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Exclusion of albumin from vesicular ingestion by isolated microvessels
MICROVASCULAR
RESEARCH
19, 127-130 (1980)
BRIEF COMMUNICATION Exclusion
of Albumin from Vesicular by Isolated Microvessels
Ingestion
ROGER C. WAGNER,* STUART K. WILLIAMS,* MAUREEN A. MATTHEWS,* AND S. BRIAN ANDREWS? *School of Life and Health Sciences, University of Delaware, Newark, Delaware 19711: TSection on Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510 Received April 4. 1979
Solutes traverse the endothelium of blood vessels by both convective and dissipative mechanisms (l-4). The relative contribution of these mechanisms to the transport of specific solutes under specific conditions is difficult to determine in intact vascular systems where all mechanisms are operative. In isolated microvessels, convective forces and concentration gradients across the endothelium are zero since the luminal and abluminal compartments are in equilibrium (5). Vesicular ingestion (micropinocytosis), the first event in dissipative transport of large molecular weight solutes (3,4,6), is a constitutive property of capillary endothelial cells (7-9) and can be quantitated in isolated microvessels without the intervention of other transport forces and mechanisms (7). We report here the differential ingestion by micropinocytosis of two different proteins into the endothelium of isolated microvessels. This implies a transport selectivity which is intrinsic to the endothelial cell which may function in the partitioning of albumin between the blood and interstitial spaces. Isolated capillary endothelium sequesters native ferritin (8,9) and rhodaminelabeled ferritin (RhF) (7) into micropinocytic vesicles in a manner similar to that of intact vascular systems. Ingestion can be quantitated by measuring the fluorescence intensity of internalized RhF and normalizing against the amount of endothelial DNA in the isolate (7). In this study, ferritin and albumin were labeled with different fluorochromes, each with specific excitation and emission maxima. This permitted the simultaneous measurement of the ingestion of more than one protein. Ferritin was labeled with rhodamine isothiocyanate (7) and the conjugate (RhF) had excitation/emission maxima of X0/580 nm and an isoelectric point of 4.2-4.6. Bovine serum albumin (fraction V) was labeled with 2-methoxy-2,4diphenyl-3(2H)furanone (10) and alternatively with rhodamine isothiocyanate; the conjugates had fluorescence maxima of 390/480 nm (MdAl) and 560/580 nm (RhAl) and both had isoelectric points of 4.7-5.0 (Fig. 1). Microvessels were isolated from rat epididymal fat (7) and incubated for 30 min at 37 and 0.5” in Hepes isotonic buffer (pH 7.2) containing RhF (88.5 p.M), MdAl 127 00262862/8W010127a4$02.00/0 Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in U.S.A.
128
BRIEF
COMMUNICATION
WAVfLfNC7H
NANOMfTfRS
FIG. 1. Fluorescence spectra of protein-fluorochrome c onjugates. Albumin labeled with 2-methoxy-2,4-diphenyl-3 (2H)furanone (MdAI) had an excitation maximum at 390 nm (-) and an emission maximum at 480 nm (--). Both albumin and ferritin labeled with rhodamine isothiocyanate (RhAl and RhF) had an excitation maximum at 560 nm (-) and an emission maximum at 580 nm h-4.
