Injecting rats with cortisone had no effect on the 5'-AMP hydrolysis by whole cells but significantly increased the activity in cell homogenates; the intracellular ...
Histochemistry63, 155-161 (1979)
Histochemistry 9 by Springer-Verlag 1979
5'-AMP Hydrolysis by Suspensions and Homogenates of Pancreatic Islet Ceils from Normal and Cortisone-Treated Rats A. Lernmark*, L.-A. S6derberg and I.-B. Tfiljedal Department of Histology, Universityof Ume~, S-90187 Ume~ 6, Sweden
Summary. Suspensions of endocrine pancreas cells were prepared by shaking collagenase-isolated rat islets of Langerhans in calcium-free buffer. When incubated with 1.0 mM substrate at pH 7.4, the cells split P~ from 5'-AMP at a rate of 87 nmol/h per ~tg DNA, and from fi-glycerophosphate at a rate of 25 nmol/h per gg DNA. Km for 5~AMP was about 54 gM. Adenosine or theophylline inhibited the 5'-AMP hydrolysis. Homogenization of the cells increased the activity toward 5'-AMP by 23% and that toward /3-glycerophosphate by 115%. Injecting rats with cortisone had no effect on the 5'-AMP hydrolysis by whole cells but significantly increased the activity in cell homogenates; the intracellular activity toward Y-AMP was more than doubled by the cortisone treatment. Staining whole islet cells for Y-AMP-splitting activity resulted in a demarcation of the cell periphery in control rats. Cells from cortisone-treated rats showed heavier deposits of reaction product, and their cell periphery did not stand out as clearly. It is suggested that 5'-nucleotidase is largely an ectoenzyme in normal rat islet cells. The cells also contain an as yet unidentified intracellnlar phosphatase that seems to be solely responsible for the increased hydrolysis of 5'-AMP in cortisone-treated rats.
Introduction Rats injected with cortisone (Gepts and Toussaint, 1964; Hellerstr6m et al., 1965) or glucagon (Johansson and Tfiljedal, 1968) exhibit an increased hydrolytic activity toward Y-adenosine monophosphate (5'-AMP) in their pancreatic islets. The effect is specific for the endocrine pancreas as compared with exocrine pancreas or heart. It is specific for the cleavage of 5'-AMP as compared with ATP, ADP, IDP, or p-nitrophenyl phosphate as substrate (Hellerstr6m et al., 1965; T/iljedal et al., 1966; Johansson and TNjedal, 1968). *
Present address: Steno Memorial Research Laboratory, Gentofle, Copenhagen,Denmark
0301-5564/79/0063/0155/$01.40
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5'-Nucleotidase is generally considered to be so typically associated with p l a s m a m e m b r a n e s as to serve as a m a r k e r for those m e m b r a n e s in the fractionation of cells, i n c l u d i n g islet cells ( L e r n m a r k et al., 1976; I d a h l et al., 1976). The p r e d o m i n a n t localization of 5'-nucleotidase to p l a s m a m e m b r a n e s is reflected in its f u n c t i o n as a n ecto-enzyme cleaving exogenous 5 ' - A M P . F o r example, in the perfused heart (Frick a n d Lowenstein, 1976) or in isolated intact fat cells ( N e w b y et al., 1975) all of the enzyme m a y be accessible to Y - A M P a d d e d externally. In the present study we have e x a m i n e d whether Y-nucleotidase f u n c t i o n s as an ecto-enzyme in rat islet cells, a n d whether cortisone stimulates such a Y-nucleotidase or a n o t h e r Y - A M P - s p l i t t i n g enzyme located w i t h i n the islet cells.
