Document jmB9Qp1mGMbpEd8gY6Eoqdq55

'<* effects that are evident are an increased slope conductance at very hyperpolarized membrane potentials and a signifi cant decrease in the NSR. Although some ofthe reduction ofNSR may result from a competing potassium conduct ance, both of these effects can be pro duced independently by varying stimulus parameters. Our results are most consistent with a cyclic AMP mediation of those inputs that act mainly via an increased slope conductance, presumed to be potassiummediated. A more mechanistic analysis of the mediation of these long-lasting synaptic events in RI5 awaits a more de tailed description of the conductance changes evoked by individual fibers within the branchial nerve. Bursting pacemaker activity is an important fea ture of normal neuronal activity; it is also an important feature of abnormal activi ty, possibly manifested in disorders such as epilepsy. The finding that the conduct ances underlying such activity may be under synaptic control suggests that complex output patterns may be pro duced or altered by relatively short input regimes. St e v e n N. Tr e is t ma n Worcester Foundationfor Experimental Biology, Shrewsbury, Massachusetts 01545 References and Notes 1. I. Pamasand F. Strumwasser, J. Neurophysiol. 37, 609(1974). 2. S. N. Triestman and I. B. Levitan, Proc. Natl, Acad. Scl. V.S.A. 73, 4689 (1976). 3. P. F. Drake and S. N. Treistman, J. Neurobioi. 11,471(1980). 4. W. A. Wilson and H. Wachtel, Science 186,932 (1974). ,, 5. _____ ibid. 202, 772 (1078). 6. D. C. Eaton, 1. M. Russel, A, M. Brown, J. Membr. Biol. 21, 353 (1,975). 7. Supported by NIH grant NS 15195-01. Part of tills work was done at the Marine Biological Laboratory at Woods Hole, Mass. 01545.1 thank Dr. Robert Conner for helpful suggestions. 11 July 1980 Lactose Facilitates the Intestinal Absorption of Lead in Weanling Rats Abstract. The milk sugar lactose is known tofacilitate calcium absorption and has been shown to enhance the uptake of essential trace metals from the intestines as well. Its physiological role as the major carbohydrate source for.suckling mammals is thus complemented by its ability tofacilitate the absorption ofnecessary minerals. The studies reported here show that the intestinal absorption oflead and its uptake into blood, liver, kidney, and bone are also increased by lactose in young weanling rats. These data extend the known range oflactosefacilitation ofmineral absorption to a nonessential, toxic element, confirming the nonspecificity of its action on the gut. In addition, they suggest an explanation for some of the conflicting evidence regarding the prophylactic efficacy of milk in lead poisoning. It is well known that children are more susceptible than adults to lead poisoning (/). This sensitivity to the toxicity of lead results in part from increased absorption and retention of ingested lead (2). En hanced absorption of lead by the young is probably a general characteristic of mammals and has been documented in nonhuman primates (J) and in rodents {4). The reasons for the subsequent de cline in lead retention with age are not completely understood but may include the development of more selective in testinal absorptive processes, more effi- LeftT>_ dent biliary and renal excretion of ab sorbed lead, and changes in the diet. Dietary changes may be particularly significant in mammals, since the pre weaning diet under normal circum stances consists almost exclusively of mother's milk, whereas the postweaning diet contains little or no milk. Milk diets have been shown to increase the reten tion of ingested lead in experimental ani mals (5), probably by facilitating in testinal absorption of the metal. The question of the prophylactic efficacy of milk in lead poisoning in humans has been debated for years but not resolved S). One difficulty associated with analyz ing the metabolic effects of milk is its chemical complexity. Hamilton <7) has argued, for example, that the facilitation of lead retention observed in rodents