(65.5 PM), or RhAl (156.5 @4) either singly or in combinations of two. The amount of protein ingested was assayed according to the method of Wagner et al. (7). Fluorescence intensity at the specific wavelengths for each conjugate was converted to micrograms or micromoles of protein from standard curves of the conjugates. The amount associated with the isolate at 0.5” (uninternalized but cell-surface-bound conjugate) was subtracted from that present at 37”. The difference represented the amount of conjugate ingested by micropinocytosis in 30 min and this was normalized against the amount of endothelial DNA in the isolate (7). Isolated microvessels ingested 10.6-12.1 pmole (6.9-7.9 wg) of RhF per microgram of endothelial DNA in 30 min. No ingestion of RhAl or MdAl was detected over the same time period (Table 1). Since all conjugates were present at saturating concentrations (increased concentration did not increase the amount of protein ingested), it was concluded that the differential uptake observed was due to a variance in the properties of the tracer proteins. If RhF and MdAl were present simultaneously in the incubation medium, 7.1 pmole (4.0 pg) of RhF but no MdAl was ingested (Table l), indicating a depression but not total inhibition of the RhF ingestion in the presence of albumin. Thus inhibition of the micropinocytic process by albumin cannot account for its lack of uptake but rather its exclusion from ingestion. The presence of the specific fluorochromes attached to the albumin cannot account for its behavior since neither RhAl nor MdAl are ingested. Exclusion on the basis of molecular size is not evidence since ferritin (A, = 6.1 nm) is ingested while albumin (A, = 3.7 nm) is not. The net charges of the ferritin and albumin conjugates (Table 1) are very similar and their difference is probably not a factor in their different uptake rates. The results on uningested, surface-bound conjugate (Table 1,O.S’) indicate that the density of total cell-surface binding sites for these two proteins are not sufficiently different to account for the results. However, the data do not exclude the possibility of significant differential affinity at specialized membrane domains (e.g., vesicle membranes). A preferred affinity for ferritin and/or repulsion of
129
BRIEF COMMUNICATION TABLE 1
VESICULAR INGESTION OF FLUORESCENT-LABELED PROTEINS BY ISOLATED MICROVESSELS
Incubation concentration
RhFc
A,” (nm)
PI*
6.1
4.2-4.6
mg/d
59.0
CLM
88.5
RhAl
3.7
4.7-5.0
10.5
156.5
RhF’
6.1
4.2-4.6
59.0
88.5
MdAl
3.7
4.7-5.0
4.4
65.5
RhFe
6.1
4.2-4.6
59.0
88.5
MdAl
3.7
4.7-5.0
4.4
65.5
Protein ingestion (pmole @g) in 30 min/pg DNA) 31”
0.5”
Net uptake
24.2 2 0.6d (15.7) 6.0 +- 0.8 (0.40)
13.6 f 0.9
10.6 + 1.1
7.1 f 1.0 (0.48)
-1.0 2 1.3 (-0.07)
20.7 2 1.4 (13.5) 5.1 + 0.4 (0.34)
8.5 + 0.5 (5.6) 5.3 k 0.9 (0.36)
12.1 * 1.5 (7.9) -0.2 2 1.0 (-0.02)
13.4 f 1.1 (8.7) 3.0 f 0.3 (0.20)
6.3 2 0.8 (4.1) 3.2 k 0.6 (0.22)
7.1 2 1.4 (4.6) -0.2 t 0.6 (-0.02)
(8.8)
(6.9)
a Molecular radii (A,, Stokes-Einstein or hydrodynamic radius) are approximate and based on values for native ferritin and albumin since any changes in dimensions due to the bound fluorochrome are not likely to be significant. * Isoelectric points @I) of the native proteins are ferritin, 4.2-4.6, and albumin, 4.6-5.0. c Microvessels were incubated separately with labeled proteins in individual uptake experiments. d Standard error of the mean. e Microvessels were incubated in RhF and MdAl simultaneously in the same experiment and concentrations were determined by measuring fluorescence at the specific wavelengths for each fluorochrome (RhF, 560/580; MdAl, 390/480).