Materials and Methods Collagenase-isolatedislets of Langerhans from Sprague-Dawleyrats, starved overnight, were broken up to a milky suspension by shaking in calcium-free Krebs-Ringer bicarbonate buffer supplemented with 20 mM Hepes (pH 7.4) and cleansed fom debris by centrifuging through dense albumin (Lernmark, 1974). Resuspended cells were incubated at 37~ C for 45 min in a medium containing 80 mM tris-maleate buffer, 30 mM NaCI, 1 mM MnCI2, and substrate and lead nitrate as required. In staining experiments based on the Gomori lead sulfide precipitation principle, the medium had pH 6.8 and contained 3 mM 5'-AMP (free acid; Sigma Chemical Co., St. Louis, Mo., USA) and 2 mM Pb (NO3)2 ; control experiments were performed in the presence of sodium/~-glycerophosphate (Boehringer, Mannheim, Germany) instead of 5'-AMP, or in the complete absence of substrate. In quantitative enzyme assays, the medium had pH 7.4, lacked lead nitrate, and unless otherwise stated contained 1.0 mM 5'-AMP or 1.0 mM ~-glycerophosphate. After incubation in the histochemical staining medium, ceils were washed in physiological saline, reacted with ammonium sulfide, washed again, and finally inspected between object and cover glasses in a microscope. After incubation for quantitative enzyme measurements, the cellcontaining medium was mixed with the Malachite Green-molybdate reagent of Itaya and Ui (1966). The dye complex was solubilized with Triton X-100 and A660 read in a Zeiss spectrophotometer. In most experiments parallel incubations were performed with whole cells in suspensions and with cell suspensions that had first been homogenized by ultrasonication. Appropriate tissue and medium blanks were run throughout. The cell mass in each reaction vessel was assayed in terms of DNA (Lernmark et al., 1975) and generally amounted to about 20 ng DNA (about l gg protein). The volume of the reaction medium was 40 gl. The rate of 5'-AMP hydrolysis was approximately linear with time and tissue concentration under the conditions employed. Rats weighing 300 360 g were subcutaneously injected with cortisone acetate (Merck, Sharp and Dohme: Cortone; 50 mg/kg body weight), or with corresponding volumes of physiological saline, once daily for i2 days. The cortisone-treated rats lost about I00 g, and the saline-injected controls gained about 25 g body weight during the treatment period. Results are presented as mean values _+SEM for indicated numbers of experiments.
Results The hydrolysis of 5 ' - A M P by dispersed islet cells was characterized by Km a r o u n d 54 g m (Fig. 1) a n d a Vmax (1.0 m M 5 ' - A M P ) of 8 7 + 9 n m o l / h per lag D N A (n = 12). T h e rate o f ~-glycerophosphate (1.0 raM) hydrolysis u n d e r identical c o n d i t i o n s was only 25_+5 n m o l / h per ~tg D N A ( n = 8 ) . W h e n the cells were i n c u b a t e d with 1.0 m M Y - A M P as substrate, 0.4 m M a d e n o s i n e i n h i b i t e d the hydrolysis by 31_+8% ( n = 3 ) ; 5 m M t h e o p h y l l i n e i n h i b i t e d it by 51 _+3%
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V-1
0.02
0.01
// -0.0185
i
i
0.01 0.02 s-1 (~M-I)
i
i
i
0.03
0.04
0.05
Fig. 1. Double-reciprocal plot of the relationship between 5'-AMP concentration and hydrolysis by whole islets cells. Four different preparations of cells were incubated with 45 gM and some other concentrations of 5'-AMP. The rate of substrate hydrolysis at each concentration was expressed in per cent of that 45 gM and the results of the 4 separate experiments averaged. Thus, each point represents the mean of 2-4 experiments Table 1. Effect of cortisone injections on the 5'-AMP-splitting activity in whole islet cells and
islet cell homogenates Treatment
Saline Cortisone Difference
Enzyme activity (nmol Pi liberated/h per gg DNA) Whole cells
Homogenates
Intracellular
95+ 10 1l0 • 15 15 _+10
141 _+14 207 _+27 66 -+ 14 *
46+
6
97 + 12 51 + 8 *
For 5 pairs of rats, one member in each pair was injected with cortisone and the other with physiological saline. For each rat the Y-AMP-splitting activities of whole islet cells and cell homogenates were measured in parallel. The results for each group of rats and each type of tissue preparation are shown together with the differences between paired rats, and between homogenates and whole cells. Mean values+ SEM for 5 observations. *P< 0.01 for equality with zero (two-tailed t-test)
(n = 3). H o m o g e n i z a t i o n o f t h e d i s p e r s e d islet cells i n c r e a s e d t h e a c t i v i t y t o w a r d 1.0 m M 5 ' - A M P b y 23 + 7 % (n = 5), w h e r e a s t h e h y d r o l y s i s o f 1.0 m M # - g l y c e r o p h o s p h a t e r o s e b y as m u c h as 115 + 3 3 % (n = 4). R e s u l t s o b t a i n e d w i t h c o r t i s o n e - t r e a t e d rats a n d t h e i r s a l i n e - i n j e c t e d c o n t r o l s are s h o w n in T a b l e 1. C o r t i s o n e h a d n o effect o n t h e r a t e o f 5 ' - A M P h y d r o l y s i s by w h o l e islet cells. H o w e v e r , t h e e n z y m e a c t i v i t y o f cell h o m o g e n a t e s was i n c r e a s e d b y 4 6 % . By s u b t r a c t i n g t h e e n z y m e a c t i v i t y r e c o r d e d w i t h w h o l e cells f r o m t h a t in a c o r r e s p o n d i n g m a s s o f h o m o g e n i z e d cells, t h e i n t r a c e l l u l a r activity toward Y-AMP was estimated for each animal. Cortisone more than d o u b l e d t h e i n t r a c e l l u l a r e n z y m e activity.
