Clin. exp. Immunol. (1991) 86, 260-265
Deficiency of purine nucleoside phosphorylase activity in thymocytes from the immunodeficient diabetic BB rat G. WU & E. B. MARLISS McGill Nutrition and Food Science Centre, Royval Victoria Hospital, Montreal, Quebec, Canada
(Accepted for publication 13 May, 1991)
SUMMARY
The spontaneously diabetic BB (BBd) rat displays marked T lymphopenia. The present study was designed to investigate whether the immunodeficiency in this animal may be associated with deficiency of purine nucleoside phosphorylase (PNP) and possibly adenosine deaminase (ADA). The activities of these two enzymes were measured in lymphoid and non-lymphoid cells from both nondiabetes-prone (BBn) and BBd rats as well as from streptozotocin-induced diabetic (STZ) BBn rats. There were no significant differences between BBn and BBd rats in ADA activities in thymocytes, skeletal muscle or brain. However, ADA activity was increased (P < 0 01) by 50%, in BBd mesenteric lymph node lymphocytes and splenocytes as compared with BBn cells, but was not altered in cells from STZ-BBn rats. On the other hand, the PNP activity in BBd thymocytes was only 61 I% (P < 0 01) of that observed in BBn cells. This PNP deficiency was not the consequence of diabetes per se, as its activity was normal in thymocytes from STZ-BBn rats. There were no significant differences in PNP activities between BBn and BBd rats in all other cell types examined. The diabetic BB rat may be a novel source of PNP-deficient thymocytes (mainly immature T cells) for studying biochemical mechanisms of immunodeficiency in association with decreased PNP activity. The findings also raise the question of whether a causal relationship exists between PNP deficiency and the recently demonstrated abnormality in T cell maturation in the thymus of the BBd rat. purine nucleoside phosphorylase Keywords lymphopenia
adenosine deaminase
thymus
BB rats
epitomized by the severe immunodeficiency diseases in humans caused by the deficiency of either ADA (Giblett & Anderson, 1972) or PNP (Giblett et al., 1975). Specifically, most patients with ADA deficiency show immunodeficiencies of both cellmediated (T cell) and humoral (B cell) immunity, whereas PNPdeficiency results in severe defects in cell-mediated immunity without altering normal humoral immunity (Kredich & Hershfield, 1989). The in titro proliferative response to mitogens is impaired in peripheral lymphocytes from patients with ADA or PNP deficiency (Luca et al., 1978; Kredich & Hershfield, 1989). Also, autoantibodies against erythrocytes have been reported in PNP-deficient patients (Luca et al., 1978; Rich et al., 1979). The relative rarity of ADA or PNP deficiency in humans or animals is a major factor limiting progress in characterizing the biochemical mechanisms involved in the development of the accompanying immunodeficiency (Kredich & Hershfield, 1989).
INTRODUCTION Both adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) participate in the salvage pathways that re-utilize purines formed from the breakdown of nucleic acids (Martin & Gelfand, 1981). Adenosine deaminase catalyses the hydrolytic cleavage of adenosine or deoxyadenosine to inosine or deoxyinosine. The products of ADA are substrates for PNP which catalyses phosphorolysis of purine ribonucleosides or 2'deoxyribonucleosides to form the free base plus ribose Iphosphate or 2'-deoxyribose 1-phosphate. Adenosine deaminase and PNP are widely distributed in mammalian cells, with the highest activities being reported in lymphoid organs (Carson, Kaye & Seegmiller, 1977; Seegmiller et al., 1980). It has been suggested that ADA and PNP play an important role in removing adenosine, deoxyadenosine, inosine, deoxyinosine and their metabolites that are potentially toxic to cells, particularly those of the immune system (Adams & Harkness, 1976; Seegmiller et al., 1980; Martin & Gelfand, 1981). This is
An alternative model system which involves the use of chemical inhibitors of ADA has been employed to study lymphospecific toxicity associated with ADA deficiency (Hovi et al., 1976; Seegmiller et al., 1980; Martin & Gelfand, 1981). However, there is not yet a suitable chemical inhibitor of PNP available. The spontaneously diabetic BB (BBd) rat is a widely studied animal model of autoimmune insulin-dependent (Type I)
Correspondence: E. B. Marliss, McGill Nutrition and Food Science Centre, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec, Canada H3A IAl.
