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Wor rat
Biochimica t't BiophysicaActa, I 118(1'491)77-82
77
•3 1991ElsevierSciencePublishersB.V. All rightsreserved0167.4838/91/$03.511
BBAPRO34(191
Molecular isoforms of Na +/K+-ATPase in the nervous system and kidney of the spontaneously diabetic BB/Wor rat Susan C. Specht, Jos~ Martin, R. Enid Gaud and Jost~ De Hoyos Department a]'Phannacology and Institute of Neurobiolo~,%Uoicersityof Puerto Rico, Sa~lJuan, PR (U.S.A.)
(Received23 April lqt~!) ( Revisedmanuscriptreceived 14 August 1~191)
Keywords: Isozyme:Isoform;Calalyticsubunil,a: Retina;AxtmalIrangport N a + / K +-ATPase was evaluated in the retina and kidney of the spontaneously diabetic BB/Wor rat after I and 4 months of insulin dependency. Retinal synthesis of the Na + / K +-ATPase was measured during a 2-h intravitreal pulse of [35S]methiunine and analyzed by SDS-PAGE and scintillation counting. Synthesis of the a-I and 'a( +)' (includes both a-2 and a-3) isofonns of the catalytic subunit was increased 123% and 69%, respectively at 4 months. Increases were also suggested at 1 month, but were not significant. The diabetes-dependent peak of synthesis m long.term diabetic rats turned over rapidly and by 3 days after intravitreai labeling, radioactively labeled enzyme was equal in both control and diabetic retinae. The amount of axonally transported, labeled enzyme recovered from endings of the optic nerve in the superior ¢olliculus paralleled retinal labeling. Significant renal hypertrophy (48%) was noted at 4 months, but not at 1 month. The strophanthidin-inhibition constant for diabetes-induced renal enzyme was the same as for control enzyme (approx. 10-4 M), indicating that diabetic renal hypertrophy does not induce a Na pump isozyme that is more sensitive to cardiotonic steroids. SDS-PAGE of the renal enzyme also failed to indicate more than one isoform of the a subunit.
Introduction
The Na+/K+-ATPase is a membrane protein which actively transports Na ÷ and K ~ across the membrane against their respective electrochemical gradients. It comprises two subunits, a and/~, the former possessing all known ligand binding sites and enzymatic activities (1). Although the physiological function of the /3 subunit remains to be established, in sequence and immunological reactivity it is homologou~ to the glial adhesion molecule AMOG (2). The Na+/K÷-ATPase is specifically inhibited by cardiac glycosides such as ouabain and strophanthidin. Several molecular isoforms of the a subunit have been described recently [3]. Of the three isoforms present in the rat, a-2 and a-3 are highly sensitive to ouabain and other cardiac glycosides (K~ = 10 -~ M to 10-7 M), whereas a-I is 10-
Abbreviations:IDDM.insulin-dependentdiabetesmellitus:DR. diabetes resistant. Correspondence: S.C. Specht. Institute of Neurobiology. 21)1 Blvd. Del Valle,San Juan, Puerto Rico1109111.USA.
1000-fold less sensitive (K i ffi 10 -5 M to 10 -3 M)[4,5]. In SDS-PAGE, the a-2 and a-3 isoforms run as a single band known as ~( + ) [4]. By the use of synthetic ot isoforms [6-8], as well as by partial tryptic digestion of ' a ( + ) ' [9] it has been shown that a-2 migrates slightly faster than ~-3, thus filling the lower part of the 'a( + )' gel band. Evidence is slowly accumulating that the ~ isoforms may be differentially induced by several physiological and pathological stimuli (reviewed in Ref. 10). For example, isoform ratios change during development in both heart [11] and brain [6,12]. Two rat models of hypertension show deinduction of mRNAs encoding for a2 and or3 with coordinate induction of mRNA for al (13). Thyroid hormone alters both isoform ratios in the adult ferret heart [14] and isoform abundance in neonatal brain [12], Several workers have shown that biosynthesis of the Na÷/K+-ATPase is up-regulated when enzymatic activity is partially inhibited by incubating cells with ouabain [15,16], lnsuIin is a well-known stimulator of Na+/K÷-ATPase enzyme activity [17] and may act in contrary fashion to down-regulate biosynthesis of Na+/K+-ATPase. Lytton reported that insulin stimu-
lates the activity of the Na+/K*-ATPase isozymes in rat adipocytes by increasing their affinity for Na ÷ [18]. The effect is greater on the "a( + r isoenzyme, with the Ko~ for Na ÷ decreasing 36% from 52 mM to 33 raM, whereas that of a-I decreases 18% from 17 mM to 14 raM. Although the change was greater for "t~(+ )', both changes were significant, it is not known whether insulin affects the Na + affinity, of both 0-2 and a-3, but Young and Lingrel have reported that adipocytes contain mRNA for only the a-2 isoform [19]. The experiments reported here were conducted to determine whether synthesis of the Na*/K *-ATPase is up-regulated when diabetes mellitus is treated with sub-optimal insulin, thus effecting a relative inhibition of Na*/K*-ATPase activity. We were particularly interested in determining whether increased synthesis would reflect the differential effect of insulin on the a and "a( + )" isoforms. In order to study isotorm synthesis, retinae of spontaneously diabetic BB and control BB-DR rats were labeled in vivo with [~S]methionine Since renal hypertrophy and increased Na*/K ÷ATPase are characteristic of diabetes meilitus induced by streptozotocin [20]. we also examined the kidneys of the BB/Wor rats to determine if renal hypertrophy occurs and whether it is associated with the appearance of a different Na'/K*-ATPase a isoform, in addition we examined Na+/K*-ATPase activity in peripheral nerve axolemma, in view of many reports of altered Na pump activity, in diabetic peripheral nerve [21]. Some of these data have been previously presented [22]. Materials and Methods
Animals. Diabetic and age-matched control diabetes-resistant female BB/Wor rats were obtained 3 weeks after detection of insulin-dependent diabetes mellitus (IDDM) from the NIH contract colony at the University of Massachusetts Medical School. Department of Pathology. The average age at detection was t 3 + 1 weeks. UrinatT glucose and ketones were checked daily by KetostLxR and insulin (NPH-40, Eli Lily) was administered daily after detection of IDDM by subcutaneous injection below the skin overlying the pectoral muscles. The treatment objective was to maintain urine sugar at 4 + with a moderate frequencT of ketosis. The daily dose varied from 0.6 to i.6 units at the onset and was increased according to need with growth. The BB/Wor rat utihzed in these studies is an inbred strain in which 40-70% of both sexes spontaneously develop IDDM, 85% of the conversions occurring between 60-120 days of age. The diabetic animals are insulin-dependent and exhibit frequent ketoacidosis. The natural control is the BB-diabetes resistant rat (BB-DR), a sub-strain with diabetic ancestors, The