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Fig. 2. Light-microscopicappearance of whole rat islet cells stained for 5'-AMP-splitting activity by the lead-sulphide precipitation technique. Left: saline-injected control rat. Right: cortisoneinjected rat
When islet cells from saline-injected rats were stained for 5'-AMP-splitting activity by the Gomori method, dark deposits of reaction product were seen to demarcate the surface of the cells (Fig. 2). Similarly stained cells from cortisoneinjected rats showed heavier deposits of reaction product, and their peripheries did not stand out in as clear contrast to the rest of the cell. Cells incubated with/%glycerophosphate or without any substrate remained virtually unstained.
Discussion
The relative substrate specificity of rat islet 5'-nucleotidase for 5'-AMP, as compared with T - A M P , 3'-AMP and ]?-glycerophosphate, has been described (Hellerstr6m et al., 1965). The present Km around 54 gM 5'-AMP in dispersed islet cells is close to the range (3-44 gM) of Km values reported for the 5'nucleotidase in extracts of heart (Frick and Lowenstein, 1976), smooth muscle (Burger and Lowenstein, 1975), or fat cells (Newby et al., 1975). The Vmax values for dispersed and sonicated islet cells agree fairly well with previous results for microdissected or collagenase-isolated islets (Table 2). As is also indicated in Table 2, the 5'-nucleotidase activity in islet cells is of the same magnitude as that in heart, a tissue rich in this enzyme (Reis, 1937). The comparison of whole islet cells with cell homogenates, as well as the histochemical staining of dispersed cells, suggests that 5'-nucleotidase is largely an ectoenzyme in normal rat islets. Whereas the non-specific enzyme activity toward/%glycerophosphate was more than doubled by homogenizing the cells, the rate of 5'-AMP cleavage rose by 23% (untreated rats) or 49% (saline-injected
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AMP in Rat Islet Cells Table 2. Comparison of
Vma x
estimates for 5'-nucleotidase in some previous studies
Study
Enzyme preparation
Present study Hellerstr6m et al. (1965) Johansson and Tfiljedal (1968) Lernmark et al. ( 1 9 7 6 )
Homogenized dispersed rat islet cells Homogenized microdissected rat islets Homogenized microdissected rat islets Homogenized collagenase-isolated rat islets Homogenized rat fat cells
Newby et al. 0975)
Enzyme activity mol/h per kg protein 2.3-2.9 a 1.8b 2.0 3.3 c
0.6 mol/h per kg dry weight
Frick and Lowenstein (1976)
Homogenized rat hearts or perfused intact hearts
1.3-2.8
a Assuming that 1 gg DNA corresponds to 47.6 ~tg protein in rat islets (Lernmark et al., 1976) u This is the value actually recorded. In that early study, however, the protein values were translated into dry weight by using a conversion factor that underestimated the protein content of rat islets c The value actually recorded was 1.4 mol/h per kg protein when using 40 ~tM 5'-AMP. When the substrate concentration is taken into account together with the present Km estimate of 54 gm 5'-AMP, Vn,x is calculated as indicated by applying Michaelis-Menten kinetics