260
Purine nucleoside phosphorylase and adenosine deaminase diabetes mellitus (Marliss et al., 1982; Yale & Marliss, 1984; Rossini, Mordes & Like, 1985). The syndrome is characterized by immunological defects including severe T cell lymphopenia (that appears to be a constant association), susceptibility to infections, the presence of autoantibodies, defective allograft rejection, and marked reduction in the in vitro proliferative response to mitogens by lymphocytes (Poussier et al., 1982; Prud'homme et al., 1984; Parfrey et al., 1989). Humoral immunity appears to be relatively normal in the BBd rat (Parfrey et al., 1989). Although the analogies between the human PNP deficiency disease and that of the BBd rat are only partial, we investigated both PNP and ADA activity in lymphoid and other cells form the BB rat. MATERIALS AND METHODS
Chemicals Adenosine, purine nucleoside phosphorylase and xanthine oxidase were obtained from Boehringer Mannheim (Montreal, Canada). Inosine was obtained from ICN Biochemicals (Mississauga, Canada). Animals Female BBd rats and sex- and age-matched non-diabetes prone (BBn) rats were obtained from the Animal Resources Division, Health Protection Branch, Ottawa, Canada. Animals were housed in a light- (12 h light-12 h dark cycles) and climatecontrolled facility in laminar flow hoods and provided ad libitum laboratory rat chow (Ralston Purina Co., Woodstock, Canada) and water. Diabetic rats (10-20 days post-onset) were maintained on a single daily injection of ultralente insulin (2-4 units).
Chemically (streptozotocin) -induced diabetes in BBn rats Female BBn rats (90 days old) were injected i.v. with streptozotocin (STZ; 65 mg/kg body weight) in 50 mm sodium citrate (pH 4-5). Rats became diabetic within 24 h of the injection, as indicated by hyperglycaemia measured with Chemstrip.bG and by glycosuria measured with Chemstrip.uG (both from Boehringer Mannheim). Diabetic rats were maintained on single daily subcutaneous injections of ultralente insulin (2-3 units) for 14 days before removal of lymphoid organs. Preparations of thymocytes, splenocytes and mesenteric lymph node cells Rats (95-115 days old) were anaesthetized with ether. The
thymus, spleen and mesenteric lymph nodes were removed from each animal. These lymphoid organs were cut individually into small pieces and passed through a 50-mesh grid in cold KrebsRinger Hepes buffer (pH 7-4) supplemented with 0-5% bovine serum albumin (BSA) (Wu et al., 199 1b). After centrifugation at 300 g for 5 min at 4°C, erythrocytes were lysed as previously described (Wu et al., 1991b). The cells were washed three times with Krebs-Ringer Hepes buffer without BSA and then suspended in this buffer at concentrations of 50 x 106 cells/ml for splenocytes and mesenteric lymph node cells or 500 x 106 cells/ ml for thymocytes. Preparations of lymphoid cell extracts for enzyme assays Cell extracts were prepared by three cycles of freezing of an aliquot (0-6 ml) of cell suspension (in ethanol on dry ice) and thawing (37°C waterbath). The extracts were centrifuged in an
261
Eppendorf Micro-Centrifuge (Fisher Scientific Co., Montreal, Canada) for 2 min. The supernatant was stored at -70'C and used within 3 days for measurements of ADA and PNP activities. In measuring thymocyte ADA activity, the supernatant was diluted 20 times. Protein in the supernatant was determined by a modified Lowry method with BSA as standard (Markwell et al., 1978). Preparations of skeletal muscle and brain Extensor digitorum longus muscles and the whole brain from each rat were isolated after the thymus, spleen and lymph nodes were removed. Muscles (0-23 + 0-01 g, mean + s.e.m., n = 