Kinetics of ascorbate transport by cultured retinal capillary pericytes ...

Kinetics of Ascorbate Transport by CulturedRetinal Capillary Pericytes

Inhibition by Glucose

Mahin Khatami, Weiye Li, and John H. Rockey

Accumulation of radioactive L-[carboxyl-14C]-ascorbic acid by cultured bovine retinal capillary pericyteswas studied. Kinetic analysis of the transport showed a time-dependent, saturable system with an apparentKm of 76.0 juM and a V ^ of 42 pmole//Ltg DNA/min. A facilitated carrier diffusion process was establishedon the basis that the system was not sensitive to 2,4-dinitrophenol, ouabain, or reduced sodium concen-tration in the incubation media, and that the carrier system demonstrated stereospecificity for an ascorbateanalogue, dehydroascorbate, and for sugar analogues such as a-D-glucose and 3-0-methyl-D-glucose(3-0-MG), but not for /3-D-fructose or L-glucose. Transport of ascorbate by cultured pericytes wasinsulin-insensitive. 3-0-Methyl-D-glucose inhibited ascorbate transport into pericytes in a non-competitivemanner with a Kt of 22 mM. These results indicate that, in cultured retinal capillary pericytes, a commonfacilitated carrier diffusion system is involved in the transport of ascorbate and sugar analogues suchas a-D-glucose or 3-0-MG. Invest Ophthalmol Vis Sci 27:1665-1671, 1986

Retinal damage by chemical insults, such as hyper-glycemia, or by light may be influenced by vital me-tabolites, such as ascorbic acid. Ascorbic acid, a carbo-hydrate vitamin, is an electron donor and antioxidant,and is involved in many hydroxylation reactions andbiosynthetic pathways, such as dopamine /3-hydrox-ylation and the synthesis of collagen and glycosami-noglycans.1"5 Its biochemical role in vascular disease(e.g., diabetic microangiopathy), however, is not com-pletely understood. Alterations in the biosynthesis ofcapillary basement membrane components (e.g., col-lagen, glycosaminoglycans) may contribute to the de-fective function and pathological changes of micro-vascular basement membranes seen in diabetic mi-croangiopathy.67

There is increasing evidence that insulin facilitatesthe transport and distribution of vitamin C in a varietyof tissues similar to its effect on glucose, and that insulin

From the Department of Ophthalmology, Scheie Eye Institute,University of Pennsylvania School of Medicine, Philadelphia, Penn-sylvania.

Supported by U.S. Public Health Service grants EY-03984 andEY-07035, by a Juvenile Diabetes Foundation International Fellow-ship (W.L.) and Grant, by Fight for Sight, Inc., New York City, byan unrestricted grant from Research to Prevent Blindness, Inc., NewYork City, and by the Elaine O. Weiner Teaching and ResearchFund.

Submitted for publication: April 23, 1985.Reprint requests: Dr. Mahin Khatami, Scheie Eye Institute, 51

North 39th Street, Philadelphia, PA 19104.

deficiency and/or hyperglycemia may interfere with thetransport and metabolism of vitamin C.8"14 Severalocular tissues may be independent of insulin,1516 andthe mechanism of ascorbate transport in tissues whichare sensitive or non-sensitive to insulin may be differ-ent.11"141718 Mann8 has suggested that species that donot synthesize ascorbic acid (e.g., human, monkey,guinea pig) and require dietary ascorbate must alsohave a mechanism for transporting this vitamin intocells. Hyperglycemia may interfere with vitamin Ctransport, and diabetic animals that require exogenousascorbic acid may have a double disadvantage.

Extensive publications deal with the mechanism ofhexose transport across the blood retinal barrier19'20

and in isolated retinal micro vessels21 or pigment epi-thelium.22"25 Recent studies also have demonstratedthe presence of a facilitated diffusion system for glucosetransport in cultured bovine retinal capillary pericytes15

and endothelial cells.26 The presence of active transportsystems for ascorbate in rat whole retina27 and in guineapig ciliary body-iris28 have been reported. However,nothing is known about the mechanism(s) by whichascorbate is transported into retinal vessel cells.Whether glucose interferes with ascorbate transport andfunction in capillary cells also is an open question.

The present studies establish the kinetics of ascorbatetransport into cultured bovine retinal microvesselpericytes. Evidence is presented for a facilitated dif-fusion, insulin-independent system which is inhibitableby a-D-glucose and its analogues.