albumin at such sites prior to ingestion could be the basis for the observed differential ingestion of these proteins. In this study, the micropinocytic uptake of two different labeled proteins was measured under identical conditions and without parallel modes of transport. The data indicate that neither molecular size nor total charge are dominant factors in the differential ingestion of ferritin and albumin. An alternative selectivity must exist which is intrinsic to the endothelial cell and which is able to discriminate between ferritin and albumin during the ingestion process. Deviations from strict permeability-molecular size relationships have been observed in intact microvascular systems. Highly anionic proteins are transported less readily than weakly charged proteins of comparable size (11). Factors other than charge may also determine transport efficiency since uncharged dextrans are transported less readily than proteins of the same hydrodynamic size (12). Autoradiographic analysis of radiolabeled albumin injected into the interstitium of skeletal muscle, an application site comparable to the incubation of isolated microvessels in albumin, indicates that no significant flux of albumin into the plasma occurs during normal blood flow (13). These data are in general agreement with those presented in this paper and may indicate an exclusion phenomenon whereby albumin is partitioned between the blood and interstitial fluids. ACKNOWLEDGMENTS Supported by PH.8 Grant HL16666 and Career Development Award HLO0270 to R.C.W.
130
BRIEF COMMUNICATION
REFERENCES 1. RENKIN, E. M., JOYNER, W. L., SLOOP, C. H., AND WATSON, P.D. (1977). Influence of venous pressure on plasma-lymph transport in the dog’s paw: Convective and dissipative mechanisms. Microvasc. Res. 14, 191-204. 2. LANDIS, E. M., AND PAPPENHEIMER, J. R. (1963). Exchange of substances through capillary walls. In “Handbook of Physiology,” Sect. 2: “Circulation” (W. F. Hamilton and P. Dow, eds.), Chap. 29, pp. 961-1034. Amer. Physiol. Sot., Washington, D.C. 3. PALADE, G. E. (1960). Transport in quanta across the endothelium of blood capillaries. Anat. Rec.
136, 274. 4. BRUNS, R. R., AND PALADE, G. E. (1968). Studies on blood capillaries. II. Transport of ferritin molecules across the wall of muscle capillaries. J. Cell Biol. 37, 277-299. 5. WAGNER, R. C., AND MATTHEWS, M. A. (1975). The isolation and culture of capillary endothelium from epididymal fat. Microvasc. Res. 10, 286-297. 6. KARNOVSKY, M. J. (1968). The ultrastructural basis of transcapillary exchanges. J. Gen. Physiol. 52, 64-95. 7. WAGNER, R. C., ANDREWS, S. B., AND MATTHEWS, M. A. (1977). A fluorescence assay for micropinocytosis in isolated capillary endothelium. Microvasc. Res. 14, 67-80. 8. SIMIONESCU, M., AND SIMIONESCU, N. (1978). Constitutive endocytosis of the endothelial cell. J. Cell Biol. 79, 381a. 9. SIMIONESCU, M., AND SIMIONESCU , N. (1978). Isolation and characterization of endothelial cells from the heart microvasculature. Microvnsc. Res. 16, 426-452. 10. WEIGELE, M., DEBERNARDO, W., LEIMGRUBER, R., CLEELAND, R., AND GELJNBERG, E. (1973). Fluorescent labelling of proteins, a new methodology. Biochem. Biophys. Res. Commun. 54, 899-906. 11. CARTER, R. D., JOYNER, W. L., AND RENKIN, E. M. (1974). Effects of histamine and some other substances on molecular selectivity of the capillary wall to plasma proteins and dextrans. Microvasc. Res. 7, 31-48. 12. JOYNER, W. L., CARTER, R. D., RAZES, G. S., AND RENKIN, E. M. (1974). Influence of histamine and some other substances on blood-lymph transport of plasma protein and dextran in the dog paw. Microvasc. Res. 7, 19-30. 1251-labelled albumin into blood 13. JOHANSSON, B. R. (1978). Movement of interstitially microinjected
capillaries Microvasc.
of rat skeletal muscle demonstrated Res.
with electron
Statementof ownership,managementand circulationrequiredby United
microscopic
autoradiography.