fed a milk diet (5) is an indirect result of the low iron content of milk, since iron defi ciency has been shown repeatedly to in crease the absorption and retention of in gested lead (7, 8). However, milk con tains other materials that directly influence lead metabolism, including cal cium, phosphorus, vitamin D, fat, and protein. Another constituent, unique to milk, is the milk sugar lactose, which al so has profound effects on mineral me tabolism in mammals. The absorption and retention of many minerals, includ ing calcium (9), iron (/0),zinc (11), man ganese (12), cobalt (13), magnesium, strontium, barium, and rubidium (14), are enhanced by dietary lactose. To our knowledge, however, the effects of lac tose on the absorption and retention of lead or other toxic metals have not been investigated. We report here that lac tose, in physiological quantities, facili tates the intestinal absorption of orally administered lead in weanling rats and thus increases its uptake by tissues, Male weanling rats (Holtzman Compa ny, Madison, Wisconsin) were housed Table 1. The effects of glucose or lactose on the absorption and tissue uptake of lead. Rats which Were 26 days old and which had fasted for 24 hours were intubated with 1.0 nil of a dosing solution containing 0,1, 3, or 6 mg of sugar per gram of body weight and 4.0 pCi of *"Fb, The rats were killed 18 to 22 hours later. Tissues were assayed for radioactivity as described in the text. Values shown are the mean ( the standard error) percent of administered dose absorbed from the gastrointestinal tract (percentage absorption) or the mean percent of administered dose.per grant of tissue (percentage uptake). Treatment Dose (mg/g) N Absorp tion (%) Femur (%) Uptake by Kidneys (%) Liver m Water (control) 0 10 .38.7 5.1 3.40 .0.59 3.89 0.65 0.76 0.13 Glucose 1 12 40.8 7.2 3.35 0.62 0.84 0.15 Glucose 3 10 44.5 2,7 4.13 0.32 4.68 0.41 0.85 0.06 Glucose 6 11 42.7 3.6 2.83 0.28 3.70 0.24 0.77 0.05 Lactose 1 12 40.0 5.2 3.35 0.52 0.87 0.14 Lactose 3 9 74.7 3.6* 6.74 0.35* 7.31 0.47* 1.52 0.16* Lactose 6 12 69.1 3.0* 5.88 0.33* 7.78 0.91* 1.36 0.10* ' Values that differ from the water control at P < .01. SCIENCE, VOL. 211, 2 JANUARY 1981 0036-8075/81/0102-0061$00.50/0 Copyright 1980 AAAS Blood (%) 0,52 0.09 0.55 0.10 0.60 0.05 D.56 0.04 0.61 0.09 0.90 0.06* 1.09 0.08* 61 TEH 0531900 Table 2. A comparison of the effects of glucose, galactose, maltose, and lactose (3 mg/g) on the absorption and tissue uptake ofsl0)Pb. AD lactose values differ from those for glucose (P < .05), whereas those for galactose and maltose do not. See Table 1 legend and text for procedures. Treat ment N Absorp tion <%) Femur (%) Uptake by Kidneys (%) Liver (%) Blood (%) Glucose 5 45.8 4.9 4.26 0.61 4.73 0.77 0.80 0.11 0.57 0.09 Galactose 6 48.9 6.3 4.30 0.68 5.23 0.81 0.94 0,16 0.51 0.07 Maltose 6 40.9 9.9 3.79 0.95 4.77 1,25 0.81 0.23 0.50 0.14 Lactose 5 70.6 4.9 6.96 0.61 7.32 0.73 1.41 0.10 0.91 0.09 singly in suspended wire cages for 5 days beginning on day 21 after birth and were maintained on freely available distilled water and a vitamin-enriched semi-purified diet containing 0.47 percent calcium and 0.3 percent phosphorus (IS). Food, but not water, was removed from the cages 24 hours prior to treatment. Treatment consisted of a single in tubation of a test solution on day 26 after birth followed 18 to 22 hours later by tis sue assay. Under ether anesthesia, a 1.0ml dosing solution containing 4.0 pCi of 2I0Pb, 0.10 ml of 0.01 mM lead acetate carrier, and a test dose of0,1,3, or 6 mg of glucose or lactose per gram of body weight (16) was administered by gavage. After dosing, animals fasted overnight and were killed the next day by ether overdose. Blood was obtained by car diac puncture; the liver, kidneys, femur, the entire gastrointestinal tract from esophagus to rectum, and all feces were collected, weighed, and placed in 20-ml plastic scintillation vials. Tissues were assayed for radioactivity by gamma-counting at the 47-KeV emis sion peak of 210Pb