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1666 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / November 1986 Vol. 27

Materials and Methods

Preparation of Bovine Retinal Microvessels

Retinal microvessels from fresh bovine eyes wereprepared as previously described,29'30 with minor mod-ifications. Retinal homogenates, prepared in DulbeccoModified Eagle Medium (DMEM) containing 20% fetalcalf serum, were applied to a 55 /nm mesh size nylonscreen under aseptic conditions and washed thoroughlywith saline. Microvessels were collected only from theback side of the nylon mesh, and assessed for purityby phase contrast and differential interference micros-copy.29'30

Pericyte Culture

Pericytes were isolated from microvessels as previ-ously described.29'30 Cells were cultured in 60 or 100mm plastic Petri dishes at a density of approximately1 X 104 cells/cm2 in DMEM supplemented with 20%fetal calf serum, 100 U/ml penicillin, and 100 fig/m\ascorbic acid (Medium). Cells at confluency were tryp-sinized with 0.25% trypsin in a Ca++-Mg++-free saltsolution containing 6 mM KC1, 154 mM NaCl, 1.2mM NaHCO3,0.83 mM NaH2PO4, 1 mM EDTA, pH8.0, at 25°C for 2 min. Trypsinized cells were addedto five volumes of Medium (over trypsin solution vol-ume) and centrifuged at 1,000 rpm for 10 min at 25 °C.Sedimented cells were taken up in Medium, replatedin Petri dishes, and incubated at 37°C under 95% air-5% CO2. The Medium was changed every 2-3 days.Confluent cells were collected every 4-6 days and re-plated as above. Pericytes at passage 9-11, grown inmultiwell plates, were used for the majority of the pres-ent studies. In selected experiments, cells were used atpassage 20-25.

Ascorbate Uptake by Pericytes

L-Ascorbic acid (sodium salt, Sigma Chemical Co.,St. Louis, MO) was dissolved in balanced salt solution(BSS) composed of 150 mM NaCl, 4.2 mM KC1, 1.0mM Na phosphate, 0.7 mM MgCl2, 2.0 mM CaCl2,and 10 mM Hepes buffer, pH 7.4. Prior to the exper-iments, 0.5-5.0 ^Ci/ml of lyophyilized L-[carboxyl-l4C]-ascorbic acid (specific activity 17 mCi/mmole,98% purity, Amersham, Arlington Heights, IL) wasdissolved in BSS and mixed with the freshly preparedunlabeled ascorbate solution (0.5-5 mM) in the pres-ence or absence of 1-10 mM thiourea. Radioactiveascorbate solutions were either used immediately orkept frozen (—20°C) under nitrogen in light-tight con-tainers for up to 7 days. Confluent pericytes werewashed with BSS (2X, 2 ml each), and the abscorbatetransport was initiated by the addition of 10-1,000 ^M(final concentration) radioactive ascorbate. Cells wereincubated for selected periods at 37°C in a water bath

with gentle automatic shaking under atmospheric ox-ygen. Additional dishes were incubated at 4°C. Ascor-bate uptake was terminated by aspirating the mediaand immediately washing (5X) the cells with ice-coldBSS. The total time for washing was 2 min or less.Cells were extracted with 0.1 ml of 0.2 N NaOH, andthe extract was transferred into 5 ml of Ultrafluor (Na-tional Diagnostic, Somerville, NJ) for radiometry. Inselected experiments, cells were incubated in the pres-ence of 150 mM LiCl or 150 mM choline-Cl in placeof 150 mM NaCl in BSS.

Effects of Sugars and Other Compounds onAscorbate Transport

Cultured pericytes at confluency were incubated inBSS in the presence of 0-20 mM 3-0-methyl-D-glu-copyranoside (3-0-MG; Sigma Chemical Co., St. Louis,MO). Radioactive L-ascorbate at selected concentra-tions (50, 200, or 500 nM) was added 30 sec after theaddition of 3-0-MG, and incubated as described above.In a series of similar experiments, either a-D-glucose,5-thio-D-glucopyranoside (5-TDG), L-glucose, (3-D-fructose or myo-inositol were used in place of 3-0-MG.In addition, the effect of phloretin and phlorizin, in-hibitors of glucose transport; dehydroascorbate (DHA),an ascorbate analogue; 2,4-dinitrophenol (DNP), ametabolic inhibitor; ouabain, an inhibitor of Na+-K+-ATPase; and insulin on ascorbate uptake were studied.

Analysis of Radioactive Samples

Radioactive samples obtained from incubation me-dia were analyzed in a liquid scintillation spectrometer.Radioactive components also were spotted on silica gelplates (20 X 20 cm, LK6DG linear-K, Whatman, Clif-ton, NJ) in the presence or absence of thiourea. Ra-dioactive and non-radioactive ascorbate, dehydroas-corbate (DHA, ICN, Nutritional Biochemical, Cleve-land, OH), and D-isoascorbate (DIA; Sigma ChemicalCo., St. Louis, MO) were run in parallel. Chromatog-raphy was performed in benzene:methanol:acetic acid:acetone (20:20:80:68), and ascorbate and DIA spotswere developed by silver nitrate (1% in acetone) spray.31

DHA spots were identifiable after 10 min heating at65°C. Plates were scraped (0.5 cm fractions), and theradioactivity was determined.

Cell Counts, DNA Measurements, andDetermination of Cellular Water

Representative cultured pericytes at confluency wereused to count viable cells with a hemocytometer in thepresence of Trypan blue.30 DNA content per culturedish also was measured in parallel as previously de-scribed.15 The intracellular water space of culturedpericytes was determined as previously described using[3H]-3-0-MG.32


No. 11 ASCORBATE TRANSPORT BY PERICYTES IS INHIBITED BY GLUCOSE / Khoromi er ol. 1667

Results

Kinetics of Ascorbate Transport byCultured Pericytes

Confluent pericytes, incubated in the presence of 200jtiM radioactive ascorbate, progressively accumulatedascorbate (Fig. 1). The uptake was essentially linear upto 10 min. The linearity of ascorbate uptake was similarwhen cells were incubated in the presence of 50 or 500nM of radioactive ascorbate (data not shown).

