results clearly show that the K + medium induced the greatest decline in phosphorylase activity, being 78 per cent (P < 0.05) of that obtained in the Na ÷ incubation medium. In order to demonstrate an activation effect of the hormone on phosphorylase in vitro it is important to have as much of the enzyme as possible in the inactive form when the incubation with the hormone is initated. The information so far obtained indicates that this may be best achieved by preincubation of the tissues in the K+ medium. The following experiment was designed to show the effect on phosphorylase activity of tissue incubation in the Na+ medium following preincubation in the K ÷ medium. Phosphorylase activity in/~moles Pi/g per 15 min was found to be 276.42 _+4.68 for unincubated tissue. However, tissue which had been preincubated in the K ÷ medium for 30 rain followed by incubation for an equal period of time in the Na + medium had an activity of 4.18 _+1.93/~moles Pi/g per 15 min (n = 4). This is a decrease in activity of 98.5% ( P < 0.01). In the light of these observations on the effect of media high in Na ÷ and K ÷ on the activity of phosphorylase in the fat body it was of interest to determine what effect, if any, these two ions might have on hormonal activation of the enzyme. Fat bodies were removed and separated into left and right halves. The tissues were rinsed quickly in the K + medium and transferred to 2.0 ml of the same and shaken gently for 30 min at 30°C in order to reduce the phosphorylase activity to the lowest possible level. Following this period of preincubation the tissues were transferred to 0.5 ml of the same medium, with or without hormone, care being taken that control and treated tissues came from the same insect. The incubation was continued for an additional 30 min. In the second part of the experiment conditions were identical with those just described except that the second incubation was carried out in the Na + medium. After the incubations with the hormone or water addition had been completed the tissues were homogenized and phosphorylase aetivity determined as described.
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The results of the experiment shown in Table 1 indicate that the activation of phosphorylase by the hormone is markedly affected by the nature of the predominant ion. Treatment of the tissues with hormone in the K + medium resulted in an increase in enzyme activity of about 100 per cent. However, those tissues which were treated with hormone in the Na + medium showed a fivefold increase in phosphorylase activity. These results are easily explained if we assume that the preincubation in the K + medium reduces the level of Na + in the tissue. Because the amount of active phosphorylase present within the cell at a given moment is a function of the opposing actions of phosphorylase kinase and phosphorylase phosphatase, loss of Na + from the cell will result in lowered inhibition of the phosphatase. This lower inhibition is reflected in the conversion of the phosphorylase to the inactive form. Therefore, when the tissues are treated with hormone in the K + medium, action of the hormone to convert inactive phosphorylase to the active form will be strongly opposed by phosphatase action. Alternatively, if the tissues are transferred to the Na + medium after preincubation in the K + medium it would be expected that the return of Na + ions to the tissue would increase the inhibition of the phosphatase. The conversion of the inactive phosphorylase to the active form, in the presence of hormone, should then proceed more or less unopposed. An attempt has been made to explain these results in terms of the Na + and K + content of the bathing media. This explanation assumes that the ionic content of the incubating media will reflect the ionic composition of the tissue. Na + and K + concentrations in fat body tissue were determined in order to find out what effect, if any, the preincubation and incubation procedure had on the intracellular concentrations of those ions. In the first part of the experiment the effect of preincubation in the K + medium on fat body Na + and K + was compared with normal (unincubated) fat body Na + and K + concentrations. The tissues were removed and the Na + and K + concentrations of the left half determined. The right half was transferred to 2.0 ml of the K + medium and shaken gently for 30 rain at 30°C. The tissues were given a 5-see rinse in distilled water and blotted before the digestions were carried out. In the second part of the experiment both left and right halves were preincubated in the K + medium for 30 rain. The Na + and K + concentrations of the left halves were determined at the conclusion of the preincubation period. Each right half was transferred to 2.0 ml of the Na + medium and the incubation continued for an additional 30 rain before determination of the Na + and K + concentrations. The results, shown in Table 2, clearly show that the preincubation and incubation procedures affect the level of Na + and K + within the tissue. The preincubation phase induces a slight rise in the level of tissue K +; however, this contrasts sharply with the decrease in Na + which is of the order of 30 per cent. On transfer to the Na + medium the level of tissue K + falls slightly but that of Na + rises to a level which is approximately three times that found in unincubated tissue. It is suggested that the striking effects which the hormone has on phosphorylase (Table 1) when the