6) and brain (1 23 + 0 07 g, mean + s.e.m., n = 6) were homogenized in 1 ml and 2 ml of extraction media (125 mm KCI, 6-25 mm MgCl2, 1-25 mm EDTA, 62 5 mm Tris/HCI, pH 7 5), respectively. The homogenates were centrifuged for 2 min in an Eppendorf Micro-Centrifuge and the supernatant was used for enzyme assays. Determination of activities of ADA and PNP The activities of ADA and PNP were determined at 37 C by coupled enzyme reactions on the basis of the principles as previously described (Kalchar, 1947; Hopkinson, Cosk & Harris, 1969). For determination of ADA activity, the reaction mixture (3 ml) contained 2-8 ml 50 mm potassium phosphate buffer (pH 7 5), 0-24 U PNP, 0 04 U xanthine oxidase, 0 I ml 0-38-6-0 mm adenosine, and 0-1 ml cell extracts. The reaction mixture without adenosine was pre-incubated at 37°C for 5 min. The reaction was initiated by the addition of adenosine. The absorption at 293 nm due to the uric acid produced from adenosine was measured by a UV/VIS Spectrophotometer (Perkin-Elmer Instruments, Montreal, Canada). The reaction was linear from 2 to 10 min and with the amount of protein used. Adenosine deaminase activities were calculated from four timepoints during the linear portion of the assay. The reagent blanks without cell extracts were run to correct for the ADA activity potentially contaminating the commercial exogenous enzyme (PNP and xanthine oxidase) preparations, which was found to be minimal (less than 7% of sample activity). For determination of PNP activity, the reaction mixture (3 ml) contained 2-85 ml 50 mm potassium phosphate buffer (pH 7 5), 0-04 U xanthine oxidase, 0-1 ml 0-38-6-0 mm inosine, and 0-05 ml cell extracts. The reaction mixture without inosine was pre-incubated at 37 C for 5 min. The reaction was initiated by the addition of inosine. The absorption at 293 nm due to the uric acid produced from inosine was measured as described above. The reaction was linear for 5 min and with the amount of protein used. Purine nucleoside phosphorylase activities were calculated from four time-points during the linear portion of the assay. The reagent blanks without cell extracts were run to correct for the PNP activity potentially contaminating the commercial exogenous enzyme (xanthine oxidase) preparations, which was found to be undetectable. The activity of ADA or PNP was expressed as the rate of formation of product (uric acid) per min per mg protein. The molar absorption coefficient of 12-5 x 103 M- 1. cm- 'for uric acid at 293 nm was used for the calculation (Kim, Cha & Parks, 1968; Zimmermann et al., 1971). It was established in preliminary experiments that maximal activities of ADA or PNP were achieved in the presence of 0-2 mm adenosine or inosine for all cell preparations studied.
262
G. Wu & E. B. Marliss Table 1. Activities of purine nucleoside phosphorylase in thymocytes, lymph node cells, splenocytes, skeletal muscle and brain from non-diabetes-prone (BBn), spontaneously diabetic BB rats (BBd) and streptozotocin-induced BBn rats
BBn rats
Streptozotocin-induced diabetic BBn rats
BBd rats
Thymocytes 64-3 + 2-6a (12) 39 4 + 1 9b (12) Lymph node cells 97-7 + 5-Oa ( 11) 91l8 + 6-3a (9) Splenocytes 122.6+6-7a (11) 119 7+5-4a (9) 54 +05a (6) 522+05a (6) EDL muscle Brain 39 7+2 7a (6) 39-7+2-8a (6)
570 + 2 2a (6) 86-9 + 4-6a (6) 116 3+3-7a (6) ND ND
Data are given as nmol product/min/mg protein and are mean + s.e.m., with the number of animals in parentheses. Means not sharing the same superscript within a row are significantly different (P lymph node cells > thymocytes > brain > extensor digitorum longus (EDL) muscle. ND, Not determined. 75
150 60 E0 cmw
125
E
45_
100 _ 0
0.