disease appears to be an autoimmune disorder and can be induced in the BB-DR rat as well as in other susceptible strains by infusion of bone marrow cells from affected animals. Susceptibility is associated with the rat class 11 major histocompatibility complex [23]. lnrravitreal radioactive labeling. Rats were lightly anes'.hetized with either pentobarbital (10 mg/kg) supplemented with dietbyl ether or diethyl ether alone. The selera was punctured below the limbus with a sterile 26 G needle and a glass pipet containing 5 #l of [~- S]metmomne (60 #Ci; approx. 1200 Ci/mmol; New England Nuclear, Boston, MA) was inserted into the vitreal chamber. The solution was injected by application of hydrostatic pressure. Labeling was always conducted in the morning, before the scheduled daily insulin treatment. Animals were killed after 2 h in studies of synthesis or after several days in studies of enzyme degradation and axonal transport. The method of killing was decapitation following diethyl, ether ~ac~'.hcsia. Preparation of enzyme and SDS-PAGE. Na+/K +ATPase was prepared from radioactive retinae according to the method of Sweadner [24]. Protein concentrations were determined [3] using bovine serum albumin as a standard. Briefly, microsomes were prepared, then treated with SDS (0.3 rag/rag protein) and centrifuged through a 7-30% sucrose gradient. The enz~ane-enriched membranes were collected from the gradient, diluted in ice-cold water and a pellet was collected by eentrifugation at 1000O0 x g for 2 h 17 rain (o~ = 6.82- 10"~). The a isoforms were separated on a 5% sDS-polyacfflamide gel [26]. In an immunoblot protocol, the hands react with isoformspecific monoclonai antibodies developed by Sweadner [5]. Incorporation of 3-~Swas determined by cutting the appropriate bands from the wet gel and dissolving them [27] for scintillation counting. The results were corrected for radioactive decay between receipt of radioactive material and scintillation counting of labeled protein bands. Retinae were analyzed individually, Since the amount of protein in one retina was too low for visualization with Coomassie blue, 20 #g of Na +/K+-ATPase isolated from non-radioactive cerebral cortex was adde.d for SDS-PAGE. Results are reported in terms of radioactivity per gel band per retina. The superior colliculus was dissected to the depth of the cerebral aqueduct, a cut that includes the entire nucleus as well as some non-visual tissue. Each gel lane was loaded with approx. 50 #g of Na'/K÷-ATPase partially purified from superior colliculus, using several lanes to accommodate the entire sample, The average amount of protein loaded (mean +_S.E.) was 1O0 +_22/tg (control, ~ = 12) and l l0 _+ 17 v-g (diabetic, n -- 12). Data are reported for the entire superior colliculus sample, although the amount of protein may differ, since ra-
79 dioactive label is largely confined to optic nerve axons in the upper layers of the superior collicuiuso Preparation of kidney enzyme. Kidneys were removed from rats at the time of death, decapsulated, weighed, sliced, frozen in liquid nitrogen and stored at -70°(; until processed. For preparation of enzyme, the grey papilla was dissected from kidney slices and discarded; the entire remaining medulla and cortex was then minced, and enzyme was prepared according to the procedure of Jorgensen and Skou [28]. Enzyme assays. The Na+/K+-ATPase was usually assayed using an associated activity, the K+-dependent p-nitrophenyi phosphatase [29]. Strophanthidin was prepared in dimethylformamide. The enzyme and reactants except KCI or water were pre-incubated for 10 rain at 30°C. The reaction was started with their addition and allowcd to proceed for 20 rain, then stopped with ice-cold, 30% trichloroacetic acid. The final reaction volume was 0.25 ml and 0.7 to 1.6 pg enzyme protein were used per tube. ,~J! reaedens were performed in duplicate. Occasionally+ true Na+/K ÷ATPase activity was assayed [30]. In that case, no preincubation was employed. The reaction was performed at 37 o C for 10 rain. Enzyme protein per tube was approx. 40 #g. Preparation of sciatic nen'e axolemma. The method of Chou et al. [31] was employed with modifications. Briefly, the nerves were cleaned, homogenized in 0.29 M sucrose and centrifuged at 2400 rpm in a Beckman JA-20 rotor for 10 rain. The pellet was rinsed twice and the resultant supematant fractions were centrifuged at 9000 rpm for 30 rain. The supernatant was then centrifuged in a SW 28 rotor at 23000 rpm for ! h. The pellet was resuspended in 0.32 M sucrose with 1 mM EDTA (pH 7.4) and either assayed as sciatic nerve 'microsomes' or u ~ d for preparation of axolcmma. To prepare axolemma, the resuspended pellet was layered on a sucrose step gradient (1.2 M, ! M, and 0. 8 M) and centrifuged in a SW 28 rotor at 20000 rpm for 17 h. The three interface fractions were collected. The interfaces were diluted in ice-cold water and pelleted in a SW 28 rotor at 27500 rpm for 1 h. The three fractions were resuspended in 0.32 M sucrose with 1 mM EDTA (pH 7.4) for enzyme assays. Pellets of the axolemmal membrane fractions were also fixed in the centrifuge tube (polypropylene) and processed for conventional electron microscopy.
TABLE ! Diebetic aeMdiahetes-redstaatrats at time of death Two periods of instdin dependencywere examined,30 days (group A) and 16 weeks(group B). Values pre~nted far the two treatment groups arc mean_+S.E, for the number of animals indicated in parentheses. Not all data were obtained from all animals Parameter Age(week)
Group Diabetic (A) t8 +- 1(I7) (B) 28 +- 1(12)
Duration of diabetes (A) (B) Last insulindose(units) (A) (B) Weight(g)
CA) (B)
Blm~dglucose(rag%) (A) (B)
30 +_ 2 days 16 +_ 1weeks 1.7+_ ILl (17) 2.2+- 0.2 (10) ¢ 210 __.!1(17) 225 +_ 5(!i)
264 +4(21) ~'a 238 +_3(10)
n.d. ~ 323 +-37(7)
80 _+5(il) "'a
Glycaledhemoglobins(%) (A) 17.2+_ 2(5) (B) ill.! + i (7) Kidneyweight(g) CA) (B)
Control 22 +1(17)~ 27 +_I (I(I)
1.7+- 0.1 (7) 2.1 _+ 0.l (7)
8,3_+1.8(5) ''° 5.2+_0A(ll) ~.0 1+7+-0.1(6) 1.4__0.l (11)ax
" Significantlydifferent, P < 0.001 or less. ~' Significantlydifferent, P < 0.01. Excludingtwo animalswith initiallylowinsulin requirementswhich had graduallydecreasedto zero overthe courseof the experiment. Their averageblood glucose and glyeatedhemoglobinlevel~were 345 rag% and 12.8%.respectively. 8 Includingthree animals for which birth data were not available. Not determined,
Results
tained as low as possible, producing significant elevations of both blood glucose and glycated hemoglobins. Two animals in group B had initially low insulin requirements which gradually diminished so that they were receiving no insulin at the end of the experiment; nonetheless, they maintained a 4 + urinary, glucose, and both blood glucose and glycated hemoglobin levels were elevated. Renal hypertrophy was present only in the long-term diabetics; many group B rats also had renal pathology apparent on gross examination. In group A, the diabetes-resistant controls were slightly, but significantly, older and heavier than the diabetic animals.