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tissues are pretreated in the manner described are indirectly related to the initial decrease in Na + and subsequent greater than normal concentrations. DISCUSSION Although a definitive study of phosphorylase and its associated enzymes in insects is not available, there is no evidence to indicate a departure from the general scheme observed in vertebrate liver and muscle. The observation that enzyme activity is much increased after treatment of the tissues with hormone suggests that it may exist in both active and inactive forms. Furthermore, the reduction in enzyme activity following incubation of the tissue in saline media supports this idea. Indeed, the demonstration in this study that reduction in activity by incubation in saline media may be reversed by subsequent treatment of the tissue with hormone strongly indicates that the enzyme probably exists in two states. If the assumption is made that the enzyme exists in both active and inactive forms, the greater reduction in phosphorylase activity observed in the K + medium when compared with the Na + medium may be explained by assuming the presence of a phosphorylase phosphatase-like enzyme as part of the phosphorylase system, with properties (i.e. ability to inactivate phosphorylase and be inhibited by Na +) similar to those described for the liver enzyme (Wosilait & Sutherland, 1956). The higher activity obtained when tissues are treated with hormone in the Na + medium in contrast to the K + medium may also be explained by the presence of this enzyme in fat body. The data presented raise the possibility that the hyperglycaemic hormone may act, partially at least, by regulating the concentration of Na + or K + or both within the cell. It is interesting to note that epinephrine has been shown to induce a liberation of K + from liver (D'Silva, 1934). However, there is no apparent change in the level of blood Na + and K + following injection of hyperglycaemic hormone into intact cockroaches, indicating no extrusion of these ions from the fat body (Steele, unpublished observations). Neither is there any change in the concentration of Na + and K + in fat body which has been incubated with the hormone in vitro (Steele, unpublished observations). The change in activity of phosphorylase when subjected to different concentrations of Na + and K + may indicate a mechanism of control which is entirely distinct from that of hormonal control. The suggestion of ionic regulation of phosphorylase is not unique, however, as Meyer et al. (1964) have already demonstrated in vitro that skeletal muscle phosphorylase kinase is activated by low concentrations of Ca ~+. More recently Friesen et al. (1967) have shown that Ca ~+ appears to be related to the activation of phosphorylase in cardiac muscle. On the basis of the present study it is suggested that the regulation of phosphorylase activity by phosphorylase phosphatase is partially dependent on the intracellular concentration of Na +. Acknowledgement--The author wishes to thank Dr. A. Vardanis for reading the manuscript and for suggesting a number of improvements.
IONIC ENVIRONMENT AND ACTIVATION OF PHOSPHORYLASE IN AN INSECT
763
REFERENCES BALLENTINER. & BURFORDD. D. (1957) Determination of metals. In Methods in Enzymology (Edited by COLOWICKS. V. ~ KAPLAN N. O.), Vol. 3, pp. 1002-1035. Academic Press, New York. BOWERS W. S. & FRIEDMAN S. (1963) Mobilization of fat body glycogen by an extract of corpus cardiacum. Nature, Lond. 198, 685. CAHILL G. F., ASHMOREJ., ZOTTU S. ~ HASTINGSA. B. (1957) Studies on carbohydrate metabolism in rat liver slices--IX. Ionic and hormonal effects on phosphorylase and glycogen, ft. biol. Chem. 224, 237-250. CARROLLN. V., LONGLEYR. W. ~ ROE J. H. (1956) The determination of glycogen in liver and muscle by use of anthrone reagent, jT. biol. Chem. 220, 586-593. D'SILVA J. L. (1934) The action of adrenaline on serum potassium, ft. Physiol., Lond. 82, 393-398. FRIESEN A. J. D., ALLEN G. ~ VALADARESJ. R. E. (1967) Calcium induced activation of phosphorylase in rat hearts. Science, N. Y. 155, 1108-1109. MEYER W. L., FISCHERE. H. & KREBSE. G. (1964) Activation of skeletal muscle phosphorylase b kinase by Ca ~+. Biochemistry 3, 1033-1039. RALPH C. L. & McCARTHY R. (1964) Effects of brain and corpus cardiacum extracts on haemolyrnph trehalose of the cockroach, Periplaneta americana. Nature, Lond. 203, 1195-1196. STEELE J. E. (1963) The site of action of insect hyperglycaemic hormone. Gen. comp. Endocrinol. 3, 46-52. STEELE J. E. (1964) The activation of phosphorylase in an insect by adenosine 3",5"-phosphate and other agents. Am. Zool. 4, 328. STEVENSONE. & WYATT G. R. (1964) Glycogen phosphorylase and its activation in silkworm fat body. Archs Biochem. Biophys. 108, 420-429. W I ~ s A. W. & GILBERT L. I. (1967) The phosphorylase system of the silkmoth, Hyalophora cecropia. Comp. Biochem. Physiol. 21, 145-159. WOSILAITW. D. & SUTmSaLANOE. W. (1956) The relationship of epinephrine and glueagon to liver phosphorylase II. Enzymatic inactivation of liver phosphorylase, ft. biol. Chem. 218, 469--481.