with a scintillation spectrometer (Packard Auto-Gamma, model 5220). To assess lead uptake by specific organs, counts were converted to counts per minute (cpm) per gram of tissue for each sample and compared to a standard composed of the initial dosing solution diluted to about 2 ml in distilled water. The percentage absorption from the gut was calculated by difference ac cording to Smith et al. (17) as follows: % absorption = |0Q /CpQlfltd cpglgut + fecei \ l cpm8td / .Group mean differences were tested for significance by an analysis of variance and by Dunnett's test for comparison of experimental means to a control (IS). The absorption of lead from the gut and the uptake by femur, kidneys, liver, and blood in 26-day-old weanling rats are shown in Table 1, for three dosages of sugar. Controls given water absorbed 38.7 percent of the administered lead, a level consistent with those reported for animals close to weaning and food-de prived for nearly 48 hours (5,19). Equiv alent amounts of lead were absorbed by all groups given glucose and by the group given 1 mg of lactose per gram. At the higher doses of lactose, however, the ab sorption of lead was nearly doubled, reaching 74.7 percent of the adminis tered dose (193 percent of the control) at 3 mg/g (P < .01). A similar pattern was obtained for tissue lead uptake values, which were approximately doubled by lactose doses of 3 and 6 mg/g (P < .01). In a second experiment, we tested the specificity of the effect for lactose by comparing the lead absorption and tissue uptake when glucose, galactose, mal tose, and lactose were used. All sugars were given at concentrations of 3 mg/g to 26-day-old weanling rats. Table 2 shows that galactose and maltose produced lead absorption and uptake values com parable to that for glucose, whereas lac tose, as before, increased lead absoiption (P < .05), Thus, neither galactose, the monosaccharide paired with glucose to form lactose, nor maltose, a disac charide composed of two glucose moi eties, was able to enhance the absorp tion and uptake of lead by weanling rats, as was lactose. These data show that lactose enhances the absorption of lead from the in testines, but this result does not rule out the possibility of further, extra-alimen tary interaction. Such an interaction in bone has been proposed (20) to account for the facilitation of calcium absorption by lactose. However, Lengemann (14) demonstrated that parenteral lactose had no effect on the femoral uptake of orally administered calcium; in fact, calcium uptake was enhanced only when lactose and calcium were administered simulta neously into the same gut segment. This result suggests an immediate effect of lactose on intestinal transport processes. Kello and Kostial (5) showed that diets containing milk markedly increased the retention of orally administered lead (30to 60-fold increases over the control) and had only minor effects on parenterally administered lead (1.2 to 1.3 times the control). These effects have been attrib uted to a secondary effect of iron deft- * ciency induced by the low iron content 1 of the milk diets used (5, 7). In light of * the data presented here, however, an ex- 4 planation of these results must now con- 4 aider the possibility that lactase in the milk diets facilitated directly the absorp tion of orally administered lead. These findings suggest that the in creases in tissue lead concentrations in the present experiments are the result of the enhanced absorption oflead from the intestine. Little is known about this pro cess; however, it may be argued that, be cause lactose acts so nonselectively in promoting the absorption of minerals (914), lead is treated similarly in this re spect. If so, then the mechanisms pro posed to explain lactose facilitation of calcium absorption should apply to lead as well. These mechanisms involve facil itation of passive ion diffusion in the dis tal small intestine and are distinct from the vitamin D-dependent active trans port system in the duodenum. In this