rats). The increase o f 5 ' - A M P hydrolysis u p o n h o m o g e n i z a t i o n m a y in part reflect the activity of the same non-specific enzymes as those acting on fl-glycerophosphate. It is also conceivable that the islet cells, like liver cells (Pletsch and Coffey, 1972), contain a small intracellular pool o f 5'-nucleotidase. However, the preferential, t h o u g h not necessarily exclusive, localization of 5'-nucleotidase to the plasma m e m b r a n e o f islet cells supports its use as one o f several markers in islet-cell fractionation experiments ( L e r n m a r k et al., 1976; Idahl et al., 1976). That the intracellular activity t o w a r d 5 ' - A M P was not mainly due to nonspecific acid phosphatase is suggested by the cortisone-injection experiments. Prolonged cortisone treatment does not enhance the acid phosphatase activity in rat (T/iljedal et al., 1966) or mouse (T/iljedal, 1969) islets. In contrast, injections o f cortisone are here shown to m a r k e d l y increase the intracellular p o r t i o n of the 5'-AMP-splitting activity. Virtually the whole effect o f cortisone appeared to be mediated by enzyme(s) other than the 5'-nucleotidase in plasma m e m branes. That treatment with cortisone stimulates Y - A M P hydrolysis in rat islets was first observed in histochemical staining experiments (Gepts and Toussaint, 1964) and was then confirmed by quantitative biochemical measurements (Hellerstr6m et al., 1965). Quantitative measurements also showed that the hydrolysis of 5 ' - A M P by islet h o m o g e n a t e s is stimulated by injections o f glucagon as well (Johansson and T/iljedal, 1968). U p to now, the simplest interpretation o f these observations has been that cortisone and glucagon increase the activity o f a specific 5'-nucleotidase in the/?-cells. This interpretation m a y still be correct. However, in view o f the present results one must add the reservations that the hormone-sensitive enzyme(s) m a y not be identical with the classical 5'-
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nucleotidase that resides in plasma membranes, and that its true identity remains to be elucidated. In this context it m a y be w o r t h mentioning that rat tissues are generally quite rich in 5'-nucleotidase (Reis, 1937). On the other hand, unlike mouse islets rat islets contain but little activity of specific glucose-6phosphatase (T/iljedal, 1969). Perhaps the cortisone-induced enhancement o f 5 ' - A M P hydrolysis in rat islet cells is s o m e h o w analogous to the cortisoneinduced increase activity o f glucose-6-phosphatase in mouse, guinea-pig (T/iljedal, 1969) and rabbit (Gepts and Toussaint, 1964) islets. A l t h o u g h the enzymes assayed with Y - A M P in n o r m a l rat islets and with glucose-6-phosphate in n o r m a l m o u s e islets are not likely to be identical, it c a n n o t be ruled out that islet cells contain a cortisone-inducible enzyme with b r o a d e r substrate specificity than has hitherto been taken for granted. In whole islet cells the 5'-nucleotidase was inhibited by the product, adenosine. As has been shown in other tissues as well (Tsuzuki and Newburgh, 1975; F r e d h o l m et al., 1979), the enzyme was also inhibited by the structurally related purine derivative, theophylline. The potentiating effect o f methylxanthines on insulin release is usually attributed to inhibition o f the cyclic nucleotide phosphodiesterase activity (Ashcroft et al., 1972; Sams and Montague, 1972) with resulting enhancement of the 3',5'-cyclic A M P concentration. Alternative mechanisms m a y involve inhibition of Ca 2 § adenosine triphosphatase ( F o r m b y et al., 1976). If at least a portion o f 5'-nucleotidase can act on endogenous Y - A M P in the islet cells, its sensitivity to theophylline m a y contribute to strangling the degradation path for 3', 5'-cyclic A M P and thus to the potentiation o f insulin release.
Acknowledgements. This work was supported by the Swedish Medical Research Council (12x-2288) and the Swedish Diabetes Association.