Key Word Index--Phosphorylase; Periplaneta americana; glycogenolysis; sodiumpotassium effect on phosphorylase; insect fat body.
A RELATIONSHIP BETWEEN IONIC E N V I R O N M E N T A N D HORMONAL ACTIVATION OF PHOSPHORYLASE IN AN INSECT* j. E. S T E E L E t Research Institute, Research Branch, Canada Department of Agriculture, University Sub Post Office, London, Ontario, Canada (Received 17 September 1968)
A b s t r a c t - - 1 . T h e rate of glycogenolysis in insect fat body tissue is greater in a medium containing Na + than one in which the Na + is replaced by K +. 2. Phosphorylase activity decreases to a lower level when the tissue is preincubated in a medium in which K + is the dominant cation than in one in which Na + is the only cation. 3. The preincubation of fat body tissue in a Na+-deficient medium results in loss of this ion from the tissue. Tissue Na + is restored to a level greater than normal when the tissue is returned to a medium containing physiological concentrations of the ion. 4. It is suggested that the effects of the high-Na + and Na+-deficient media on phosphorylase are the result of changes in the intracellular concentration of Na + and consequent effects on phosphorylase phosphatase. INTRODUCTION
STUDIES on glycogen phosphorylase in a variety of animals have led to the conclusion that it may occur within the cell in two forms, one of which usually shows little or no activity. The quantitative relationship between the two forms appears to depend on the physiological state of the animal. Unfortunately, phosphorylase and the enzymes associated with its conversions have not received much attention in insects. However, it is possible that both active and inactive forms may exist since it has been shown that phosphorylase activity is increased in the presence of adenosine-5'-monophosphate (AMP) (Steele, 1964; Stevenson & Wyatt, 1964; Wiens & Gilbert, 1967). This possibility is further strengthened by the observations of Steele (1963) who demonstrated that there is a considerable increase in phosphorylase activity of cockroach fat body incubated with extracts prepared from the corpora cardiaca of the same insect. Additional evidence for the dual nature of phosphorylase in insects is provided by the work of Wiens & Gilbert (1967), who showed a decrease in active phosphorylase of silkmoth fat body upon incubation * Contribution No. 390, Research Institute, Research Branch, Canada Department of Agriculture, University Sub Post Office, London, Ontario, Canada. Present address: Department of Zoology, University of Western Ontario, London, Ontario, Canada. 755
756
J . E . STEELE
without a decrease in total phosphorylase. Furthermore, they showed an increase in activity of the enzyme attendant u p o n the addition of A T P and M g e+ which is indicative of a kinase-type enzyme. T h e reasons for the current study were twofold. First, to examine the possibility that fat b o d y phosphorylase activity in the intact cell, and its activation b y the carbohydrate mobilizing hormone, is affected b y the ionic composition within the cell since earlier studies b y Cahill et al. (1957) had shown that glycogen metabolism in rat liver slices incubated in vitro was strongly influenced b y the ionic environment. A n d secondly, to determine whether it is possible to regulate at will the level of phosphorylase activity. T h i s objective is of considerable importance to in vitro studies on the m e c h a n i s m of hormonal activation of the enzyme.