model, lactose nonselectively increases the permeability of the intestinal mem brane to divalent cations, probably by al tering a sodium-dependent electrical po tential across the intestinal wall which normally opposes passive cationic dif fusion to the serosa (21). In vitro studies have demonstrated that other sugars facilitate ileal (but not duodenal) absorption of calcium in adult rats (22). These effects are not apparent in vivo because of the rapid hydrolysis and absorption of these sugars in the up per small intestine (23). Lactose, which is hydrolyzed and absorbed much more slowly, thus passes into the ileum where it can influence the absorption of metals present. The rapid hydrolysis of maltose into glucose, and the rapid absorption of glucose and galactose, can account for the lower levels of lead absorption asso ciated with these sugars, as compared to lactose. Milk in the diet of the young mammal plays a complex role in determining the sensitivity to lead poisoning. On the one hand, its lactose content would enhance absorption of lead, as shown here, whereas its high concentrations of cal cium, phoshonrs, zinc, and protein would be expected to reduce the reten tion of lead (24). The fact that milk con tains such "protective" species suggests that it should protect against lead poison ing. However, for such prophylaxis to be fully realized, lead (and possibly other toxic heavy metals including mercury and cadmium) must be absent from the ingested milk and, in fact, removed from the milk meal to prevent the immediate effects of lactose on the intestinal trans- 62 SCIENCE, VOL. 211 TEH 0531901 DUP050032397 J ' *port of metals. Because of this com plexity, it is not surprising that earlier 9.F. W. Lengemana, C. L. Comar, R. H. Was herman, J. Nutr. 61, 571 (1937); H. J. Armbrccht and R. H. Wasserman, ibid. 106, 1265 we found that the fluorescence was lo cated at or near the upper and lower sur- ' experiments with lead and milk should (1976). 10. E. K. Amine and D. M. Hegsted, ibid. 101,927 faces of the cell, as would be expected for ` have yielded conflicting results (6). (1971); D, Bouvet, C.R. Acad. Sci. Ser. D 270, binding to the plasma membrane. When Ph il ip J. Bu s h n e l j . Hec t o r F. DeLu c a Department ofBiochemistry, College of 1264 (1970). 11. P. Fournier and A. Digaud,C.R. Acad. Sci. Ser. Dm, mi (1971). 12. P. Fournier and A. Fournier, C.R. Sac. Biol. 166,39(1972). grown at high density the cells become somewhat rounded, and this allows one to see clear evidence for membrane flu Agricultural and Life Sciences, University of Wisconstn-Madison, 13. A. Fournier, ibid. 168, 244 (1974). 14. F. W. Lengomaan, J. Nutr. 69, 23 (1959). orescence because the optical path at the 15. T. Suda, H. F. DeLuca. Y. Tanaka, ibid. 100, boundary between cells contains more of Madison 53706 ,1049(1970). 16. That these doses tie in the physiological range the plasma membrane than the optical for a 21-day-old rat may be determined by the path away from the boundary. Whereas References and Notes 1. J. J. Chisolm, M. B. Barrett, E. D. Mellits. J. Pediatr. 87, 1152(1975). 2. E. E. Ziegler, B. B. Edwards, R. L. Jensen, K. R. Mahaffey, S. J. Fomoa, Pediatr., Res. 12, 29 (1978); F. W. Alexander, H. T. Delves, B. E. Clayton, in Environmental Health Aspects of Lead (Proceedings of an International Sym posium oo Lead 0972), Commission of Euro pean Communities Directorate General for Dis semination of Knowledge, Centre for Informstion and Documentation, Luxembourg, 1973), p. .319. 3. R. F. Willes, E. Lok, J. F. Truelove, A. Sunda- following considerations. Assume that a 50-g rat consumes 10 ml of milk in 24 hours. Rat milk contains a lactose concentration of SO mM, which converts to 28,8 mg/ml or 288 mg per 19 ml. In a 50-g rat, 288 mg yields a "dose" of 5.76 mg of lactose per gram of rat. 17. C. M. Smith. H. F. DeLuca, Y. Tanaka. K. R. Mahaffey, J. Nutr. 108, 843 (1978). 18. J. Myers, Fundamentals of Experimental De sign (Aliya & Bacon, Boston, 1971). 