References Ashcroft, S.J.H., Randle, P.J., T/iljedal, I.-B. : Cyclic nucleotide phosphodiesterase activity in normal mouse pancreatic islets. FEBS Lett. 20, 263-266 (1972) Burger, R.M., Lowenstein, J.M.: 5'-Nucleotidase from smooth muscle of small intestine and from brain. Inhibition by nucleotides. Biochemistry 14, 2362-2366 (1975) Formby, B., Capito, K., Egeberg, J., Hedeskov, C.J.: Ca-activated ATPase activity in subcellular fractions of mouse pancreatic islets. Am. J. Physiol. 230, 441448 (1976) Fredholm, B.B., Hedqvist, P., Vernet, L.: Effect of theophylline and other drugs on rabbit renal cyclic nucleotide phosphodiesterase, 5'-nucleotidase and adenosine deaminase. Biochem. Pharmacol. 27, 2845 2850 (1979) Frick, G.P., Lowenstein, J.M.: Studies of 5'-nucleotidase in the perfused rat heart. Including measurements of the enzyme in perfused skeletal muscle and liver. J. Biol. Chem. 251, 6372 6378 (1976) Gepts, W., Toussaint, D. : Effect of cortisone, growth hormone and hypophysectomy on the enzymatic activity of the pancreatic islets. In: The structure and metabolism of the pancreatic islets. S.E. Brolin, B. Hellman, H. Knutson, (eds.), pp. 357-376. Oxford: Pergamon Press 1964 Hellerstr6m, C., Tfiljedal, I.-B., Hellman, B.: Quantitative studies on isolated pancreatic islets of mammals. 5-Nucleotidase and adenosine triphosphatase activities in normal and cortisonetreated rats. Endocrinology 76, 315-322 (1965)
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Idahl, L.-A., Lernmark, A., Sehlin, J., T/iljedal, I.-B.: Studies on the function of pancreatic islet cell membranes. J. Physiol. (Paris) 72, 729 746 (1976) Itaya, K., Ui, M. : A new micromethod for the colorimetric determination of inorganic phosphate. Clin. Chim. Acta 17, 361-366 (1966) Johansson, S., T/iljedal, I.-B.: Stimulation of 5-nucleotidase activity in the pancreatic islets of rats by glucagon. Endocrinology 82, 173-174 (1968) Lernmark, A.: The preparation of, and studies on, free cell suspensions from mouse pancreatic islets. Diabetologia 10, 431-438 (1974) Lernmark, A., Sehtin, J., T/iljedal, I.-B. : The use of dispersed pancreatic islet cells in measurements of transmembrane transport. Anal. Biochem. 63, 73-79 (1975) Lernmark, ~., Nathans, A., Steiner, D.F.: Preparation and characterization of plasma membraneenriched fractions from rat pancreatic islets. J. Cell Biol. 71, 606-623 (1976) Newby, A.C., Luzio, J.P., Hales, C.N. : The properties and extracellular location of 5'-nucleotidase of the rat fat-cell plasma membrane. Biochem. J. 146, 625-633 (1975) Pletsch, Q.A., Coffey, J.W.: Studies on 5'-nucleotidases of rat liver. Biochim. Biophys. Acta 276, 192 205 (1972) Reis, J.: f,Jber die Aktivitfit der 5-Nukleotidase in den tierischen und menschlichen Geweben. Enzymologia 2, 183-190 (1937) Sams, D.J., Montague, W.: The role of adenosine 3':5'-cyclic monophosphate in the regulation of insulin release. Properties of islet-cell adenosine 3' :5'-cyclic monophosphate phosphodiesterase. Biochem. J. 129, 945-952 (1972) Tfiljedal, I.-B., Hellman, B., Hellerstr6m, C.: Quantitative studies on isolated pancreatic islets of mammals: enzymic hydrolysis of nucIeoside diphosphates and p-nitrophenyt phosphate in normal and cortisone-treated rats. J. Endocrinol. 36, 115-124 (1966) T/iljedal, I.-B.: Presence, induction and possible role of glucose 6-phosphatase in mammalian pancreatic islets. Biochem. J. 114, 387-394 (1969) Tsuzuki, J., Newburgh, R.W.: Inhibition of 5'-nucleotidase in rat brain by methylxanthines. J. Neurochem. 25, 895-896 (1975)
Received June 5, 1979