Cells incubated in the presence of varying concen-trations of radioactive ascorbate exhibited saturationkinetics (Fig. 2). From the initial velocities, it was foundthat the uptake followed Michaelis-Menton kinetics,assuming a steady state. The kinetic Constance (Km)for the ascorbate uptake was determined from a Line-weaver-Burk transformation of the Michaelis-Mentonequation (Fig. 2, inset). From the plots of the reciprocalof the initial velocity vs the reciprocal of the ascorbateconcentration, an apparent half maximal velocity wasfound at 76.0 A*M of ascorbate (Km) with a maximalvelocity (Vmax) of 42 pmole/^g DNA/min. In addition,a plot of the ratio of the ascorbate concentration to thecorresponding velocity ([S]/V) vs the ascorbate con-centration was linear, and gave a Km/Vmax (interceptof the [S]/V axis) ratio of 1.92. From this plot, Vmax

(1/slope) was 40 pmole/^g DNA/min.Cells incubated at 4°C in the presence of 50 or 200

nM ascorbate showed a significant reduction in theirability to accumulate ascorbate when compared tocontrol experiments carried out at 37°C (Table 1).

Analysis of Radioactive Samples

Radioactive ascorbate from the incubation mediaremained in the reduced form (>70%) when analyzedup to 24 hr (Fig. 3). Radioactive stock solutions ofascorbate, as well as non-radioactive samples at higherconcentrations (^100 mg%), were found to be morestable during the storage period than diluted samples(1-10 mg%). The TLC analysis showed that ascorbatein stock solutions, when kept frozen (-20°C) up to 7days under nitrogen, remained in its reduced form(>90%). Longer storage of ascorbate, particularly indilute solutions, produced oxidized products detectedby TLC (Fig. 3). Freshly prepared radioactive ascorbatein the presence or absence of thiourea showed similaruptake by pericytes (Table 1).

Effects of Sugars and Other Compounds onAscorbate Uptake

Confluent pericytes, incubated at 37°C in the pres-ence of increasing concentrations of 3-0-MG, showeda progressive reduction in the uptake of ascorbate atfixed concentrations (50, 200, or 500 /JM) present in

Fig. 1. Time course of as-corbate accumulation bypericytes. Cultured pericyteswere incubated in the pres-ence of 200 nM (2 mCi/mmole) radioactive ascorbatefor various periods of timesfollowed by washing withbalanced salt solution. Eachpoint is the mean of 3-6 de-terminations ± SD.

0.4

o 0.3 -

0.2

10 20Minutes

30

the media. Figure 4 is a Dixon plot of the reciprocalvelocities for ascorbate uptake plotted against 3-0-MGconcentration. The y-axis of Figure 4 shows the recip-rocal velocities in the absence of 3-0-MG. The inhi-bition of ascorbate uptake, at fixed concentrations, byincreasing concentrations of 3-0-MG was characteristicof a non-competitive inhibition.

Alpha-D-Glucose similarly inhibited the transportof ascorbate into cultured pericytes. Figure 5 shows the.extent of ascorbate uptake (50 /xM extracellular con-centration) in the presence of increasing concentrationsof a-D-glucose or 3-0-MG. Confluent cells at passage20-25 also showed a similar pattern of uptake of as-corbate and inhibition by glucose.

Phloretin (0.1 mM) or phlorizin (0.4 mM) signifi-cantly reduced the net ascorbate uptake by pericytes(Table 1). In contrast to the effect of a-D-glucose or 3-

0 200 400 600 800 1000Ascorbate concentration

Fig. 2. Ascorbate transport by pericytes as a function of extracellularradioactive ascorbate concentrations. Cultured pericytes were incu-bated in the presence of varying radioactive ascorbate concentrationsfor 5 min at 37°C. From Lineweaver-Burk plcjt a kinetic Constance(Km) of 76 fiM was obtained (inset). Maximum velocity (Vmax) forascorbate uptake was found to be 42 pmole/min//tgDNA. Each pointis the mean of 3-6 determinations ± SD.



Table 1. Effect of various compounds on ascorbate uptake by cultured retinal capillary pericytes

Additions Concentration Relative uptake Comments

1) NoneThioureaInsulinDehydroascorbate

5-Thio-D-glucosePhloretin2,4-DinitrophenolOuabainNone (4°C)t

2) NonePhlorizinwyolnositolDehydroascorbate2,4-Dinitrophenol

InsulinLiClijCholine-d£0-D-FructoseL-GlucoseNone (4°QJ

10 mM1000 MlU/ml

1 mM10 mM5mM

0.1 mM0.2 mM

1 mM

0.4 mM10 mM10 mM1 mM2mM

2500 jiIU/ml150 mM150 mM10 mM10 mM

1.001.09 ±0.100.98 ±0.050.77 ±0.05*0.39±0.09f0.60 ±0.10*0.11 ±0.06f0.99 ±0.080.88 ±0.090.13±0.09t

1.000.56 ±0.09*0.94 ±0.080.27±0.08f1.1 ±0.151.09 ±0.101.2 ±0.121.05 ±0.090.91 ±0.081.05 ±0.120.89 ±0.090.17±0.10f

1) Extracellular concentration of radioactive ascorbatewas 50 MM.