Basic parameters of diabetic rats Diabetic rats were examined after either 4 weeks (group A} or 16 weeks (group B) of insulin dependency. Group B mortality during insulin treatment was 31%. Parameters of the experimental and control rats are presented in Table I. Insulin levels were main-
Studies of synthesis and degradation Synthesis and degradation of the Na +/K+-ATPase was studied in the retina of diabetic and control, diabetes-resistant rats. In group A, synthesis of both ~-1 and ' a ( + )' isoforms was apparently slightly higher in the diabetic rats than in controls, hut the difference
80 TABLE II
TABLE Iv
Retinal ~.ntfwsis of Na "/K +-ATPase afwr 4 weeks of itmdm-dependent diabetes melfims
.4xmzaU.r-tratul~rted Na "/K "-ATPase a st&unit isafonns recocercd fr~mz the SUl~'riorcoil(cullof rats dial had been diabeticfor four weeks and diabetes-resistantc+mtrolrats
Control and diabetic rats v,ere anesthetized vdth diethyl ether. [3sSiMethionine1251JgCi) was injected into the vitreal chamber ¢dth l'6"dr~tatic pressure. Rats ,~'ere killed alter L 18. 24 or 48 h and retinae ~+ere removed. Retinal Na"/K "-ATPa.~egas prepared and separated b~ $DS-PAGE. Da~aarc iulal ~SScpm imcan + S.E.) in gel bands of isolated Na "/K "-ATPasc frvm indMdual retinae analyzed SDS-PAGE and scinlillatlon counting Su~.-i',altim~ ~
CPM/gel band per retina alpha-I
alpha-2_'~
gel front b
Data are ~5S cpm (mean +_S.E.) in gel bands of isolated Na"/K ÷ATPa_se from indi,ddual superior colliculi analyzed by SDS-PAGE and scintillalhm counting Sm~b,al lime
CPM/gel band per retina alpha-I
alpha-23,
gel front
2h Control Diabelie
3~1-+ 8tl 5211+_200
1611+_ t0 3211+_110
490.+2611 15611+-850
2h Control Diabetic
53511*_ 8fill 35211± bt0 73310_+12320 6Iq~)+_i810 39211+_11~1 86320_+20410
24 h Control Diabetic
370+ 70 430-+ 811
1811_+ 20 L~! _+ 50
8611_+320 1I1211-+480
Ib h Control Diabetic
34411_+1320 65611+25111
23711_+ 7ill} 47770_+233111 48411+19511 996811+-411271)
~h Control Diabetic
4411_+ 511a 720_+ 160 +
1911_+ 20 290+_ 61)
17lgl+_~l} 2 130_+7511
24h Control Diabetic
54111+_111211 61~t-L- 1460
3591)+ 66t} 6702|1+_115811 3860+_ 84!1 82391)±!8 l.!!l
J Significantb"different. P < 0.05.
48 h Control Diabetic
241111+_ 8~1 c 16711± 7711 33"2.2(1_+13211} 53211± 7711'- 3310± 530 64100_+14800
Time elapsed bet~een intta~'itrcalinjection of [35Slmcthionineand sacrifice. Umesolved proteins M, < 7011110running at the gel front. Significantlydifferent. P < 0.05.
was not statistically significant. The effect of increased synthesis was maintained and became significant in early diabetics after 48 h (Table !I), possibly due to
TABLE Iii S~thesL~ and degradation of retinal Na +/K "-ATPa~w a subunit isofi~mts after I8 weeksof insulin-dependent diabetes mellirtts The data are as in Table !I. except for sur~'ivaltimes Sur~'i~'ai
time 2h Control Diabetic I day Control Diabetic 3 da)'s Control Diabetic 7 days Control Diabetic
CPM/gel band per retina alpha-I
alpha-22,
gel front
! 461)-+2911" 2 4711__.4110~
8011_+180 a 19911+_3311b
24750±47~) " 49 370 ±-7 350 b
1610+_45IIa 291(1.-+41t0 ~
1350_+350 19211+-430
Iq3311-+ 445[) 33650+59~1 a
1730+-450 13811+_260
128t1+460 950+_180
18690_+5170 15221)±3250
16~)±3fd) 2070+_531)
12211+_2511 1280+_390
172111+_451111 195911+ 6170
~ Sigcificantlydifferent. P < t1.115. " Significantlydifferent. P < 0.01.
slightly slower turnover in the diabetic retina. In rats that had been diabetic for 16 weeks, synthesis of a - I and ' a ( + )" was significantly increased 19_3% ( p < 0.011 and 69% ( P < 0 . 0 5 ) a t 2 h after intraretinal labeling (Table liD. Increased labeling was also apparent at 1 day, but the difference disappeared at 3 and 7 days after intraretinal labeling. T h e labeling ratio o f the two protein bands ( ' a ( + Y / a - t ) was lower than controls at 2 h, but higher at later times. Incorporation into proteins tanning at the gel front, M, < 70000, was also significantly increased ( P < 0.05) (Tables I! and 1111. The specific peptides whose synthesis was increased have not been identified. Higher labeling was also seen in N a + / K * - A T P a s e ~sonally-transported in the optic nerve from retinal ganglion cells to the superior eolliculus in group A diabetics (Table IV). The difference became significant for the "a( + )" isoform at 48 h after intraretinal labeling, reflecting the increased retinal synthesis and possibly slower degradation. Studies o f Na +/ K *-ATPase actM~" in tile kidno' The diabetic kidneys were significantly hypertrophic after 16 weeks (Table I). The activity of renal N a * / K + - A T P a s e assayed using the associated activity, the K*-dependent p-nitrophenyl phosphatase, was 53.4 tzmol p - n i t r o p h e n o l / h per mg protein (diabetic) compared to 19.8 ~tmol (control). Only the a - I isoform is present in the adult rat kidney, although the presence of other isoforms in the fetus is controversial [10]. In o r d e r to determine if the new activity induced by hypertrophy represented a different molecular isoform, the strophanthidin inhibition constants were determined (Fig. 1). Inhibition constants for control anti
>-
100-
80
<
60 x
o _ _ . ~
. . . .
2
_+.__,__...._,__,_____~__
3 4 -tOG [STROPHANTHIDIN]
Fig, 1. S t r o p h a n t h i d i n inhibition o f N a * / K ' ÷ A T P a s e isolated f r o m l h e kidneys o f rats that h a d b e e n d i a b e t i c for 16 w e e k s ( o ) a n d their d i a b e t e s - r e s i s t a n t e o n t m l s ( ' i t T h e d a t a a r e m e a n _ S . E . o f a mintm u m o f t h r e e d e t e r m i n a t i o n s al e a c h c o n c e n t r a t i o n p e r f o r m e d in duplicate.
diabetic hypertrophic kidney were not significantly different, 10-2 M and 4.10-3M, respectively. SDS-PAGE of the renal enzymes also showed no indication of a second isoform (Fig. 2). The gel does show that the preparation was contaminated with low molecular weight proteins. Since the strophanthidin inhibition constants determined for the K+-dependent p-nitrophenyl phosphatase were somewhat higher than those reported previously for Na*/K+-ATPase [4], we re-assayed enzyme activity with 10 -t M to 10 -~ M strophanthidin using the Na+/K÷-ATPase assay. The inhibition conslant shifted down as expected, and the K~ was approx. 6" 10 -4
M.
Na */ K +-ATPaseacth'ity in axolemma The Na+/K*-ATPase is less active when measured in vivo in peripheral nerves of diabetic rats [21], in order to determine if this difference might be partially l
Z
/=.'!". !:
3
4
:" ::i-:,.!:::::~-~ : :~::i::i~,~
:i/i~.
:
•
~:~.~,::
.