16, 354-361.
the Act of October
23. 1%2, Section 4369, Title
39,
States Code: of MICROVASCULAR
RESEARCH
Published bimonthly by Academic Press, Inc., III Fifth Avenue, New York, N.Y. 10003. Number of issues published annually: 6. Editors: David Shepro, Boston University, Boston, Massachusetts and Benjamin Zweifach, University of California, San Diego, La Jolla, California 92037. Owned by Academic Press, Inc., 111 Fifth Avenue, New York, N.Y. 10003. Know” bondholders. mortgagees, and other security holders owning or holding percent or more of total amount of bonds, mortgages or other securities: None. Paragraphs 2 and 3 include, in cases where the stockholder or security holder appears upon the bwks of the company as trust.% or in any other fiduciary relation, the name of the person or corporation for whom such “ustee is acting, also the statements in the hvo paragraphs show the aBiant’s Ml knowledge and belief as to the circumstances and conditions under which stockholders and security holders who do not appear upon the books of the company as trustees. hold stock and securities in a caI.ucity other than that of a bona fide owner. Names and addresses of individuals who are stockholders of a corporation which itself is a stockholder or holder of bonds, mortgages or other securities of the publishing corporation have bee” included in paragraphs 2 and 3 when the interests of such individuals are equivalent to of the total amount of the stock or securities of the publishing corporation. Total no. copies printed: average no. copies each issue during preceding 12 months: 1048; single issue nearest to filing date: 1006.Paid circulation (a) to term subscribers by mail, carrier delivery or by other means: average no. copies each issue during preceding 12 months: 643; single issue nearest to tiling date: 644; (b) Sales through agents, news dealers, or otherwise: average no. copies each issue during preceding 12 months: 0; single issue nearest to tiling date: 0. Free distribution by mail, carrier delivery, or by other means: average no. copies each issue during preceding 12 months: 58; single issuenearest to tiling date: 58. Total no. of copies distributed: average no. copies each issue during preceding 12 months: 701; single issue nearest to tilii date: 702. (Signed) Roselle Coviello, Senior Vice President
I
I percent ormore
RESEARCH
19, 127-130 (1980)
BRIEF COMMUNICATION Exclusion
of Albumin from Vesicular by Isolated Microvessels
Ingestion
ROGER C. WAGNER,* STUART K. WILLIAMS,* MAUREEN A. MATTHEWS,* AND S. BRIAN ANDREWS? *School of Life and Health Sciences, University of Delaware, Newark, Delaware 19711: TSection on Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510 Received April 4. 1979
Solutes traverse the endothelium of blood vessels by both convective and dissipative mechanisms (l-4). The relative contribution of these mechanisms to the transport of specific solutes under specific conditions is difficult to determine in intact vascular systems where all mechanisms are operative. In isolated microvessels, convective forces and concentration gradients across the endothelium are zero since the luminal and abluminal compartments are in equilibrium (5). Vesicular ingestion (micropinocytosis), the first event in dissipative transport of large molecular weight solutes (3,4,6), is a constitutive property of capillary endothelial cells (7-9) and can be quantitated in isolated microvessels without the intervention of other transport forces and mechanisms (7). We report here the differential ingestion by micropinocytosis of two different proteins into the endothelium of isolated microvessels. This implies a transport selectivity which is intrinsic to the endothelial cell which may function in the partitioning of albumin between the blood and interstitial spaces. Isolated capillary endothelium sequesters native ferritin (8,9) and rhodaminelabeled ferritin (RhF) (7) into micropinocytic vesicles in a manner similar to that of intact vascular systems. Ingestion can be quantitated by measuring the fluorescence intensity of internalized RhF and normalizing against the amount of endothelial DNA in the isolate (7). In this study, ferritin and albumin were labeled with different fluorochromes, each with specific excitation and emission maxima. This permitted the simultaneous measurement of the ingestion of more than one protein. Ferritin was labeled with rhodamine isothiocyanate (7) and the conjugate (RhF) had excitation/emission maxima of X0/580 nm and an isoelectric point of 4.2-4.6. Bovine serum albumin (fraction V) was labeled with 2-methoxy-2,4diphenyl-3(2H)furanone (10) and alternatively with rhodamine isothiocyanate; the conjugates had fluorescence maxima of 390/480 nm (MdAl) and 560/580 nm (RhAl) and both had isoelectric points of 4.7-5.0 (Fig. 1). Microvessels were isolated from rat epididymal fat (7) and incubated for 30 min at 37 and 0.5” in Hepes isotonic buffer (pH 7.2) containing RhF (88.5 p.M), MdAl 127 00262862/8W010127a4$02.00/0 Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in U.S.A.