2) Extracellular concentration of radioactive ascorbatewas 200 MM.

* Differed significantly from control, P<0.0l.t^<0.001.i Cells were incubated with radioactive ascorbate at 4°C.

| The 150 mM NaCl in the incubation media was replaced with either LiClor choline-Cl.

0-MG, jS-fructose and L-glucose did not have a signif-icant effect on the uptake of ascorbate (Table 1). De-hydroascorbate significantly reduced the accumulationof ascorbate, and the inhibitory effect of DHA wasconcentration dependent (Table 1). rayo-Inositol at 10mM did not change the level of radioactive ascorbateuptake by pericytes (Table 1). The transport of ascor-

bate into pericytes was energy-independent, and a re-duced sodium ion concentration, or the presence ofeither ouabain (1 mM) or DNP at different concentra-tions in the incubation media, did not significantly af-fect ascorbate uptake (Table 1). When pericytes wereincubated for up to 40 min with low concentrations ofascorbate (5 /xM), the ratio of intracellular to extracel-lular concentration of radioactive ascorbate never ex-

8r

0 | 5 10Dehydroascorbate

15 20

Distance

25 30Ascorbate or

D-isoascorbate

35

Fig. 3. Analysis of radioactive ascorbate by thin layer chromatog-raphy (TLC). Radioactive samples obtained from incubation media,as well as stock solutions of radioactive and non-radioactive ascorbate,D-isoascorbate, or dehydroascorbate were analyzed by TLC. 1, As-corbate standard solutions (100 mg%) analyzed up to 7 days. 2, Ra-dioactive ascorbate (200 nM) taken from the incubation media. 3,Ascorbate solution stored for more than 3 weeks. Identification ofascorbate, D-isoascorbate, and dehydroascorbate spots also are shownafter color development.

-20 -15 -10 - 5 0 53-0-Methyl-glucose (mM)

10 15 20

Fig. 4. Dixon Plots of inhibition of ascorbate uptake by 3-0-MG.Pericytes were incubated at 37°C in the presence of increasing con-centrations of 3-0-MG for 30 sec. Additional 10 min incubation oc-curred in the presence of radioactive ascorbate at 50, 200, or 500/iM. Plots of reciprocal velocities of ascorbate uptake at fixed con-centrations vs inhibitor (3-0-MG) at increasing concentrations. TheK; for inhibition of ascorbate uptake was 22 mM. Each point is themean of 3-6 determinations ± SD.


No. 11 ASCORBATE TRANSPORT BY PERICYTES IS INHIBITED BY GLUCOSE / Khatami er al. 1669

ceeded unity, indicating that ascorbate was not accu-mulating against a concentration gradient.

Discussion

The present studies indicate that a facilitated diffu-sion system mediates the transport of L-ascorbate intocultured retinal capillary pericytes on the basis of thefollowing observations: 1) the system is saturable; 2)the carrier requires physiological temperature to me-diate ascorbate transport; 3) the carrier demonstratesstereospecificity for ascorbate analogues, such as de-hydroascorbate, and for sugars, such as a-D-glucoseand its non-metabolizable analogue, 3-0-methyl-D-glucose, but not for fructose or L-glucose; 4) the systemdoes not require metabolic energy and is not dependenton the sodium concentration in the external media;and 5) ascorbate does not accumulate against a con-centration gradient.

The time course of ascorbate transport, shown inthe present studies, as well as in those reported for cul-tured chromaffin cells,33 adrenal cortex and brain cor-tex,34 and cultured retinal pigment epithelial cells,35 ismuch slower than the time course reported for trans-port of dehydroascorbate into red blood cells,18 neu-trophils, and fibroblasts.12 The oxidized form of as-corbate, dehydroascorbate, is a non-ionized carbohy-drate and may cross the plasma membrane of cellsmore rapidly than the reduced ascorbate. The timecourse for dehydroascorbate transport into several tis-sues is comparable to that of glucose.1214'36 Reducedascorbate is an anion and may enter the cellular barriersas a neutral species by its interaction with monovalentcations, such as Na+ or K+ ions. In the present studies,we have presented evidence that the replacement of150 mM NaCl in the media by 150 raM LiCl or cholineCl did not affect the ascorbate transport into pericytes.This reflects the fact that the uptake does not involvean active transport system that depends on a sodiumgradient established by an Na+-K+-ATPase system.However, since the ascorbate concentration in the me-dia is in the micromolar range, the reduced extracellularsodium concentrations may not affect the interactionof the remaining Na+ (> 1 mM) present in the mediain the form of Na phosphate, as well as Na ascorbate,which were used in the experiments. The neutral so-dium (or potassium)-ascorbate species perhaps bindsto the carrier, which then is internalized. Alternatively,ascorbate-free radical, semidehydroascorbate, which isformed by partial autoxidation of ascorbic acid in so-lutions, may electrostatically bind to specific site(s) onthe carrier molecule.37"39