~
__~-
:--~
- -__
. ! :~::I:¸ L~
--
:
Fig. 2. Co~m~ssie blue-stained SDS-PAGE of Na~/K*-ATPa~e. Lane I is enzymeisolatedfrom kidneysof rats that had been diabetic for 16 ~'eeks: lane 2 is from kidneysof the diabetes-resistant controis. Lane 3 is Na~/K'-ATPase isolated from the cerebral cortex. which contains both the "a( + )" (upper) and a-I (lower) protein band. Lane 4 has molecularmassstandards. 116.5kD (upper) and 77 kDa (lo~er).
due to decreased enzyme activity in the axolemma, enzyme activity was measured in axolemma and axolemmal 'microsomes' isolated from control and diabetic sciatic nerves. The three fractions of axolemma prepared from 16-week diabetic and control rats appeared to be free of myelin by electron microscopy (not shown). No significant difference in total specific activity was measured between control and diabetic purified axolemma in two experiments, although the distribution differed. In control rats, 100% of ouabain-inhibited activity was recovered from the i). 8/1 M sucrose interface, whereas in diabetic rats, activity was divided 40:55 between the 0.8/1 M and 1/1.2 M interfaces, with 5% in the light 0.32/0.8 M interface. The significance of this difference is not clear at present. There was insufficient tissue to permit analysis of strophanthidin inhibition, Axolemmal 'microsomes' that probably contained some myelin were used to analyze p-nitrophenyl phosphatase activity in peripheral nerve after 4 weeks of insulin-dependent diabetes mellitus. Ouabain-inhibited activity was 6.45 + 1.12 nmol/h per mg protein (mean + S.E.) for 13 diabetic nerves and 4.95 + 0.47 for 15 diabetes-resistant nerves; these data are not significantly different. Discussion
The data demonstrate that retinal synthesis of Na pump isoforms is slowly altered when sub-optimal levels of insulin are administered to diabetic BB/Wor rats, supporting the hypothesis that insulin lack stimulates Na pump synthesis, although the effect is probably indirect. Synthesis of unidentified co-isolating lower molecular weight proteins was also increased. The trend was apparent after 1 month of insulin dependence and was significant by 4 months. Synthesis of both a isoform protein bands and 'front" proteins was increased after chronic insulin lack, with the larger effect being on synthesis of a-l. This suggests that although insulin has a greater effect on the sodium affinity of 'a(+)', a-I may be more responsive to synthesis regulation, preferential stimulation of a-I synthesis was also observed in studies with streptozotocin-diabetic rats after 6 weeks of disease (Specht, S.C. and del Vaile, E., unpublished data). The data are compatible with a hypothesis of decreased activity of both Na+/K*-ATPase isozymes in the BB/Wor rat, i.e., synthesis is increased in response to decreased activity. MacGregor and Matschinsky have measured Na+/K+-ATPase in individual retinal cell layers of alloxan-diabetic rabbits. Although no difference in activity was noted in retinal homogenates, enzyme activity is significantly decreased in the outer nuclear and plexiform layers [32]. in the rat, these layers contain both a-I and '¢d + )" [33].
Insulin is known to exert rapid, direct effects on gene transcription through upstream insulin response elements (IREs) [34]. The prolonged latency of changes in synthesis of Na+/K+-ATPase catalytic subunits in diabetic B B / W o r rats makes it unlikely that insulin is directly involved in these changes. Studies are ip progress with isoform-specific monoclonal antibodies to determine the abundance of the three isoforms by immunoprecipitation. Renal hypertrophy and increased Na+/K+-ATPase are observed within a few days of the induction of diabetes with streptozotocin. Khadouri et al. [20] have demonstrated that stimulation of collecting tubule Na~/K~-ATPase is secondary to increased levels of plasma aldosterone. Both hypertrophy and increased renal N a * / K + - A T P a s e developed more slowly in the diabetic B B / W o r rat; plasma aldosterone levels were not determined. We found no evidence that renal hype~rophy and increased N a * / K ~ - A T P a s e activity was associated with the appearance of a new renal isoform. Evidence abounds of impaired N a * / K ÷ - A T P a s e function in diabetic peripheral nerve, both in excised nerve segments and in nerve homogenates [21]. Axonal transport of new proteins into peripheral nerve is also impaired [35]. In order to examine whether the axolemmal Na+/K~-ATPase is intrinsically defective or whether its activity is impaired only by endogenous factors, such as the defective sorbitol/myo-inostol cycle, we measured N a ' / K * - A T P a s e activity in isolated axolemma. Although our conclusions are limited by the low yield of material from the peripheral nerves studied, the data suggest that once the enzyme and its lipo-protein environment is isolated from the soluble cellular contents, its activity is not different from that of control, diabetes-resistant rats. Acknowledgements We are grateful to Dennis Guberski, University of Massachusetts B B / W o r project, for invaluable advice on management of the diabetic rats. Ms. Sonia Soto provided excellent technical assistance. Electron microscopy was performed by Dr. Paula Orkand. The investigation was supported in part by National Institutes of Health (NIH) grants NS-07464 to Dr. R. Orkand and SO6-PR-08224 to Dr. E. Santiago Delpin. Facilities of the NIH Research Center for Minority Institutions (RR-03051) and the RIMI Neurochemistry Laboratory (NSF RII 8705802) were also utilized.