128
BRIEF
COMMUNICATION
WAVfLfNC7H
NANOMfTfRS
FIG. 1. Fluorescence spectra of protein-fluorochrome c onjugates. Albumin labeled with 2-methoxy-2,4-diphenyl-3 (2H)furanone (MdAI) had an excitation maximum at 390 nm (-) and an emission maximum at 480 nm (--). Both albumin and ferritin labeled with rhodamine isothiocyanate (RhAl and RhF) had an excitation maximum at 560 nm (-) and an emission maximum at 580 nm h-4.
(65.5 PM), or RhAl (156.5 @4) either singly or in combinations of two. The amount of protein ingested was assayed according to the method of Wagner et al. (7). Fluorescence intensity at the specific wavelengths for each conjugate was converted to micrograms or micromoles of protein from standard curves of the conjugates. The amount associated with the isolate at 0.5” (uninternalized but cell-surface-bound conjugate) was subtracted from that present at 37”. The difference represented the amount of conjugate ingested by micropinocytosis in 30 min and this was normalized against the amount of endothelial DNA in the isolate (7). Isolated microvessels ingested 10.6-12.1 pmole (6.9-7.9 wg) of RhF per microgram of endothelial DNA in 30 min. No ingestion of RhAl or MdAl was detected over the same time period (Table 1). Since all conjugates were present at saturating concentrations (increased concentration did not increase the amount of protein ingested), it was concluded that the differential uptake observed was due to a variance in the properties of the tracer proteins. If RhF and MdAl were present simultaneously in the incubation medium, 7.1 pmole (4.0 pg) of RhF but no MdAl was ingested (Table l), indicating a depression but not total inhibition of the RhF ingestion in the presence of albumin. Thus inhibition of the micropinocytic process by albumin cannot account for its lack of uptake but rather its exclusion from ingestion. The presence of the specific fluorochromes attached to the albumin cannot account for its behavior since neither RhAl nor MdAl are ingested. Exclusion on the basis of molecular size is not evidence since ferritin (A, = 6.1 nm) is ingested while albumin (A, = 3.7 nm) is not. The net charges of the ferritin and albumin conjugates (Table 1) are very similar and their difference is probably not a factor in their different uptake rates. The results on uningested, surface-bound conjugate (Table 1,O.S’) indicate that the density of total cell-surface binding sites for these two proteins are not sufficiently different to account for the results. However, the data do not exclude the possibility of significant differential affinity at specialized membrane domains (e.g., vesicle membranes). A preferred affinity for ferritin and/or repulsion of
129
BRIEF COMMUNICATION TABLE 1
VESICULAR INGESTION OF FLUORESCENT-LABELED PROTEINS BY ISOLATED MICROVESSELS
Incubation concentration
RhFc
A,” (nm)
PI*
6.1
4.2-4.6
mg/d
59.0
CLM
88.5
RhAl
3.7
4.7-5.0
10.5
156.5
RhF’
6.1
4.2-4.6
59.0
88.5
MdAl
3.7
4.7-5.0
4.4
65.5
RhFe
6.1
4.2-4.6
59.0
88.5
MdAl
3.7
4.7-5.0
4.4
65.5
Protein ingestion (pmole @g) in 30 min/pg DNA) 31”
0.5”
Net uptake
24.2 2 0.6d (15.7) 6.0 +- 0.8 (0.40)
13.6 f 0.9
10.6 + 1.1
7.1 f 1.0 (0.48)
-1.0 2 1.3 (-0.07)
20.7 2 1.4 (13.5) 5.1 + 0.4 (0.34)
8.5 + 0.5 (5.6) 5.3 k 0.9 (0.36)
12.1 * 1.5 (7.9) -0.2 2 1.0 (-0.02)
13.4 f 1.1 (8.7) 3.0 f 0.3 (0.20)