D-glucose and its non-metabolizable analogues, 3-O-methyl-D-glucose and 5-thio-D-glucose, but notfructose or L-glucose, inhibited the transport of ascor-

Fig. 5. Effect of a-D-glu-cose or 3-0-MG on ascorbatetransport in pericytes. Theextent of transport of radio-active ascorbate (50 /xM ex-tracellular concentration)into pericytes in the pres-ence of increasing concen-trations of either a-D-glucose(A A) or 3-0-MG(• •)inBSS(37°C,airatmosphere, 10 min incuba-tion). Each point is the meanof 3-6 determinations ± SD.

100

80

60

40

20

5 10 15[a-D-glucose] or[3-0-MG] mM

20

bate into pericytes. The fact that the non-metabolizableanalogues of glucose similarly inhibited ascorbate up-take into pericytes demonstrated that the inhibitoryeffect was not due to the enhanced metabolic productsof glucose, but was due to a stereospecific inhibitionof the ascorbate transporter by glucose. Ascorbate andD-glucose, therefore, may share a common carriermechanism for transport into retinal microvascularpericytes. The kinetics of inhibition of ascorbate by 3-0-MG indicated that the inhibition was noncompeti-tive. Binding of glucose to the carrier may allostericallyinterfere with subsequent ascorbate binding. The Km

values for the uptake of glucose and ascorbate are verydifferent (76 nM for ascorbate and 1.53 mM for glu-cose15). Glucose at 200 times the concentration of as-corbate was able to inhibit ascorbate uptake 50-60%.Therefore, the affinity and/or efficiency of the carrierfor ascorbate appears to be greater than for glucose.Normal plasma concentrations of glucose are in the4-6 mM range, and of ascorbate are in the 30-100 fiMrange.

Transport of ascorbate into cultured bovine capillarypericytes was not dependent on insulin. Similarly, aswe have recently reported, insulin did not have anysignificant effect on the glucose transport system incultured pericytes.15 This further supports the conclu-sion that, in pericytes, ascorbate and glucose maybe taken up by the same facilitated diffusion carriersystem.

We have recently reported that glucose, but not itsnon-metabolizable analogues, inhibited rayo-inositoluptake by cultured pericytes in a non-competitivemanner, and that the inhibition of myoinositol uptakewas reversed by Sorbinil, and we have concluded thatglucose inhibitied myoinositol uptake through thesorbitol pathway.32 In the present studies, we have ob-served that mj/oinositol at 10 mM concentration didnot have any significant effect on ascorbate uptake,whereas glucose and its non-metabolizable analoguessignificantly inhibited ascorbate transport into peri-



cytes. These observations support the conclusion that,in pericytes, glucose and ascorbate may share the samecarrier system, which is different from that for myo-inositol.

Glucose at physiological concentrations inhibited thetransport of ascorbate, but a significantly higher percentinhibition of uptake was obtained when the glucoseconcentrations were increased to those occurring indiabetes mellitus. The ascorbate transport system, inthe presence of hyperglycemic concentrations of glu-cose, may operate at levels below its Km value, which,perhaps, is vital to the cellular function of this vitamin.

Ascorbate supplementation has been shown to pro-vide a degree of protection against retinal photo-dam-age in rats.40 Scorbutic guinea pigs suffered a greaterlight-induced retinal damage than normal animals.41

Light-induced oxidative changes in rod outer segmentshave been reported to produce hydroperoxide radicals,which may be detrimental to visual cells.42 Ascorbate,therefore, may protect against the potentially damagingeffects of light in the retina through its role as a scav-enger of free radicals. Hyperglycemia in diabetes maycause a relative ascorbate deficiency in the retina,thereby enhancing retinal photo-sensitivity anddamage.

Although ascorbate is the most efficient reducingcofactor for collagen synthesis (e.g., ascorbate-depen-dent prolyl hydroxylation), in a variety of pathologicalstates and experimental studies, other endogenous fac-tors (e.g., tetrahydrofolate, glutathione, lactate) or con-ditions (e.g., glucose composition of the growth media,cell density) are known to regulate collagen synthesisindependent of, or in the absence of, ascorbate (ascor-bate-independent prolyl hydroxylation).43"45 High glu-cose levels in the growth media stimulated the rate ofcellular collagen/protein synthesis in cultured retinalcapillary pericytes30'46 and cultured retinal pigmentepithelial cells (Khatami, Slysh, Landsburg, Li, andRockey: unpublished observations). The hyperglycemiaof diabetes, therefore, may induce factor(s) capable ofderegulating the cells from their dependency on ascor-bate for collagen synthesis. Increased collagen produc-tion and thickening of basement membranes in dia-betes may be the result of such a deregulation phenom-enon induced by hyperglycemia.