'5 30
75
50 z
25 0
50
00
I S0
200
250
Inosine concentration (AuM)
Fig. 1. The activity of purine nucleoside phosphorylase (PNP) in thymocytes from non-diabetes-prone (BBn) and spontaneously diabetic (BBd) BB rats. The activity of PNP in thymocytes was measured in the presence of 12 5-250 gm inosine. Data represent mean+s.e.m., n =7. The activity of PNP is significantly (P < 0-01) lower in BBd than in BBn cells at all substrate concentrations used. 0, BBn; 0, BBd.
0
20
60
40 I
[Inosine]
80
(mm-')
Fig. 2. Double-reciprocal plot of I/S versus 1/ V for purine nucleoside phosphorylase. The rate of enzyme activity (V) is expressed as imol product/min/mg protein. Inosine concentration (S) is expressed as mm. Data represent mean + s.e.m., n = 7. 0, non-diabetes-prone (BBn) and
, spontaneously diabetic BB rats (BBd). Statistical analysis Data were analysed by two-way analysis of variance with the SNK multiple range test (Steel & Torrie, 1980).
RESULTS Distribution of PNP activities Table I shows PNP activities in lymphoid and non-lymphoid cells from normal and diabetic rats. In BBn rats, the order of
PNP activity was as follows (P lymph node cells> thymocytes> brain > skeletal muscle. There were no differences between BBn and BBd rats in PNP activities in peripheral lymphoid cells (splenocytes and lymph node lymphocytes) and non-lymphoid cells (skeletal muscle and brain). However, the PNP activity in BBd thymocytes was only 61'S, (P extensor digitorum longus (EDL) muscle> brain. ND, Not determined.
differences in thymocyte, lymph node lymphocyte or splenocyte PNP activities between BBn and STZ-induced diabetic BBn
cellular composition. Nevertheless, our findings may have potential implications in exploring mechanisms of immunodefi-
rats.
ciencies.
The PNP activity was consistently lower (P
Deficiency of purine nucleoside phosphorylase activity in thymocytes from the immunodeficient diabetic BB rat G. WU & E. B. MARLISS McGill Nutrition and Food Science Centre, Royval Victoria Hospital, Montreal, Quebec, Canada
(Accepted for publication 13 May, 1991)
SUMMARY
The spontaneously diabetic BB (BBd) rat displays marked T lymphopenia. The present study was designed to investigate whether the immunodeficiency in this animal may be associated with deficiency of purine nucleoside phosphorylase (PNP) and possibly adenosine deaminase (ADA). The activities of these two enzymes were measured in lymphoid and non-lymphoid cells from both nondiabetes-prone (BBn) and BBd rats as well as from streptozotocin-induced diabetic (STZ) BBn rats. There were no significant differences between BBn and BBd rats in ADA activities in thymocytes, skeletal muscle or brain. However, ADA activity was increased (P < 0 01) by 50%, in BBd mesenteric lymph node lymphocytes and splenocytes as compared with BBn cells, but was not altered in cells from STZ-BBn rats. On the other hand, the PNP activity in BBd thymocytes was only 61 I% (P < 0 01) of that observed in BBn cells. This PNP deficiency was not the consequence of diabetes per se, as its activity was normal in thymocytes from STZ-BBn rats. There were no significant differences in PNP activities between BBn and BBd rats in all other cell types examined. The diabetic BB rat may be a novel source of PNP-deficient thymocytes (mainly immature T cells) for studying biochemical mechanisms of immunodeficiency in association with decreased PNP activity. The findings also raise the question of whether a causal relationship exists between PNP deficiency and the recently demonstrated abnormality in T cell maturation in the thymus of the BBd rat. purine nucleoside phosphorylase Keywords lymphopenia
adenosine deaminase
thymus
BB rats
epitomized by the severe immunodeficiency diseases in humans caused by the deficiency of either ADA (Giblett & Anderson, 1972) or PNP (Giblett et al., 1975). Specifically, most patients with ADA deficiency show immunodeficiencies of both cellmediated (T cell) and humoral (B cell) immunity, whereas PNPdeficiency results in severe defects in cell-mediated immunity without altering normal humoral immunity (Kredich & Hershfield, 1989). The in titro proliferative response to mitogens is impaired in peripheral lymphocytes from patients with ADA or PNP deficiency (Luca et al., 1978; Kredich & Hershfield, 1989). Also, autoantibodies against erythrocytes have been reported in PNP-deficient patients (Luca et al., 1978; Rich et al., 1979). The relative rarity of ADA or PNP deficiency in humans or animals is a major factor limiting progress in characterizing the biochemical mechanisms involved in the development of the accompanying immunodeficiency (Kredich & Hershfield, 1989).