19. J. Ouarterman, J. N. Morrison, W. R. Hum phries, Environ. Res. 12, 180(1976). 20. P. Fournier, C. R. Acad. Sci. 240, 115 (1955). 21. D. L. Martin and H. F. DeLuca, Am. J. Physiol. bright fluorescence occurs at the bound ary between two cells after incubation with Rho-Ta in the presence of 20 mA# methylamine, such incubation in the ab sence of methylamine leads to fluores cence appearing in endocytic vesicles but not, at least in quantity, at the plasma membrane (Fig. 1, C and EX To show that binding was saturable. ram, J, Toxicol. Environ. Health 3, 395 (1975). 4. K. Kostial, I. Simonovic, M. Pisonic, Nature (London) 233,564 (1971); G. B. Forties and J. C. Reina, J. Nutr. 102,647 (1972). .5.. D. Keflo and K. Kostial, Environ. Res. 6, 355 (1973). 6. R. Stephens and H. A. Waldron, Food Cosmet. Toxicol 13, 555 (1975). 7. D. L. Hamilton, Toxicol Appl. Pharmacol. 46, 651 0978). 8. J. C. Barton, M. E. Conrad, S. Nuby, L. Harri son, J, Lab. Clin. Med. 92, 536 (1978); K. R. Mahaffey-SiX and R. A. Goyer, ibid. 79, 128 (1972), 22. D. w. Vaughan and L. j , Faer, Jr, j . Nutr. 7t, J0 (I960). 23. A- Dahlqvist and D. L. Thomson, Acta Physiol, Scand. 61,20(1964); R. H. Wa9serman,,Vtffwrr (London) 201, 997 (1964). 24. D. Barltrop and H. E. Khoo, Postgrad. Med.J. 51,795(1975). 25. This research was supported by postdoctoral fellowship ES-0S147 to P.J.B., Food and Drug Administration contract RFP 223-77-2166, and the Harry Steenbock Research Fund of the Wis consin Alumni Research Foundation. 14 May 1980; revised 25 August 1980 we used amicroscope fluorescence spectrophotpmeter to quantify the fluores cence intensity observed after incubating the cells with Rho-T3 and methylamine in the presence or absence of unlabeled T3 (20 yM) (see legend to Fig. 1). Fluores cence intensities were measured on 20 randomly selected cells in each dish (7). Untreated cells gave an average fluores cence intensity of 33 arbitrary units 7 (standard deviation); this intensity was due to cellular autofluorescence. Cells treated With Rho-T3 alone had an in Binding and Mobility of the Cell Surface Receptors for 3,3' ,5-Truodo-L-Thyronine tensity of 103 42 units and cells in cubated with Rho-T3 and excess unla beled T3 had an intensity of 54 13 units. Thus, unlabeled T3 competed for Abstract. A fluorescent derivative of the thyroid hormone 3,3',5-triiodo-L-thyro 70 percent of the Rho-Ta binding. The nine binds to cultured mouse fibroblasts; such binding is saturable, Video in standard deviations in these measure tensification fluorescence microscopy indicates that binding occurs at the plasma ments are an indication of cellular hetero membrane. Diffusion coefficients, obtained by fluorescence photobleaching recov geneity. Duplicate dishes gave the same ery, are consistent with binding to a protein receptor on the cell surface. average values within 10 percent. Since Rho-T3 remains associated with Although much evidence suggests that 20 minutes at 23 or 37C, fluorescence is the plasma membrane in the presence of thyroid hormone action is initiated by rapidly localized in endocytic vesicles methylamine, we were able to measure binding of 3,3',5-triiodo-L-thyronine (Ta) that appear as bright points of light (Fig. the diffusion coefficient, D, in the plane to nuclear receptors (/), the mechanism IE) (SX Only background fluorescence is of the plasma membrane using fluores for delivery of Ta to the nucleus is not observed in cells incubated with rhoda- cence photobleaching recovery (FPR). fully understood. It was long thought mine-thyronine (Rho-Ta). In double-la In this method (8), fluorescence from a that Ta enters cells by passive diffu beling experiments with fluorescein-la small region (approximately 1 jam in di sion through the plasma membrane (2), beled a2-macroglobulin and Rho-T3, we ameter) on the plasma membrane is par but recent studies with [mI]T3 sug found that these endocytic vesicles are tially bleached by a brief exposure to la gest that there are cell-surface receptors the same as those which take up insulin, ser light