Hexose transport systems are subject to metabolicregulation. The number of sugar carriers of differentcell types in culture was reduced as the glucose con-centration in the growth media increased.47'48 Such adown-regulation of glucose carrier density as a resultof hyperglycemia may additionally suppress ascorbatetransport into cells (e.g., pericytes) which share the samecarrier system for ascorbate and glucose transport. Theimpairment of ascorbate transport by hyperglycemia,

therefore, may be a factor to be considered in the de-velopment of diabetic angiopathy.

Key words: ascorbate transport, retinal capillary pericytes,culture, glucose inhibition, diabetic retinopathy

Acknowledgments

Hasan Ziaie and Renuka Kumarasamy provided excellenttechnical assistance. The authors are grateful to VirginiaMesibov and Bridgett Dent for the preparation of the manu-script.

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30. Li W, Shen S, Khatami M, and Rockey JH: Stimulation of retinalcapillary pericyte protein and collagen synthesis in culture byhigh glucose concentration. Diabetes 33:785, 1984.

31. Levine, M, Asher A, Pollard H, and Zinder O: Ascorbic acidand catecholamine secretion from cultured chromaffin cells. JBiolChem 258:13111, 1983.

32. Li W, Chan LS, Khatami M, and Rockey JH: Non-competitiveinhibition of myo-\nos\to\ transport in cultured bovine retinalcapillary pericytes by glucose and reversal by Sorbinil. BiochimBiophysActa 857:198, 1986.

33. Diliberto EJ Jr, Hackman GD, and Daniels AJ: Characterizationof ascorbic acid transport by adrenomedullary chromaffin cells.J BiolChem 258:12886, 1983.

34. Sharma SK, Johnstone RM, and Quastel JH: Active transportof ascorbic acid in adrenal cortex and brain cortex and the effectsof ACTH and steroids. Can J Biochem Phys 41:597, 1963.

35. Khatami M, Stramm LE, and Rockey JH: Transport of ascorbate

by primary cultures of cat retinal pigment epithelial cells: Inhi-bition by glucose. ARVO Abstracts. Invest Ophthalmol Vis Sci26(Suppl):64, 1985.

36. Goldstein MS: Glucose transport theory of insulin action. AnnNY Acad Sci 82:378, 1959.

37. Bensch KG, Korner O, and Lohman W: On a possible mecha-nism of action of ascorbic acid: Formation of ionic bonds withbiological molecules. Biochim Biophys Res Commun 101:312,1981.

38. Khatami M, Roel LE, Li W, and Rockey JH: Ascorbate regen-eration in bovine ocular tissues by NADH dependent semide-hydroascorbate reductase. Exp Eye Res, 43:167, 1986.

39. Loewen PC and Richter HE: Inhibition of sugar uptake byascorbic acid in Escherichia coli. Arch Biochem Biophys 226:657, 1983.

40. Organisciak DT, Wang HN, and Kou AL: Ascorbate and glu-tathione levels in the developing normal and dystrophic rat retina.Curr Eye Res 3:257, 1984.

41. Woodford BJ and Tso MOM: Exaggeration of photic injury inscorbutic guinea pig retina. ARVO Abstracts. Invest OphthalmolVis Sci 25(SuppI):90, 1984.

42. Armstrong D, Hiramitsu T, Gutteridge J, and Nilsson SE: Studieson experimentally induced retinal degeneration. 1. Effect of lipidperoxides on electroretinographic activity in the albino rabbit.Exp Eye Res 35:157, 1982.

43. McGee JO'D, Patrick RS, Rodger MC, and Luty CM: Collagenproline hydroxylase activity and 35S sulfate uptake in humanliver biopsies. Gut 15:260, 1974.

44. Peterkofsky B, Kalwinsky D, and Assad R: A substance in L-929 cell extracts which replaces ascorbate requirement for pro-lylhydroxylase in a tritium release assay for reducing cofactor;correlation of its concentration with the extent of ascorbate-in-dependent proline hydroxylation and the level of prolylhydrox-ylase activity in these cells. Arch Biochem Biophys 199:362, 1980.

45. Chauhan U, Assad R, and Peterkofsky B: Cysteinyl-cysteine andthe microsomal protein from which it is derived act as reducingcofactor for prolylhydroxylase. Biochem Biophys Res Commun131:277, 1985.

46. Li W, Khatami M, and Rockey JH: The effect of glucose andan aldose reductase inhibitor on the sorbitol content and collagensynthesis of bovine retinal capillary pericytes in culture. Exp EyeRes 40:439, 1985.

47. Moran A, Turner RJ, and Handler JS: Regulation of sodium-coupled glucose transport by glucose in a cultured epithelium.J BiolChem 258:15087, 1983.

48. Ullrey D, Gammon MT, and Kalckar HM: Uptake patterns andtransport enhancements in cultures of hamster cells deprived ofcarbohydrates. Arch Biochem Biophys 167:410, 1985.


Announcements

ARVO Resolution on the Use of Animals in Research

The Visual-Science community has long recognizeda scientific and ethical responsibility to provide ap-propriately for the welfare of animals used for researchand education in biology and medicine.1

The Association for Research in Vision and Oph-thalmology strongly endorses the continued conser-vative and humane use of animals in vision research.The vast majority of the major advances made inthis field over the past several decades have comefrom animal studies—advances that have saved orrestored the vision of millions of people. The recentdevelopment of new animal models for human diseaseoffers hope for those now suffering from currentlyincurable eye problems or for the thousands who willsoon encounter unpreventable and untreatable dis-eases.