INTRODUCTION Both adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) participate in the salvage pathways that re-utilize purines formed from the breakdown of nucleic acids (Martin & Gelfand, 1981). Adenosine deaminase catalyses the hydrolytic cleavage of adenosine or deoxyadenosine to inosine or deoxyinosine. The products of ADA are substrates for PNP which catalyses phosphorolysis of purine ribonucleosides or 2'deoxyribonucleosides to form the free base plus ribose Iphosphate or 2'-deoxyribose 1-phosphate. Adenosine deaminase and PNP are widely distributed in mammalian cells, with the highest activities being reported in lymphoid organs (Carson, Kaye & Seegmiller, 1977; Seegmiller et al., 1980). It has been suggested that ADA and PNP play an important role in removing adenosine, deoxyadenosine, inosine, deoxyinosine and their metabolites that are potentially toxic to cells, particularly those of the immune system (Adams & Harkness, 1976; Seegmiller et al., 1980; Martin & Gelfand, 1981). This is
An alternative model system which involves the use of chemical inhibitors of ADA has been employed to study lymphospecific toxicity associated with ADA deficiency (Hovi et al., 1976; Seegmiller et al., 1980; Martin & Gelfand, 1981). However, there is not yet a suitable chemical inhibitor of PNP available. The spontaneously diabetic BB (BBd) rat is a widely studied animal model of autoimmune insulin-dependent (Type I)
Correspondence: E. B. Marliss, McGill Nutrition and Food Science Centre, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec, Canada H3A IAl.
260
Purine nucleoside phosphorylase and adenosine deaminase diabetes mellitus (Marliss et al., 1982; Yale & Marliss, 1984; Rossini, Mordes & Like, 1985). The syndrome is characterized by immunological defects including severe T cell lymphopenia (that appears to be a constant association), susceptibility to infections, the presence of autoantibodies, defective allograft rejection, and marked reduction in the in vitro proliferative response to mitogens by lymphocytes (Poussier et al., 1982; Prud'homme et al., 1984; Parfrey et al., 1989). Humoral immunity appears to be relatively normal in the BBd rat (Parfrey et al., 1989). Although the analogies between the human PNP deficiency disease and that of the BBd rat are only partial, we investigated both PNP and ADA activity in lymphoid and other cells form the BB rat. MATERIALS AND METHODS
Chemicals Adenosine, purine nucleoside phosphorylase and xanthine oxidase were obtained from Boehringer Mannheim (Montreal, Canada). Inosine was obtained from ICN Biochemicals (Mississauga, Canada). Animals Female BBd rats and sex- and age-matched non-diabetes prone (BBn) rats were obtained from the Animal Resources Division, Health Protection Branch, Ottawa, Canada. Animals were housed in a light- (12 h light-12 h dark cycles) and climatecontrolled facility in laminar flow hoods and provided ad libitum laboratory rat chow (Ralston Purina Co., Woodstock, Canada) and water. Diabetic rats (10-20 days post-onset) were maintained on a single daily injection of ultralente insulin (2-4 units).