References I Robinson.J.D. and Flashner. M.S. (19791Biochim. Biophys.Acta 549. 145-176. 2 Sweadner. KJ.. Antonicek, H. and Schachacr, M. (1989) Sac. Ncurosci. Abstr, 15. 568. 3 Shull, G,E.. Gteeb. J. and Lingrel, J.B. (1986). Biochemistry 25, 812-8132, 4 Sweadner. KJ. (1979)J. BioLChem. 254, 6060-6067. 5 Sweadner. K3. (1985)L Biol. Chem. 260. !1508-11513. 6 Urayama. O_ Shun, H. and Sweadner, KJ. (19891J. Biol. Chem. 264, 8271-8280. 7 Hsu. Y.-M. and Guidotti. G. (19891 Biochemistry28, 569-573. 8 Hara, Y., Nika.'noto,A., Kojima, 1"., Matsummo. A. and Nakao, M (19881FEBS Lett. 238, 27-30. g Urayama. O. and Sv,'eadner, KJ. (1988) Biachcm. Biophys. Res. Commun. 156.786-800. i0 Swcadner, KJ. (1989) Biochim. Biophys.Acza 988, 185-220. II Sweadner, KJ. and Farshi, S.K. (19871 Proc. Natl. Acad. Sei. USA 84, 8404-8407. 12 Schmitl, CA. and McDonough, A.A. (19881J. Biol. Chem. 263. 17643-17649. 13 Ng. Y.-C., Yap. A.Z. and Akera, T. (19891 Am. J. Physiol. 257, H534-H539. 14 Hen.era, V.L.M., Chobanian, A.V. and Ruiz-Opazo, N. (1988) Science 241,221-223. 1:5 Kim, D., Barry, W.H. and Smith, T.W. (19841J. Pharmacol. Exp. Ther. 231,326-333. 16 Fambrough, D.M (19831Cold Spring Harbor SymposiumQuantilative Biology48, 297-304. 17 Clausen, T. (1986) Physio|. Rev. 66, 542-580. 18 L~ton, J. (19851J. Biol. Chem. 260, 10075-10080. t9 Young. R.M. and I ingrel, J.B. (19871 Biach,'m. Biophys. Res. Commun. 145.52-58. 20 Khadouri, C., Barlet-Bas, C. and Doucet, A. (1987) Pflugcrs Arch. 409, 296-301. 2t Greene. D.A., Lanimer. S.A. and Sima, A.A.F. (1988) Diabetes 37. 688-693. 22 Specht, S. (19891 Neurosci. Abstr. 15. 93. 23 Nakano. K., Mordes. J.P.. Handler, E.S., Greiner, D.U and Rossini. A.A. (1988) Diabetes. 37, 520-525. 24 S~'eadocr, KJ. (19781 Biochim. Biophys.Acta 508, 486-499. 25 Lov,w. O.H.. Rosebrough, NJ.. Fan'. A.L and Randall. R..I. (1951) J. BioL Chem. 193. 265-275. 26 Lacmmli,U~K.(19701Nature 227, 680-685. 27 Wilson, D.L., Hall, M.E., Stone, G.C. and Rubin, R.W (19771 Anal- Biochem.83.33-44. 28 Jorgensen, P.L and Skou. J.C. (19711 Biochim. Biophys. Acta 233. 366-380. 29 Robinson. J.D. (1969) Biochemistry8. 3348-3355. 30 Robinson. J.D_ (Iq67) Biochemistry6. 3250-3258. 31 Chou. ILH., Nolan, C.E. and Jungalwala, F.B. (19851 J. Neurochem. 44. 1898-t912. 32 MacGregor. L.C. and MaLschinsky,F.M. (t9861 L Biol. Chem. 261, 4052-4058. 33 McGrait. ILM. and Sweadner. KJ. (19891 Ear..L Neurosci. 2, 170-176. 34 Magnuson, M.A., Andreone. T.L, Prints. R.L. Koch, S. and Granner. D.IL (19891Proc. Natl. Acad. ,$ci. USA 86, 4838-4842. 35 Medori, R.. Jenich. H., Autilio-Gambetti, L. and Gambetti, P. (19881J. Nearosci. 8. 1814-1821.
77
•3 1991ElsevierSciencePublishersB.V. All rightsreserved0167.4838/91/$03.511
BBAPRO34(191
Molecular isoforms of Na +/K+-ATPase in the nervous system and kidney of the spontaneously diabetic BB/Wor rat Susan C. Specht, Jos~ Martin, R. Enid Gaud and Jost~ De Hoyos Department a]'Phannacology and Institute of Neurobiolo~,%Uoicersityof Puerto Rico, Sa~lJuan, PR (U.S.A.)
(Received23 April lqt~!) ( Revisedmanuscriptreceived 14 August 1~191)
Keywords: Isozyme:Isoform;Calalyticsubunil,a: Retina;AxtmalIrangport N a + / K +-ATPase was evaluated in the retina and kidney of the spontaneously diabetic BB/Wor rat after I and 4 months of insulin dependency. Retinal synthesis of the Na + / K +-ATPase was measured during a 2-h intravitreal pulse of [35S]methiunine and analyzed by SDS-PAGE and scintillation counting. Synthesis of the a-I and 'a( +)' (includes both a-2 and a-3) isofonns of the catalytic subunit was increased 123% and 69%, respectively at 4 months. Increases were also suggested at 1 month, but were not significant. The diabetes-dependent peak of synthesis m long.term diabetic rats turned over rapidly and by 3 days after intravitreai labeling, radioactively labeled enzyme was equal in both control and diabetic retinae. The amount of axonally transported, labeled enzyme recovered from endings of the optic nerve in the superior ¢olliculus paralleled retinal labeling. Significant renal hypertrophy (48%) was noted at 4 months, but not at 1 month. The strophanthidin-inhibition constant for diabetes-induced renal enzyme was the same as for control enzyme (approx. 10-4 M), indicating that diabetic renal hypertrophy does not induce a Na pump isozyme that is more sensitive to cardiotonic steroids. SDS-PAGE of the renal enzyme also failed to indicate more than one isoform of the a subunit.
Introduction
The Na+/K+-ATPase is a membrane protein which actively transports Na ÷ and K ~ across the membrane against their respective electrochemical gradients. It comprises two subunits, a and/~, the former possessing all known ligand binding sites and enzymatic activities (1). Although the physiological function of the /3 subunit remains to be established, in sequence and immunological reactivity it is homologou~ to the glial adhesion molecule AMOG (2). The Na+/K÷-ATPase is specifically inhibited by cardiac glycosides such as ouabain and strophanthidin. Several molecular isoforms of the a subunit have been described recently [3]. Of the three isoforms present in the rat, a-2 and a-3 are highly sensitive to ouabain and other cardiac glycosides (K~ = 10 -~ M to 10-7 M), whereas a-I is 10-
Abbreviations:IDDM.insulin-dependentdiabetesmellitus:DR. diabetes resistant. Correspondence: S.C. Specht. Institute of Neurobiology. 21)1 Blvd. Del Valle,San Juan, Puerto Rico1109111.USA.
1000-fold less sensitive (K i ffi 10 -5 M to 10 -3 M)[4,5]. In SDS-PAGE, the a-2 and a-3 isoforms run as a single band known as ~( + ) [4]. By the use of synthetic ot isoforms [6-8], as well as by partial tryptic digestion of ' a ( + ) ' [9] it has been shown that a-2 migrates slightly faster than ~-3, thus filling the lower part of the 'a( + )' gel band. Evidence is slowly accumulating that the ~ isoforms may be differentially induced by several physiological and pathological stimuli (reviewed in Ref. 10). For example, isoform ratios change during development in both heart [11] and brain [6,12]. Two rat models of hypertension show deinduction of mRNAs encoding for a2 and or3 with coordinate induction of mRNA for al (13). Thyroid hormone alters both isoform ratios in the adult ferret heart [14] and isoform abundance in neonatal brain [12], Several workers have shown that biosynthesis of the Na÷/K+-ATPase is up-regulated when enzymatic activity is partially inhibited by incubating cells with ouabain [15,16], lnsuIin is a well-known stimulator of Na+/K÷-ATPase enzyme activity [17] and may act in contrary fashion to down-regulate biosynthesis of Na+/K+-ATPase. Lytton reported that insulin stimu-