At the same time, ARVO applauds the efforts ofthose who seek alternatives to animals for certaintypes of research. However, animal research will ofnecessity continue to be of vital importance in thestruggle against human blindness.2

The NIH and several major biomedical researchsocieties have been working together to insure theadequate care and humane treatment of laboratoryanimals. Therefore, ARVO directs its Governmentand Public Relations Committee to work with theNational Eye Institute, other NIH components con-cerned with the use of animals in research, and otherbiomedical research societies in formulating policiesand procedures in this area to assure that recognitionof the essentiality of the continued humane use ofanimals in research is included in any federal, state,or local legislation or university edicts on this subject.

References

1. US Department of Health, Education and Welfare: Guide forthe Care and Use of Laboratory Animals. NIH PublicationNo. 80-23, p 1.

2. Adapted from the 1983 Report of the National Advisory EyeCouncil, Vision Research—A National Plan: 1983-1987. NIHPublication No. 83-2470, p 85.

Association Update

The year 1986 has proven to be full of administra-tive changes for ARVO. In May, the membershipelected a new Secretary/Treasurer and six new Trus-tees including, for the first time, a Trustee from theClinical Research Section. A new Executive Directorwas hired and the Association Headquarters hasmoved from New Rochelle, New York, to Bethesda,Maryland.

Board of Trustees

Harry A. Quigley, MD, Professor of Ophthalmologyand Director of the Glaucoma Section at the WilmerEye Institute, was elected as Secretary/Treasurer ('87-'91). A graduate from Harvard College and JohnsHopkins Medical School, Dr. Quigley was the ARVOGlaucoma Section Chairperson in 1985 and serveson the editorial board of 10 VS. Although Dr. Quig-ley's term of office does not officially begin untilJanuary 1987, he has already begun working with theSection Chairpersons and the new Executive Directorin developing the program for the 1987 AnnualMeeting.

Douglas R. Anderson, MD, the Glaucoma Trustee,was elected by the Board to serve as President for1987. Dr. Anderson is Professor of Ophthalmologyat the Bascom Palmer Eye Institute, Miami, Florida.

Daniel M. Albert, MD, the Anatomy PathologyTrustee, will serve as the 1987 Vice President. Dr.Albert is the David G. Cogan Professor of Ophthal-mology and Director of the Eye Pathology Laboratory,Harvard Medical School Massachusetts Eye and EarInfirmary, Boston, Massachusetts.

The newly elected Trustees are as follows:

Biochemistry—Richard N. Lolley, PhD—Chief,Laboratory of Developmental Neurology, VA MedicalCenter, Sepulveda, California.

Clinical Research—Barbara E. K. Klein, MD—Associate Professor of Ophthalmology at the Univer-sity of Wisconsin, Madison, Wisconsin.

Cornea—Henry F. Edelhauser, PhD—Professor ofPhysiology and Ophthalmology at the Medical Collegeof Wisconsin, Milwaukee, Wisconsin.

Electrophysiology—Daniel G. Green, PhD—Pro-fessor of Physiological Optics at the University ofMichigan, Ann Arbor, Michigan.

1672


No. 11 Announcements 1673

Eye Movements—Robert D. Reinecke, MD—Pro-fessor of Ophthalmology and Director of the EthelBrown Foerderer Center for the Study of Eye Move-ment Disorders in Children, Wills Eye Hospital, andJefferson Medical College, Thomas Jefferson Univer-sity, Philadelphia, Pennsylvania.

Lens—Joram Piatigorsky, PhD—Chief, Laboratoryof Molecular and Developmental Biology, NationalEye Institute, Bethesda, Maryland.

ARVO Headquarters Move

During the summer, ARVO moved its headquartersfrom New Rochelle, New York, to Bethesda, Mary-land. The new office is located on the Federation ofAmerican Societies for Experimental Biology (FASEB)Campus, approximately 1/2 mile from the NationalEye Institute. Kathleen C. McCasland was selectedby the Search Committee, appointed by the Board ofTrustees, to serve as Executive Director. Prior toaccepting this position, Ms. McCasland was the Ex-ecutive Director of the California Association forMedical Laboratory Technology. In addition, she wasa Program Director at a Washington, DC, basedconsulting firm, where she wrote proposals and di-rected several Department of Health and HumanServices and Association related projects.

The new address and telephone number are:

ARVO9650 Rockville PikeBethesda, Maryland 20814301-530-7000

Looking Ahead

Based on results of the 1986 ARVO MeetingSurvey, coupled with suggestions and comments of-fered by many members, a few changes worth notinghave already taken place:

• Poster Boards—Twenty-five new poster panels arebeing constructed which should allow for the accep-tance of approximately 250-300 more abstracts thatare judged to be of high scientific value.