MATERIALS AND METHODS The cockroaches (Periplaneta americana L.) used in this study were obtained from a colony maintained in the laboratory for many generations. They were used approximately 2 months after the adult moult. The Ringer's solutions used in the study were of the following eomposition--K + medium: 160raM KCI; 2 r a M CaCll; 2 m M KHCO~; 30raM glucose, 5 mM Trishydroxymethylamino-methane, pH 7"5. Na + medium: 150 mM NaC1; 10 mM KCI; 2 mM CaC12; 2 m M NaHCOs; 30 mM glucose, 5 mM Tris-hydroxymethylamino-methane, pH 7"5. Glycogen determinations were made according to the anthrone method of Carroll et al. (1956). The fat bodies were blotted dry, weighed to the nearest milligram and quickly transferred to 3'0 ml of 3 0 ~ KOH. After boiling for 15 rain the digest was centrifuged at 10,000g for 10 rain. A sample of the clear supernatant was removed and the glycogen precipitated by the addition of 1"2 vol. of 95 % ethanol. For the determination of phosphorylase activity each half fat body (right or left side) was homogenized in 1"0 ml 0.1 M NaF +0-01 M ethylenediaminetetraacetate (EDTA) using a glass tissue grinder with Teflon pestle. The temperature was maintained close to 0°C by immersing the homogenizer in a bath of crushed ice. The homogenate was centrifuged at 27,000 g for 10 rain at 4°C and a sample of the clear supematant taken for assay of phosphorylase activity. The phosphorylase assay reagent and technique employed have been described previously (Steele, 1963). Sodium and potassium were determined using a Beckman DK-1 spectrophotometer with a Beckman 9125 Flame Attachment. Fat bodies were prepared for analysis by the wet ashing technique as described by Ballentine & Burford (1957) with only minor modification. Each half fat body (approximately 50 rag) was digested in a 30-ml Kjeldahl flask containing 9.0 ml of concentrated HNO8 and 1"5 ml of HCIO~-H~O (1 : 1). The final residue was made up to 10"0 ml with deionized water. All values are presented as the mean + the standard error of the mean and the number of determinations indicated by the letter n. The results have been tested for significance using the standard t-test.
RESULTS Glycogen has long been recognized as the principal carbohydrate reserve in animals. Both the position occupied by glycogen with respect to the glycolytic pathway and the relationship between its utilization and the physiologicalstate o~ the animal indicate the prime importance of this substrate as a source of energy. The ability of hormones to regulate the rate at which glycogen is degraded in
IONIC ENVIRONMENT AND ACTIVATION OF PHOSPHORYLASE IN AN INSECT
757
vertebrate animals is well known. A comparable situation has now been shown to exist in insects (Bowers & Friedman, 1963; Steele, 1963; Ralph & McCarthy, 1964). Cahill et al. (1957) have succeeded in demonstrating that the rate of glycogenolysis, in liver slices incubated in vitro, may be modified by using a Ringer's solution in which the usually dominant Na + ion is replaced by K +. Under these conditions the rate of glycogenolysis was found to be appreciably reduced. We have repeated some of these experiments using insects, not only to determine whether glycogenolysis in the insect fat body is affected by the ionic environment but also to determine whether there is any effect on hormone-mediated glycogenolysis by certain of the ions present. The tissues were removed and divided into left and right halves, one half being placed in 2.0 ml of the Na + medium and the other in 2.0 ml of the K + medium. Glucose was omitted from both media. Incubations were carried out at 30°C with gentle shaking. Following the period of incubation the tissues were blotted and the glycogen content of the tissue determined. The concentration of glycogen in fat body incubated in the Na + medium was found to be 17.56 + 4.43 mg/g wet wt. as compared with 23.28 + 6-43 mg/g wet wt. (n = 4) for tissue incubated in the K + medium. The results suggest more rapid glycogenolysis in the Na + medium since tissue incubated in this medium contains only 75 per cent as much glycogen as that incubated in the K+ medium ( P < 0.01). The higher rate of glycogenolysis in the medium in which Na + is the predominant cation is in complete agreement with the findings of Cahill et al. (1957) for liver slices. In the preliminary studies of the effects of hyperglycaemic hormone on fat body phosphorylase the tissues were first preincubated in a buffered NaC1 medium on the assumption that phosphorylase would be converted to the inactive form. The tissue was then transferred to fresh medium to which had been added hyperglycaemic hormone or control extracts. The following experiment was designed to show that preincubation results in decreased phosphorylase activity. The left half of the fat body was homogenized in the N a F + E D T A mixture immediately upon removal and its phosphorylase activity represents the initial value. The right half was incubated for 30 min at 30°C in 0.5 ml of the Na + medium before determination of phosphorylase activity of the unincubated tissue which was 267.75 + 30.00/xmoles Pi/g per 15 min whereas that of the tissue incubated in the Na + medium was 140"75 + 30.23 /xmoles Pi/g per 15 rain (n = 8). This represents a decline in activity of 47 per cent