lates the activity of the Na+/K*-ATPase isozymes in rat adipocytes by increasing their affinity for Na ÷ [18]. The effect is greater on the "a( + r isoenzyme, with the Ko~ for Na ÷ decreasing 36% from 52 mM to 33 raM, whereas that of a-I decreases 18% from 17 mM to 14 raM. Although the change was greater for "t~(+ )', both changes were significant, it is not known whether insulin affects the Na + affinity, of both 0-2 and a-3, but Young and Lingrel have reported that adipocytes contain mRNA for only the a-2 isoform [19]. The experiments reported here were conducted to determine whether synthesis of the Na*/K *-ATPase is up-regulated when diabetes mellitus is treated with sub-optimal insulin, thus effecting a relative inhibition of Na*/K*-ATPase activity. We were particularly interested in determining whether increased synthesis would reflect the differential effect of insulin on the a and "a( + )" isoforms. In order to study isotorm synthesis, retinae of spontaneously diabetic BB and control BB-DR rats were labeled in vivo with [~S]methionine Since renal hypertrophy and increased Na*/K ÷ATPase are characteristic of diabetes meilitus induced by streptozotocin [20]. we also examined the kidneys of the BB/Wor rats to determine if renal hypertrophy occurs and whether it is associated with the appearance of a different Na'/K*-ATPase a isoform, in addition we examined Na+/K*-ATPase activity in peripheral nerve axolemma, in view of many reports of altered Na pump activity, in diabetic peripheral nerve [21]. Some of these data have been previously presented [22]. Materials and Methods
Animals. Diabetic and age-matched control diabetes-resistant female BB/Wor rats were obtained 3 weeks after detection of insulin-dependent diabetes mellitus (IDDM) from the NIH contract colony at the University of Massachusetts Medical School. Department of Pathology. The average age at detection was t 3 + 1 weeks. UrinatT glucose and ketones were checked daily by KetostLxR and insulin (NPH-40, Eli Lily) was administered daily after detection of IDDM by subcutaneous injection below the skin overlying the pectoral muscles. The treatment objective was to maintain urine sugar at 4 + with a moderate frequencT of ketosis. The daily dose varied from 0.6 to i.6 units at the onset and was increased according to need with growth. The BB/Wor rat utihzed in these studies is an inbred strain in which 40-70% of both sexes spontaneously develop IDDM, 85% of the conversions occurring between 60-120 days of age. The diabetic animals are insulin-dependent and exhibit frequent ketoacidosis. The natural control is the BB-diabetes resistant rat (BB-DR), a sub-strain with diabetic ancestors, The
disease appears to be an autoimmune disorder and can be induced in the BB-DR rat as well as in other susceptible strains by infusion of bone marrow cells from affected animals. Susceptibility is associated with the rat class 11 major histocompatibility complex [23]. lnrravitreal radioactive labeling. Rats were lightly anes'.hetized with either pentobarbital (10 mg/kg) supplemented with dietbyl ether or diethyl ether alone. The selera was punctured below the limbus with a sterile 26 G needle and a glass pipet containing 5 #l of [~- S]metmomne (60 #Ci; approx. 1200 Ci/mmol; New England Nuclear, Boston, MA) was inserted into the vitreal chamber. The solution was injected by application of hydrostatic pressure. Labeling was always conducted in the morning, before the scheduled daily insulin treatment. Animals were killed after 2 h in studies of synthesis or after several days in studies of enzyme degradation and axonal transport. The method of killing was decapitation following diethyl, ether ~ac~'.hcsia. Preparation of enzyme and SDS-PAGE. Na+/K +ATPase was prepared from radioactive retinae according to the method of Sweadner [24]. Protein concentrations were determined [3] using bovine serum albumin as a standard. Briefly, microsomes were prepared, then treated with SDS (0.3 rag/rag protein) and centrifuged through a 7-30% sucrose gradient. The enz~ane-enriched membranes were collected from the gradient, diluted in ice-cold water and a pellet was collected by eentrifugation at 1000O0 x g for 2 h 17 rain (o~ = 6.82- 10"~). The a isoforms were separated on a 5% sDS-polyacfflamide gel [26]. In an immunoblot protocol, the hands react with isoformspecific monoclonai antibodies developed by Sweadner [5]. Incorporation of 3-~Swas determined by cutting the appropriate bands from the wet gel and dissolving them [27] for scintillation counting. The results were corrected for radioactive decay between receipt of radioactive material and scintillation counting of labeled protein bands. Retinae were analyzed individually, Since the amount of protein in one retina was too low for visualization with Coomassie blue, 20 #g of Na +/K+-ATPase isolated from non-radioactive cerebral cortex was adde.d for SDS-PAGE. Results are reported in terms of radioactivity per gel band per retina. The superior colliculus was dissected to the depth of the cerebral aqueduct, a cut that includes the entire nucleus as well as some non-visual tissue. Each gel lane was loaded with approx. 50 #g of Na'/K÷-ATPase partially purified from superior colliculus, using several lanes to accommodate the entire sample, The average amount of protein loaded (mean +_S.E.) was 1O0 +_22/tg (control, ~ = 12) and l l0 _+ 17 v-g (diabetic, n -- 12). Data are reported for the entire superior colliculus sample, although the amount of protein may differ, since ra-
79 dioactive label is largely confined to optic nerve axons in the upper layers of the superior collicuiuso Preparation of kidney enzyme. Kidneys were removed from rats at the time of death, decapsulated, weighed, sliced, frozen in liquid nitrogen and stored at -70°(; until processed. For preparation of enzyme, the grey papilla was dissected from kidney slices and discarded; the entire remaining medulla and cortex was then minced, and enzyme was prepared according to the procedure of Jorgensen and Skou [28]. Enzyme assays. The Na+/K+-ATPase was usually assayed using an associated activity, the K+-dependent p-nitrophenyi phosphatase [29]. Strophanthidin was prepared in dimethylformamide. The enzyme and reactants except KCI or water were pre-incubated for 10 rain at 30°C. The reaction was started with their addition and allowcd to proceed for 20 rain, then stopped with ice-cold, 30% trichloroacetic acid. The final reaction volume was 0.25 ml and 0.7 to 1.6 pg enzyme protein were used per tube. ,~J! reaedens were performed in duplicate. Occasionally+ true Na+/K ÷ATPase activity was assayed [30]. In that case, no preincubation was employed. The reaction was performed at 37 o C for 10 rain. Enzyme protein per tube was approx. 40 #g. Preparation of sciatic nen'e axolemma. The method of Chou et al. [31] was employed with modifications. Briefly, the nerves were cleaned, homogenized in 0.29 M sucrose and centrifuged at 2400 rpm in a Beckman JA-20 rotor for 10 rain. The pellet was rinsed twice and the resultant supematant fractions were centrifuged at 9000 rpm for 30 rain. The supernatant was then centrifuged in a SW 28 rotor at 23000 rpm for ! h. The pellet was resuspended in 0.32 M sucrose with 1 mM EDTA (pH 7.4) and either assayed as sciatic nerve 'microsomes' or u ~ d for preparation of axolcmma. To prepare axolemma, the resuspended pellet was layered on a sucrose step gradient (1.2 M, ! M, and 0. 8 M) and centrifuged in a SW 28 rotor at 20000 rpm for 17 h. The three interface fractions were collected. The interfaces were diluted in ice-cold water and pelleted in a SW 28 rotor at 27500 rpm for 1 h. The three fractions were resuspended in 0.32 M sucrose with 1 mM EDTA (pH 7.4) for enzyme assays. Pellets of the axolemmal membrane fractions were also fixed in the centrifuge tube (polypropylene) and processed for conventional electron microscopy.