focused on the membrane. The for T3 and that entry of Ta is, at least in epidermal growth factor, and the serum diffusion coefficient is determined from part, energy-dependent (3). We have protein Oj-macrOglobulin (5). We demon the rate at which fluorescent molecules synthesized a rhodaminc derivative of Ta strated previously (6) that aj-macroglob- diffuse into the bleached area. In gener (Rho-Tj) that binds specifically to the nu ufin-occupied receptors cluster over coated al, the diffusion coefficients ofmembrane clear receptor for Ta with a dissociation pits before they are internalized and that proteins are 5 x 10-10 cm2/sec or less, constant (/C<j) of 20 nM (4), and in this the clustering can be inhibited by primary whereas the diffusion coefficients of lip report we show that Rho-T3 also binds to alkylamines. Figure 1 shows that methyi- ids and lipid probes are about 10-8 cm2/ a membrane component that has a mobil amine also affects the uptake of Rho-Ta. sec (9). By using FPR we could deter ity in the plasma membrane similar to the On a flat cell (Fig. 1 A) the fluorescence ap mine whether Rho-T3 behaves as if it is mobility of polypeptide hormone recep pears diffusely distributed after 20 min simply dissolved in the membrane lipid tors. utes of incubation with Rho-Ta at 23"C in phase and diffuses like a lipid probe, or When 3T3 fibroblasts from Swiss al the presence of 20 mAf methylamine. By whether its diffusion is more character bino mice are incubated with Rho-T3 for focusing up and down through the cell istic ofa membrane protein. As shown in SCIENCE, VOL. 211, 2 JANUARY 1981 0026-8075/81/0102-0063$00.50/0 Copyright 1980 AAAS 63 TEH 0531902 DUP050032398 ? Table 1. Lateral diffusion coefficients determined for Rho-Ts and other membrane-associated molecules on 3T3 cells by fluorescence photobleacbing recovery (8), Ligand p (cm2/sec) x 10' Fractional recovery {%) Reference Rho-Ta Rho-insufin Rho-epidermal growth factor Rho-armacrog]obuUn Dil* 2.8 + 1.5 4.8 1.6 3.4 0.5 7.8, 3.5 100 69 29 50 to 80 50 to 85 53 7 -100 This report VD VD V2) VI) *3,3'-Dioctadecylindocarbocyanme iodide, a fluorescent lipid probe. Table 1, the diffusion coefficient of (2.8 1.5) x 10~10 cmVsec for Rho-T3 was indistinguishable from the diffusion coefficients previously measured for the rhodamine derivatives of insulin, epider mal growth factor, and o^macroglobulin on these cells. This, together with the saturability and specificity of the bind ing, suggests that Rho-Ts binds to a cellsurface protein receptor on 3T3 cells. The similarities observed in the uptake of Rho-T3, Rho-a2-macroglobulin, Rhoinsulin, and Rho-epidermal growth fac tor also suggest that a cell-surface pro tein receptor is involved in the uptake of Rho-T-,. We have not observed Rho-Ts fluores cence in nuclei, perhaps because of a change in the fluorescent properties of Rho-Ts after it binds to the nuclear re ceptor, Alternatively, the T3 receptors may be diffusely distributed in the nucle us, making them difficult to observe against the cytoplasmic fluorescence. The physiological significance of bind ing to a cell surface receptor and recep tor-mediated uptake of T3- is not clear. Although 70 percent of the Rho-Ts bind ing could be blocked by unlabeled T3, only 20 percent of [,S5IJTs uptake could be blocked by Rho-Ts (5). This raises the possibilities that Rho-T3 has a decreased affinity for the receptor and thus is un able to compete effectively with Ts, or Fig. !. Binding of Rho-T, to 3T3 cells. The cells were rinsed five tiroes with Pulbeceo-Vogf's modi fied Eagle's medium and then incubated with 10~rAf Rho-T, for 20 minutes at 23C. When present, methylamine (20 mM) was added 30 minutes before the addition of Rho-Tj. After incu bation, the cells were rinsed five tiroes with : serum-free medium, three times with se rum-free medium con taining bovine serum albumin (1 mg/ml), and five times with phosphate-buffered sa line (each 1 ml). The cells were then ob served with a silicon intensifier target tele vision camera