(P< 0.01). The inactivation of phosphorylase in liver requires the participation of a phosphorylase phosphatase. Wosilait & Sutherland (1956) have shown that the latter enzyme is inhibited 78 per cent by 0-15 M NaC1 while the studies of Cahill et al. (1957) indicate that the inhibition is due to the Na + ion. The studies of Cahill et al. (1957) showed that liver slices incubated in a medium high in K + and lacking Na + exhibited lower phosphorylase activity than that of liver slices incubated in a medium in which Na + was predominant and K + absent. One
758
J.E. STEELE
explanation may be that phosphorylase activity is reduced as a result of enhanced phosphorylase phosphatase activity due to loss of Na + from the tissue. Should insect fat body contain a phosphorylase phosphatase similar to that found in mammalian liver, inhibition of this enzyme by Na ÷ ion would be expected. Furthermore, incubation of the tissue in a medium lacking this ion ought to result in greater conversion of phosphorylase to the inactive form. Because of the higher rate of glycogenolysis shown to occur in the Na + medium it was of considerable importance to determine the relative abilities of Na + and K + to reduce the level of phosphorylase activity in isolated fat body. The fat bodies were removed and separated into left and right halves. The left half was rinsed briefly in the Na ÷ medium, blotted dry and transferred to 2.0 ml of the same medium. The right half was treated in a similar manner using the K+ medium. The tissues were incubated for 30 min at 30°C in a shaker bath, following which phosphorylase activities were determined. Enzyme activity of the tissue which had been incubated in the Na ÷ medium was equivalent to 194.25 _+16.08 /~moles Pi/g per 15 min while that of tissue incubated in the K÷ medium was 152.25 _+10.46/~moles Pi/g per 15 min (n = 4). The results clearly show that the K + medium induced the greatest decline in phosphorylase activity, being 78 per cent (P < 0.05) of that obtained in the Na ÷ incubation medium. In order to demonstrate an activation effect of the hormone on phosphorylase in vitro it is important to have as much of the enzyme as possible in the inactive form when the incubation with the hormone is initated. The information so far obtained indicates that this may be best achieved by preincubation of the tissues in the K+ medium. The following experiment was designed to show the effect on phosphorylase activity of tissue incubation in the Na+ medium following preincubation in the K ÷ medium. Phosphorylase activity in/~moles Pi/g per 15 min was found to be 276.42 _+4.68 for unincubated tissue. However, tissue which had been preincubated in the K ÷ medium for 30 rain followed by incubation for an equal period of time in the Na + medium had an activity of 4.18 _+1.93/~moles Pi/g per 15 min (n = 4). This is a decrease in activity of 98.5% ( P < 0.01). In the light of these observations on the effect of media high in Na ÷ and K ÷ on the activity of phosphorylase in the fat body it was of interest to determine what effect, if any, these two ions might have on hormonal activation of the enzyme. Fat bodies were removed and separated into left and right halves. The tissues were rinsed quickly in the K + medium and transferred to 2.0 ml of the same and shaken gently for 30 min at 30°C in order to reduce the phosphorylase activity to the lowest possible level. Following this period of preincubation the tissues were transferred to 0.5 ml of the same medium, with or without hormone, care being taken that control and treated tissues came from the same insect. The incubation was continued for an additional 30 min. In the second part of the experiment conditions were identical with those just described except that the second incubation was carried out in the Na + medium. After the incubations with the hormone or water addition had been completed the tissues were homogenized and phosphorylase aetivity determined as described.