TABLE ! Diebetic aeMdiahetes-redstaatrats at time of death Two periods of instdin dependencywere examined,30 days (group A) and 16 weeks(group B). Values pre~nted far the two treatment groups arc mean_+S.E, for the number of animals indicated in parentheses. Not all data were obtained from all animals Parameter Age(week)
Group Diabetic (A) t8 +- 1(I7) (B) 28 +- 1(12)
Duration of diabetes (A) (B) Last insulindose(units) (A) (B) Weight(g)
CA) (B)
Blm~dglucose(rag%) (A) (B)
30 +_ 2 days 16 +_ 1weeks 1.7+_ ILl (17) 2.2+- 0.2 (10) ¢ 210 __.!1(17) 225 +_ 5(!i)
264 +4(21) ~'a 238 +_3(10)
n.d. ~ 323 +-37(7)
80 _+5(il) "'a
Glycaledhemoglobins(%) (A) 17.2+_ 2(5) (B) ill.! + i (7) Kidneyweight(g) CA) (B)
Control 22 +1(17)~ 27 +_I (I(I)
1.7+- 0.1 (7) 2.1 _+ 0.l (7)
8,3_+1.8(5) ''° 5.2+_0A(ll) ~.0 1+7+-0.1(6) 1.4__0.l (11)ax
" Significantlydifferent, P < 0.001 or less. ~' Significantlydifferent, P < 0.01. Excludingtwo animalswith initiallylowinsulin requirementswhich had graduallydecreasedto zero overthe courseof the experiment. Their averageblood glucose and glyeatedhemoglobinlevel~were 345 rag% and 12.8%.respectively. 8 Includingthree animals for which birth data were not available. Not determined,
Results
tained as low as possible, producing significant elevations of both blood glucose and glycated hemoglobins. Two animals in group B had initially low insulin requirements which gradually diminished so that they were receiving no insulin at the end of the experiment; nonetheless, they maintained a 4 + urinary, glucose, and both blood glucose and glycated hemoglobin levels were elevated. Renal hypertrophy was present only in the long-term diabetics; many group B rats also had renal pathology apparent on gross examination. In group A, the diabetes-resistant controls were slightly, but significantly, older and heavier than the diabetic animals.
Basic parameters of diabetic rats Diabetic rats were examined after either 4 weeks (group A} or 16 weeks (group B) of insulin dependency. Group B mortality during insulin treatment was 31%. Parameters of the experimental and control rats are presented in Table I. Insulin levels were main-
Studies of synthesis and degradation Synthesis and degradation of the Na +/K+-ATPase was studied in the retina of diabetic and control, diabetes-resistant rats. In group A, synthesis of both ~-1 and ' a ( + )' isoforms was apparently slightly higher in the diabetic rats than in controls, hut the difference
80 TABLE II
TABLE Iv
Retinal ~.ntfwsis of Na "/K +-ATPase afwr 4 weeks of itmdm-dependent diabetes melfims
.4xmzaU.r-tratul~rted Na "/K "-ATPase a st&unit isafonns recocercd fr~mz the SUl~'riorcoil(cullof rats dial had been diabeticfor four weeks and diabetes-resistantc+mtrolrats
Control and diabetic rats v,ere anesthetized vdth diethyl ether. [3sSiMethionine1251JgCi) was injected into the vitreal chamber ¢dth l'6"dr~tatic pressure. Rats ,~'ere killed alter L 18. 24 or 48 h and retinae ~+ere removed. Retinal Na"/K "-ATPa.~egas prepared and separated b~ $DS-PAGE. Da~aarc iulal ~SScpm imcan + S.E.) in gel bands of isolated Na "/K "-ATPasc frvm indMdual retinae analyzed SDS-PAGE and scinlillatlon counting Su~.-i',altim~ ~
CPM/gel band per retina alpha-I
alpha-2_'~
gel front b
Data are ~5S cpm (mean +_S.E.) in gel bands of isolated Na"/K ÷ATPa_se from indi,ddual superior colliculi analyzed by SDS-PAGE and scintillalhm counting Sm~b,al lime
CPM/gel band per retina alpha-I
alpha-23,
gel front
2h Control Diabelie
3~1-+ 8tl 5211+_200
1611+_ t0 3211+_110
490.+2611 15611+-850
2h Control Diabetic
53511*_ 8fill 35211± bt0 73310_+12320 6Iq~)+_i810 39211+_11~1 86320_+20410
24 h Control Diabetic
370+ 70 430-+ 811
1811_+ 20 L~! _+ 50
8611_+320 1I1211-+480
Ib h Control Diabetic
34411_+1320 65611+25111
23711_+ 7ill} 47770_+233111 48411+19511 996811+-411271)
~h Control Diabetic
4411_+ 511a 720_+ 160 +
1911_+ 20 290+_ 61)
17lgl+_~l} 2 130_+7511
24h Control Diabetic
54111+_111211 61~t-L- 1460
3591)+ 66t} 6702|1+_115811 3860+_ 84!1 82391)±!8 l.!!l
J Significantb"different. P < 0.05.
48 h Control Diabetic
241111+_ 8~1 c 16711± 7711 33"2.2(1_+13211} 53211± 7711'- 3310± 530 64100_+14800
Time elapsed bet~een intta~'itrcalinjection of [35Slmcthionineand sacrifice. Umesolved proteins M, < 7011110running at the gel front. Significantlydifferent. P < 0.05.
was not statistically significant. The effect of increased synthesis was maintained and became significant in early diabetics after 48 h (Table !I), possibly due to
TABLE Iii S~thesL~ and degradation of retinal Na +/K "-ATPa~w a subunit isofi~mts after I8 weeksof insulin-dependent diabetes mellirtts The data are as in Table !I. except for sur~'ivaltimes Sur~'i~'ai
time 2h Control Diabetic I day Control Diabetic 3 da)'s Control Diabetic 7 days Control Diabetic
CPM/gel band per retina alpha-I
alpha-22,
gel front
! 461)-+2911" 2 4711__.4110~
8011_+180 a 19911+_3311b
24750±47~) " 49 370 ±-7 350 b
1610+_45IIa 291(1.-+41t0 ~
1350_+350 19211+-430
Iq3311-+ 445[) 33650+59~1 a
1730+-450 13811+_260
128t1+460 950+_180
18690_+5170 15221)±3250
16~)±3fd) 2070+_531)
12211+_2511 1280+_390
172111+_451111 195911+ 6170
~ Sigcificantlydifferent. P < t1.115. " Significantlydifferent. P < 0.01.
slightly slower turnover in the diabetic retina. In rats that had been diabetic for 16 weeks, synthesis of a - I and ' a ( + )" was significantly increased 19_3% ( p < 0.011 and 69% ( P < 0 . 0 5 ) a t 2 h after intraretinal labeling (Table liD. Increased labeling was also apparent at 1 day, but the difference disappeared at 3 and 7 days after intraretinal labeling. T h e labeling ratio o f the two protein bands ( ' a ( + Y / a - t ) was lower than controls at 2 h, but higher at later times. Incorporation into proteins tanning at the gel front, M, < 70000, was also significantly increased ( P < 0.05) (Tables I! and 1111. The specific peptides whose synthesis was increased have not been identified. Higher labeling was also seen in N a + / K * - A T P a s e ~sonally-transported in the optic nerve from retinal ganglion cells to the superior eolliculus in group A diabetics (Table IV). The difference became significant for the "a( + )" isoform at 48 h after intraretinal labeling, reflecting the increased retinal synthesis and possibly slower degradation. Studies o f Na +/ K *-ATPase actM~" in tile kidno' The diabetic kidneys were significantly hypertrophic after 16 weeks (Table I). The activity of renal N a * / K + - A T P a s e assayed using the associated activity, the K*-dependent p-nitrophenyl phosphatase, was 53.4 tzmol p - n i t r o p h e n o l / h per mg protein (diabetic) compared to 19.8 ~tmol (control). Only the a - I isoform is present in the adult rat kidney, although the presence of other isoforms in the fetus is controversial [10]. In o r d e r to determine if the new activity induced by hypertrophy represented a different molecular isoform, the strophanthidin inhibition constants were determined (Fig. 1). Inhibition constants for control anti
>-
100-
80
<
60 x
o _ _ . ~
. . . .