Chemically (streptozotocin) -induced diabetes in BBn rats Female BBn rats (90 days old) were injected i.v. with streptozotocin (STZ; 65 mg/kg body weight) in 50 mm sodium citrate (pH 4-5). Rats became diabetic within 24 h of the injection, as indicated by hyperglycaemia measured with Chemstrip.bG and by glycosuria measured with Chemstrip.uG (both from Boehringer Mannheim). Diabetic rats were maintained on single daily subcutaneous injections of ultralente insulin (2-3 units) for 14 days before removal of lymphoid organs. Preparations of thymocytes, splenocytes and mesenteric lymph node cells Rats (95-115 days old) were anaesthetized with ether. The
thymus, spleen and mesenteric lymph nodes were removed from each animal. These lymphoid organs were cut individually into small pieces and passed through a 50-mesh grid in cold KrebsRinger Hepes buffer (pH 7-4) supplemented with 0-5% bovine serum albumin (BSA) (Wu et al., 199 1b). After centrifugation at 300 g for 5 min at 4°C, erythrocytes were lysed as previously described (Wu et al., 1991b). The cells were washed three times with Krebs-Ringer Hepes buffer without BSA and then suspended in this buffer at concentrations of 50 x 106 cells/ml for splenocytes and mesenteric lymph node cells or 500 x 106 cells/ ml for thymocytes. Preparations of lymphoid cell extracts for enzyme assays Cell extracts were prepared by three cycles of freezing of an aliquot (0-6 ml) of cell suspension (in ethanol on dry ice) and thawing (37°C waterbath). The extracts were centrifuged in an
261
Eppendorf Micro-Centrifuge (Fisher Scientific Co., Montreal, Canada) for 2 min. The supernatant was stored at -70'C and used within 3 days for measurements of ADA and PNP activities. In measuring thymocyte ADA activity, the supernatant was diluted 20 times. Protein in the supernatant was determined by a modified Lowry method with BSA as standard (Markwell et al., 1978). Preparations of skeletal muscle and brain Extensor digitorum longus muscles and the whole brain from each rat were isolated after the thymus, spleen and lymph nodes were removed. Muscles (0-23 + 0-01 g, mean + s.e.m., n = 6) and brain (1 23 + 0 07 g, mean + s.e.m., n = 6) were homogenized in 1 ml and 2 ml of extraction media (125 mm KCI, 6-25 mm MgCl2, 1-25 mm EDTA, 62 5 mm Tris/HCI, pH 7 5), respectively. The homogenates were centrifuged for 2 min in an Eppendorf Micro-Centrifuge and the supernatant was used for enzyme assays. Determination of activities of ADA and PNP The activities of ADA and PNP were determined at 37 C by coupled enzyme reactions on the basis of the principles as previously described (Kalchar, 1947; Hopkinson, Cosk & Harris, 1969). For determination of ADA activity, the reaction mixture (3 ml) contained 2-8 ml 50 mm potassium phosphate buffer (pH 7 5), 0-24 U PNP, 0 04 U xanthine oxidase, 0 I ml 0-38-6-0 mm adenosine, and 0-1 ml cell extracts. The reaction mixture without adenosine was pre-incubated at 37°C for 5 min. The reaction was initiated by the addition of adenosine. The absorption at 293 nm due to the uric acid produced from adenosine was measured by a UV/VIS Spectrophotometer (Perkin-Elmer Instruments, Montreal, Canada). The reaction was linear from 2 to 10 min and with the amount of protein used. Adenosine deaminase activities were calculated from four timepoints during the linear portion of the assay. The reagent blanks without cell extracts were run to correct for the ADA activity potentially contaminating the commercial exogenous enzyme (PNP and xanthine oxidase) preparations, which was found to be minimal (less than 7% of sample activity). For determination of PNP activity, the reaction mixture (3 ml) contained 2-85 ml 50 mm potassium phosphate buffer (pH 7 5), 0-04 U xanthine oxidase, 0-1 ml 0-38-6-0 mm inosine, and 0-05 ml cell extracts. The reaction mixture without inosine was pre-incubated at 37 C for 5 min. The reaction was initiated by the addition of inosine. The absorption at 293 nm due to the uric acid produced from inosine was measured as described above. The reaction was linear for 5 min and with the amount of protein used. Purine nucleoside phosphorylase activities were calculated from four time-points during the linear portion of the assay. The reagent blanks without cell extracts were run to correct for the PNP activity potentially contaminating the commercial exogenous enzyme (xanthine oxidase) preparations, which was found to be undetectable. The activity of ADA or PNP was expressed as the rate of formation of product (uric acid) per min per mg protein. The molar absorption coefficient of 12-5 x 103 M- 1. cm- 'for uric acid at 293 nm was used for the calculation (Kim, Cha & Parks, 1968; Zimmermann et al., 1971). It was established in preliminary experiments that maximal activities of ADA or PNP were achieved in the presence of 0-2 mm adenosine or inosine for all cell preparations studied.