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The results of the experiment shown in Table 1 indicate that the activation of phosphorylase by the hormone is markedly affected by the nature of the predominant ion. Treatment of the tissues with hormone in the K + medium resulted in an increase in enzyme activity of about 100 per cent. However, those tissues which were treated with hormone in the Na + medium showed a fivefold increase in phosphorylase activity. These results are easily explained if we assume that the preincubation in the K + medium reduces the level of Na + in the tissue. Because the amount of active phosphorylase present within the cell at a given moment is a function of the opposing actions of phosphorylase kinase and phosphorylase phosphatase, loss of Na + from the cell will result in lowered inhibition of the phosphatase. This lower inhibition is reflected in the conversion of the phosphorylase to the inactive form. Therefore, when the tissues are treated with hormone in the K + medium, action of the hormone to convert inactive phosphorylase to the active form will be strongly opposed by phosphatase action. Alternatively, if the tissues are transferred to the Na + medium after preincubation in the K + medium it would be expected that the return of Na + ions to the tissue would increase the inhibition of the phosphatase. The conversion of the inactive phosphorylase to the active form, in the presence of hormone, should then proceed more or less unopposed. An attempt has been made to explain these results in terms of the Na + and K + content of the bathing media. This explanation assumes that the ionic content of the incubating media will reflect the ionic composition of the tissue. Na + and K + concentrations in fat body tissue were determined in order to find out what effect, if any, the preincubation and incubation procedure had on the intracellular concentrations of those ions. In the first part of the experiment the effect of preincubation in the K + medium on fat body Na + and K + was compared with normal (unincubated) fat body Na + and K + concentrations. The tissues were removed and the Na + and K + concentrations of the left half determined. The right half was transferred to 2.0 ml of the K + medium and shaken gently for 30 rain at 30°C. The tissues were given a 5-see rinse in distilled water and blotted before the digestions were carried out. In the second part of the experiment both left and right halves were preincubated in the K + medium for 30 rain. The Na + and K + concentrations of the left halves were determined at the conclusion of the preincubation period. Each right half was transferred to 2.0 ml of the Na + medium and the incubation continued for an additional 30 rain before determination of the Na + and K + concentrations. The results, shown in Table 2, clearly show that the preincubation and incubation procedures affect the level of Na + and K + within the tissue. The preincubation phase induces a slight rise in the level of tissue K +; however, this contrasts sharply with the decrease in Na + which is of the order of 30 per cent. On transfer to the Na + medium the level of tissue K + falls slightly but that of Na + rises to a level which is approximately three times that found in unincubated tissue. It is suggested that the striking effects which the hormone has on phosphorylase (Table 1) when the
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tissues are pretreated in the manner described are indirectly related to the initial decrease in Na + and subsequent greater than normal concentrations. DISCUSSION Although a definitive study of phosphorylase and its associated enzymes in insects is not available, there is no evidence to indicate a departure from the general scheme observed in vertebrate liver and muscle. The observation that enzyme activity is much increased after treatment of the tissues with hormone suggests that it may exist in both active and inactive forms. Furthermore, the reduction in enzyme activity following incubation of the tissue in saline media supports this idea. Indeed, the demonstration in this study that reduction in activity by incubation in saline media may be reversed by subsequent treatment of the tissue with hormone strongly indicates that the enzyme probably exists in two states. If