2
_+.__,__...._,__,_____~__
3 4 -tOG [STROPHANTHIDIN]
Fig, 1. S t r o p h a n t h i d i n inhibition o f N a * / K ' ÷ A T P a s e isolated f r o m l h e kidneys o f rats that h a d b e e n d i a b e t i c for 16 w e e k s ( o ) a n d their d i a b e t e s - r e s i s t a n t e o n t m l s ( ' i t T h e d a t a a r e m e a n _ S . E . o f a mintm u m o f t h r e e d e t e r m i n a t i o n s al e a c h c o n c e n t r a t i o n p e r f o r m e d in duplicate.
diabetic hypertrophic kidney were not significantly different, 10-2 M and 4.10-3M, respectively. SDS-PAGE of the renal enzymes also showed no indication of a second isoform (Fig. 2). The gel does show that the preparation was contaminated with low molecular weight proteins. Since the strophanthidin inhibition constants determined for the K+-dependent p-nitrophenyl phosphatase were somewhat higher than those reported previously for Na*/K+-ATPase [4], we re-assayed enzyme activity with 10 -t M to 10 -~ M strophanthidin using the Na+/K÷-ATPase assay. The inhibition conslant shifted down as expected, and the K~ was approx. 6" 10 -4
M.
Na */ K +-ATPaseacth'ity in axolemma The Na+/K*-ATPase is less active when measured in vivo in peripheral nerves of diabetic rats [21], in order to determine if this difference might be partially l
Z
/=.'!". !:
3
4
:" ::i-:,.!:::::~-~ : :~::i::i~,~
:i/i~.
:
•
~:~.~,::
.
~
__~-
:--~
- -__
. ! :~::I:¸ L~
--
:
Fig. 2. Co~m~ssie blue-stained SDS-PAGE of Na~/K*-ATPa~e. Lane I is enzymeisolatedfrom kidneysof rats that had been diabetic for 16 ~'eeks: lane 2 is from kidneysof the diabetes-resistant controis. Lane 3 is Na~/K'-ATPase isolated from the cerebral cortex. which contains both the "a( + )" (upper) and a-I (lower) protein band. Lane 4 has molecularmassstandards. 116.5kD (upper) and 77 kDa (lo~er).
due to decreased enzyme activity in the axolemma, enzyme activity was measured in axolemma and axolemmal 'microsomes' isolated from control and diabetic sciatic nerves. The three fractions of axolemma prepared from 16-week diabetic and control rats appeared to be free of myelin by electron microscopy (not shown). No significant difference in total specific activity was measured between control and diabetic purified axolemma in two experiments, although the distribution differed. In control rats, 100% of ouabain-inhibited activity was recovered from the i). 8/1 M sucrose interface, whereas in diabetic rats, activity was divided 40:55 between the 0.8/1 M and 1/1.2 M interfaces, with 5% in the light 0.32/0.8 M interface. The significance of this difference is not clear at present. There was insufficient tissue to permit analysis of strophanthidin inhibition, Axolemmal 'microsomes' that probably contained some myelin were used to analyze p-nitrophenyl phosphatase activity in peripheral nerve after 4 weeks of insulin-dependent diabetes mellitus. Ouabain-inhibited activity was 6.45 + 1.12 nmol/h per mg protein (mean + S.E.) for 13 diabetic nerves and 4.95 + 0.47 for 15 diabetes-resistant nerves; these data are not significantly different. Discussion
The data demonstrate that retinal synthesis of Na pump isoforms is slowly altered when sub-optimal levels of insulin are administered to diabetic BB/Wor rats, supporting the hypothesis that insulin lack stimulates Na pump synthesis, although the effect is probably indirect. Synthesis of unidentified co-isolating lower molecular weight proteins was also increased. The trend was apparent after 1 month of insulin dependence and was significant by 4 months. Synthesis of both a isoform protein bands and 'front" proteins was increased after chronic insulin lack, with the larger effect being on synthesis of a-l. This suggests that although insulin has a greater effect on the sodium affinity of 'a(+)', a-I may be more responsive to synthesis regulation, preferential stimulation of a-I synthesis was also observed in studies with streptozotocin-diabetic rats after 6 weeks of disease (Specht, S.C. and del Vaile, E., unpublished data). The data are compatible with a hypothesis of decreased activity of both Na+/K*-ATPase isozymes in the BB/Wor rat, i.e., synthesis is increased in response to decreased activity. MacGregor and Matschinsky have measured Na+/K+-ATPase in individual retinal cell layers of alloxan-diabetic rabbits. Although no difference in activity was noted in retinal homogenates, enzyme activity is significantly decreased in the outer nuclear and plexiform layers [32]. in the rat, these layers contain both a-I and '¢d + )" [33].
Insulin is known to exert rapid, direct effects on gene transcription through upstream insulin response elements (IREs) [34]. The prolonged latency of changes in synthesis of Na+/K+-ATPase catalytic subunits in diabetic B B / W o r rats makes it unlikely that insulin is directly involved in these changes. Studies are ip progress with isoform-specific monoclonal antibodies to determine the abundance of the three isoforms by immunoprecipitation. Renal hypertrophy and increased Na+/K+-ATPase are observed within a few days of the induction of diabetes with streptozotocin. Khadouri et al. [20] have demonstrated that stimulation of collecting tubule Na~/K~-ATPase is secondary to increased levels of plasma aldosterone. Both hypertrophy and increased renal N a * / K + - A T P a s e developed more slowly in the diabetic B B / W o r rat; plasma aldosterone levels were not determined. We found no evidence that renal hype~rophy and increased N a * / K ~ - A T P a s e activity was associated with the appearance of a new renal isoform. Evidence abounds of impaired N a * / K ÷ - A T P a s e function in diabetic peripheral nerve, both in excised nerve segments and in nerve homogenates [21]. Axonal transport of new proteins into peripheral nerve is also impaired [35]. In order to examine whether the axolemmal Na+/K~-ATPase is intrinsically defective or whether its activity is impaired only by endogenous factors, such as the defective sorbitol/myo-inostol cycle, we measured N a ' / K * - A T P a s e activity in isolated axolemma. Although our conclusions are limited by the low yield of material from the peripheral nerves studied, the data suggest that once the enzyme and its lipo-protein environment is isolated from the soluble cellular contents, its activity is not different from that of control, diabetes-resistant rats. Acknowledgements We are grateful to Dennis Guberski, University of Massachusetts B B / W o r project, for invaluable advice on management of the diabetic rats. Ms. Sonia Soto provided excellent technical assistance. Electron microscopy was performed by Dr. Paula Orkand. The investigation was supported in part by National Institutes of Health (NIH) grants NS-07464 to Dr. R. Orkand and SO6-PR-08224 to Dr. E. Santiago Delpin. Facilities of the NIH Research Center for Minority Institutions (RR-03051) and the RIMI Neurochemistry Laboratory (NSF RII 8705802) were also utilized.
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