262
G. Wu & E. B. Marliss Table 1. Activities of purine nucleoside phosphorylase in thymocytes, lymph node cells, splenocytes, skeletal muscle and brain from non-diabetes-prone (BBn), spontaneously diabetic BB rats (BBd) and streptozotocin-induced BBn rats
BBn rats
Streptozotocin-induced diabetic BBn rats
BBd rats
Thymocytes 64-3 + 2-6a (12) 39 4 + 1 9b (12) Lymph node cells 97-7 + 5-Oa ( 11) 91l8 + 6-3a (9) Splenocytes 122.6+6-7a (11) 119 7+5-4a (9) 54 +05a (6) 522+05a (6) EDL muscle Brain 39 7+2 7a (6) 39-7+2-8a (6)
570 + 2 2a (6) 86-9 + 4-6a (6) 116 3+3-7a (6) ND ND
Data are given as nmol product/min/mg protein and are mean + s.e.m., with the number of animals in parentheses. Means not sharing the same superscript within a row are significantly different (P lymph node cells > thymocytes > brain > extensor digitorum longus (EDL) muscle. ND, Not determined. 75
150 60 E0 cmw
125
E
45_
100 _ 0
0.
'5 30
75
50 z
25 0
50
00
I S0
200
250
Inosine concentration (AuM)
Fig. 1. The activity of purine nucleoside phosphorylase (PNP) in thymocytes from non-diabetes-prone (BBn) and spontaneously diabetic (BBd) BB rats. The activity of PNP in thymocytes was measured in the presence of 12 5-250 gm inosine. Data represent mean+s.e.m., n =7. The activity of PNP is significantly (P < 0-01) lower in BBd than in BBn cells at all substrate concentrations used. 0, BBn; 0, BBd.
0
20
60
40 I
[Inosine]
80
(mm-')
Fig. 2. Double-reciprocal plot of I/S versus 1/ V for purine nucleoside phosphorylase. The rate of enzyme activity (V) is expressed as imol product/min/mg protein. Inosine concentration (S) is expressed as mm. Data represent mean + s.e.m., n = 7. 0, non-diabetes-prone (BBn) and
, spontaneously diabetic BB rats (BBd). Statistical analysis Data were analysed by two-way analysis of variance with the SNK multiple range test (Steel & Torrie, 1980).
RESULTS Distribution of PNP activities Table I shows PNP activities in lymphoid and non-lymphoid cells from normal and diabetic rats. In BBn rats, the order of
PNP activity was as follows (P lymph node cells> thymocytes> brain > skeletal muscle. There were no differences between BBn and BBd rats in PNP activities in peripheral lymphoid cells (splenocytes and lymph node lymphocytes) and non-lymphoid cells (skeletal muscle and brain). However, the PNP activity in BBd thymocytes was only 61'S, (P extensor digitorum longus (EDL) muscle> brain. ND, Not determined.
differences in thymocyte, lymph node lymphocyte or splenocyte PNP activities between BBn and STZ-induced diabetic BBn
cellular composition. Nevertheless, our findings may have potential implications in exploring mechanisms of immunodefi-
rats.
ciencies.
The PNP activity was consistently lower (P