the assumption is made that the enzyme exists in both active and inactive forms, the greater reduction in phosphorylase activity observed in the K + medium when compared with the Na + medium may be explained by assuming the presence of a phosphorylase phosphatase-like enzyme as part of the phosphorylase system, with properties (i.e. ability to inactivate phosphorylase and be inhibited by Na +) similar to those described for the liver enzyme (Wosilait & Sutherland, 1956). The higher activity obtained when tissues are treated with hormone in the Na + medium in contrast to the K + medium may also be explained by the presence of this enzyme in fat body. The data presented raise the possibility that the hyperglycaemic hormone may act, partially at least, by regulating the concentration of Na + or K + or both within the cell. It is interesting to note that epinephrine has been shown to induce a liberation of K + from liver (D'Silva, 1934). However, there is no apparent change in the level of blood Na + and K + following injection of hyperglycaemic hormone into intact cockroaches, indicating no extrusion of these ions from the fat body (Steele, unpublished observations). Neither is there any change in the concentration of Na + and K + in fat body which has been incubated with the hormone in vitro (Steele, unpublished observations). The change in activity of phosphorylase when subjected to different concentrations of Na + and K + may indicate a mechanism of control which is entirely distinct from that of hormonal control. The suggestion of ionic regulation of phosphorylase is not unique, however, as Meyer et al. (1964) have already demonstrated in vitro that skeletal muscle phosphorylase kinase is activated by low concentrations of Ca ~+. More recently Friesen et al. (1967) have shown that Ca ~+ appears to be related to the activation of phosphorylase in cardiac muscle. On the basis of the present study it is suggested that the regulation of phosphorylase activity by phosphorylase phosphatase is partially dependent on the intracellular concentration of Na +. Acknowledgement--The author wishes to thank Dr. A. Vardanis for reading the manuscript and for suggesting a number of improvements.
IONIC ENVIRONMENT AND ACTIVATION OF PHOSPHORYLASE IN AN INSECT
763
REFERENCES BALLENTINER. & BURFORDD. D. (1957) Determination of metals. In Methods in Enzymology (Edited by COLOWICKS. V. ~ KAPLAN N. O.), Vol. 3, pp. 1002-1035. Academic Press, New York. BOWERS W. S. & FRIEDMAN S. (1963) Mobilization of fat body glycogen by an extract of corpus cardiacum. Nature, Lond. 198, 685. CAHILL G. F., ASHMOREJ., ZOTTU S. ~ HASTINGSA. B. (1957) Studies on carbohydrate metabolism in rat liver slices--IX. Ionic and hormonal effects on phosphorylase and glycogen, ft. biol. Chem. 224, 237-250. CARROLLN. V., LONGLEYR. W. ~ ROE J. H. (1956) The determination of glycogen in liver and muscle by use of anthrone reagent, jT. biol. Chem. 220, 586-593. D'SILVA J. L. (1934) The action of adrenaline on serum potassium, ft. Physiol., Lond. 82, 393-398. FRIESEN A. J. D., ALLEN G. ~ VALADARESJ. R. E. (1967) Calcium induced activation of phosphorylase in rat hearts. Science, N. Y. 155, 1108-1109. MEYER W. L., FISCHERE. H. & KREBSE. G. (1964) Activation of skeletal muscle phosphorylase b kinase by Ca ~+. Biochemistry 3, 1033-1039. RALPH C. L. & McCARTHY R. (1964) Effects of brain and corpus cardiacum extracts on haemolyrnph trehalose of the cockroach, Periplaneta americana. Nature, Lond. 203, 1195-1196. STEELE J. E. (1963) The site of action of insect hyperglycaemic hormone. Gen. comp. Endocrinol. 3, 46-52. STEELE J. E. (1964) The activation of phosphorylase in an insect by adenosine 3",5"-phosphate and other agents. Am. Zool. 4, 328. STEVENSONE. & WYATT G. R. (1964) Glycogen phosphorylase and its activation in silkworm fat body. Archs Biochem. Biophys. 108, 420-429. W I ~ s A. W. & GILBERT L. I. (1967) The phosphorylase system of the silkmoth, Hyalophora cecropia. Comp. Biochem. Physiol. 21, 145-159. WOSILAITW. D. & SUTmSaLANOE. W. (1956) The relationship of epinephrine and glueagon to liver phosphorylase II. Enzymatic inactivation of liver phosphorylase, ft. biol. Chem. 218, 469--481.
Key Word Index--Phosphorylase; Periplaneta americana; glycogenolysis; sodiumpotassium effect on phosphorylase; insect fat body.