• Audio Visual Requirements—As in the past, ARVOwill provide dual 35 mm slide projectors in allrooms. This year the Association will also provideoverhead projectors and 16 mm movie projectors,if it is requested on the abstract form. A "specialrequirements" box has been added to the form forauthors to indicate their special A/V needs. AlthoughARVO will not provide video equipment, everyeffort will be made to facilitate cost sharing amongthe presenters.

• Membership Renewal Notices—Transferring Head-quarters to,Bethesda, Maryland, necessitated newinputting of all the membership data into thecomputer. The Membership Renewal Notices weredesigned to let the members know exactly whatinformation is on record at .the Association Office.If you have not already mailed your renewal notice,be sure and make any additions or corrections thatare needed. Don't let your membership lapse!

• Discount on Car Rentals—AVIS and Budget RentalCars are both offering special discounted rates toattendees at ARVO's Annual Meeting. Look fordetails in the Pre-Registration material that will besent to members in December.

Fight For Sight Scientific Awards Program

The Fight For Sight Scientific Awards Program for1987-88 has been announced by Mildred Weisenfeld,who is the founder and executive director of FightFor Sight, Inc. The awards program and peer reviewof applications is administered by the Association forResearch in Vision and Ophthalmology.

Applications are available for the following pro-grams in ophthalmic and vision research:

1. Grants-in-aid for research projects, stressing pilotand feasibility projects, with awards of $1,000 to$10,000;

2. Postdoctoral fellowships, with maximum awardsof $12,000;

3. Student (summer) fellowships, with maximumawards of $400 per month.

The deadline for receipt of completed applicationswill be March 1, 1987. Starting dates are July 1 orSeptember 1, 1987, and June 1, 1987, for studentfellowships. For further information and applicationforms (indicate category desired) write to: Fight ForSight, Inc., Box 474, 601 N. Broadway, Baltimore,MD 21205.


1674 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / November 1986 Vol. 27

Ludwig von Sallmann Prize

Gerald Westheimer, PhD, professor of physiologyat the University of California in Berkeley, has wonthe prestigious Ludwig von Sallmann Prize for signif-icant contributions to vision research and ophthal-mology. The $30,000 international prize, awarded ev-ery two years, was presented at the International Con-gress for Eye Research held in September in Japan.

A Berkeley faculty member since 1960, Dr. West-heimer has shown through his research how the eye

processes optical signals and how the human brainchannels visual information.

The von Sallmann Prize, which is administered bythe College of Physicians and Surgeons of ColumbiaUniversity, is named for Ludwig von Sallmann, whowas professor of ophthalmology there and one of theworld's leading authorities on the lens of the eye andits diseases, particularly the development of cataracts.

National Institutes of Health—100 Anniversary

In October of this year, the National Institutes ofHealth (NIH) began a year-long observance of its 100thAnniversary. All components of NIH are participatingin this celebration of a century of excellence inbiomedical research. It is anticipated that individualgrantees, academic institutions, voluntary organiza-tions, and groups representing physicians and otherhealth care providers will become involved in a mean-ingful way. In addition, NIH hopes that medical jour-nals, medical news publications, newsletters, and otherpublications prepared by and for these groups will pro-vide information about the Centennial and the impactof NIH-suppbrted research advances on the welfare ofall humankind. Local and national coverage of Cen-tennial events by the general news media is expectedas well.

It is hoped that all members of ARVO will join NIHin commemorating its Centennial Year. For example,one could make announcements about the Centennialat meetings, seminars, and workshops. One might beable to arrange for organizations to formally dedicatetheir next annual meeting or portion of it to NIH andthe many research advances it has supported. A mes-sage about the Centennial also might be incorporated

into the printed programs for these meetings. And ofcourse it is always appropriate to mention the sourceof support for research when the results are published,or presented at meetings, news conferences, and hos-pital rounds.

The National Eye Institute's Office of Scientific Re-porting is assembling a "How to participate in the NIHCentennial" kit for friends of the NEI. The kit willcontain background information on NIH and the NEI;Centennial Year objectives; a list of available photo-graphs, slides, and film segments; brochures for laymencontaining information on various eye diseases andrelevant research findings; and a description of an NEIexhibit for laymen that will be available for display atappropriate locations. If you would like to receive anyof these materials, please contact Ms. Marsha Corbett,Chief, Office of Scientific Reporting, National Eye In-stitute, Building 31, Room 6A32, Bethesda, MD 20892or call (301) 496-5248.

The NEI's kit will also contain a timetable for Cen-tennial events. In the timetable established by NIH, allInstitutes have been assigned specific months to providea focal point for their Centennial participation. TheNEI's month is January 1987.

ISCEV Annual Symposium

The International Society for the Electrophysiologyof Vision (ISCEV) announces its XXV annual sym-posium to be held April 26-30, 1987, at the Lido BeachHoliday Inn, Sarasota, Florida. For more informationcontact Dr. William Biersdorf, Department of Oph-

thalmology, University of South Florida and JAH Vet-erans Hospital, 13000 N. 30th Street, Tampa, FL 33618or Dr. William W. Dawson, Department of Ophthal-mology, University of Florida, Box J-284 JHMHC,Gainesville, FL 32610.


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