mount ed on a Zeiss standard epifluoresccnce micro scope with a x 63 (N.A. 1.4) objective as described (10). (A, C, and E) Fluores cence micrographs; (B, D, and F) the same fields under phase-contrast illumination. (A and B) A flat cell incubated with Rho-Ts in the presence of methylamine shows a diffuse pattern of fluorescence. (C and D) Cells incubated with Rho-Ts in the presence of methylamine show a heavy concentration at fluorescence at the boundary between cells. (E and F) Cells incubated with Rho-Ts without methylamine show the concentration of Rho-Ts in endocytic vesicles (magnification; x 2500 in A, B, E, and F; x 800 in C and D). 64 , a that there is more than one mechanism of uptake of T3 into the cells and Rho-Ts can use only one of these mechanisms (that is, receptor-mediated uptake). The * consequences of T$ accumulating in endocytic vesicles are also unclear. We have observed (data not shown) that ves icles containing Rho-Ts or a3-macro- globulin (10) undergo rapid saltatory mo tion inside the cell. These vesicles might be responsible for delivering T3 rapidly to other locations in the cell. F. R. Ma x f ie l d * M. C. Wil l in g h a m I. Pas t a n National Cancer Institute, Laboratory of Molecular Biology, National Institutes of Health, Bethesda, Maryland 20205 P. Dr a g s t e n Laboratory of Theoretical Biology, National Cancer Institute S.-Y. CHENGt Clinical Endocrinology Branch, National Institute ofArthritis, Metabolic and Digestive Diseases, National Institutes of Health References and Notes 1. J. H. Oppenheimer. Science 203,971 (1979). 2. S. H. Ingbar and N. Freinkel, Recent Prog. Horn. Res. IS, 333 (1960); J. R. Tata, in The Thyroid Gland. R. Pitt-Rivers and W. B. Trot ter, Eds. (Butterworth, London, 1964), vol. 1, pp. 163-168. 3. N. B, Pliam and I. D. Goldfine, Biochem. Biophys. Res. Commun. 19, 166 (1977); E. P. Krenning, R. Doctor, H. F. Bernard, T. J. Visser. G. Henneman, FEBS Lett. *1,113(1978); J, Eckel, G. S. Rao, M. L. Rao, H. Breuer, Bio chem. J. 182,473.(1979). 4. S.-Y. Cheng, N. L. Eberhardt, J. Robbins, J. D. Baxter, I. Pastan, FEBS Lett. 100, 113 (1979). 5. S.-Y, Chet. F. R. Maxfield, J. Robbins, M. C. Willingham, t. Pastan, Froc. Natl. Acad. Set. U.S.A. 77, 3425 (1980). 6. F. R. Maxfield, M. C. Willingham, P. 1. A. Davies, I. Pastan, Nature (London) 277, 661 (1979); M. C. Willingham, F. R. Maxfield, I. Pastan, J. Cell Biol. 82,614 (1979). 7. Fluorescence intensities were measured with an EMI 9638 R photomultiplier mounted on a Zeiss standard microscope equipped for incident light fluorescence. Incident light from a 50-W mercury lamp was passed through an Instruments S.A. model H 10 monochromator with a spectral bandpass of 8 nm at 546 mu. An FI 546 exciter filter, FI 580 reflector, aud-LP 590 barrier filter were used. A x 63 (N.A. 1.4) objective was used- After random selection of each cell, fluo rescence intensities were measured Rom a circle approximately 40 pm in diameter centered on the nucleus of the ceil. 8. The instrument used in these studies is de scribed in P. Dragsten, P. Henkart, R. Blumenthal, j. Weinstein, J. Schlessinger IProc, Natt. Acad. Sci. V.SJt. 16, 5163 (1979)). A 530-nm laser beam was focused on the upper membrane of cells after incubation with 1Q~'M Rho-T, in the presence of 20 mM tnethylamine for 20 min utes at 23C. After monitoring the fluorescence intensity at low levels of illumination, we in creased the intensity to 500-fold for 50 to 100 msec and then returned to the monitoring in tensity. Recovery of fluorescence intensity was monitored until a stable level'was achieved. The diffusion coefficient, D, is related to the half time for recovery, txa, by D -- yK'I/(4r,a) where y is a factor (--1.2) that accounts for the extent of bleaching when one uses a gaussiaa beam profile, and w is the focused laser beam radius at the e~* intensity point (0.52 pm in this study) [D. Axelrod. D. E. Koppel, J. Schlessinger, E. L. Elson, W. W. Webb, Biophys J. 14, 1055 (1976)). SCIENCE, VOL. 211 TEH 0531903 DUP050032399