6.3 2 0.8 (4.1) 3.2 k 0.6 (0.22)
7.1 2 1.4 (4.6) -0.2 t 0.6 (-0.02)
(8.8)
(6.9)
a Molecular radii (A,, Stokes-Einstein or hydrodynamic radius) are approximate and based on values for native ferritin and albumin since any changes in dimensions due to the bound fluorochrome are not likely to be significant. * Isoelectric points @I) of the native proteins are ferritin, 4.2-4.6, and albumin, 4.6-5.0. c Microvessels were incubated separately with labeled proteins in individual uptake experiments. d Standard error of the mean. e Microvessels were incubated in RhF and MdAl simultaneously in the same experiment and concentrations were determined by measuring fluorescence at the specific wavelengths for each fluorochrome (RhF, 560/580; MdAl, 390/480).
albumin at such sites prior to ingestion could be the basis for the observed differential ingestion of these proteins. In this study, the micropinocytic uptake of two different labeled proteins was measured under identical conditions and without parallel modes of transport. The data indicate that neither molecular size nor total charge are dominant factors in the differential ingestion of ferritin and albumin. An alternative selectivity must exist which is intrinsic to the endothelial cell and which is able to discriminate between ferritin and albumin during the ingestion process. Deviations from strict permeability-molecular size relationships have been observed in intact microvascular systems. Highly anionic proteins are transported less readily than weakly charged proteins of comparable size (11). Factors other than charge may also determine transport efficiency since uncharged dextrans are transported less readily than proteins of the same hydrodynamic size (12). Autoradiographic analysis of radiolabeled albumin injected into the interstitium of skeletal muscle, an application site comparable to the incubation of isolated microvessels in albumin, indicates that no significant flux of albumin into the plasma occurs during normal blood flow (13). These data are in general agreement with those presented in this paper and may indicate an exclusion phenomenon whereby albumin is partitioned between the blood and interstitial fluids. ACKNOWLEDGMENTS Supported by PH.8 Grant HL16666 and Career Development Award HLO0270 to R.C.W.
130
BRIEF COMMUNICATION
REFERENCES 1. RENKIN, E. M., JOYNER, W. L., SLOOP, C. H., AND WATSON, P.D. (1977). Influence of venous pressure on plasma-lymph transport in the dog’s paw: Convective and dissipative mechanisms. Microvasc. Res. 14, 191-204. 2. LANDIS, E. M., AND PAPPENHEIMER, J. R. (1963). Exchange of substances through capillary walls. In “Handbook of Physiology,” Sect. 2: “Circulation” (W. F. Hamilton and P. Dow, eds.), Chap. 29, pp. 961-1034. Amer. Physiol. Sot., Washington, D.C. 3. PALADE, G. E. (1960). Transport in quanta across the endothelium of blood capillaries. Anat. Rec.
136, 274. 4. BRUNS, R. R., AND PALADE, G. E. (1968). Studies on blood capillaries. II. Transport of ferritin molecules across the wall of muscle capillaries. J. Cell Biol. 37, 277-299. 5. WAGNER, R. C., AND MATTHEWS, M. A. (1975). The isolation and culture of capillary endothelium from epididymal fat. Microvasc. Res. 10, 286-297. 6. KARNOVSKY, M. J. (1968). The ultrastructural basis of transcapillary exchanges. J. Gen. Physiol. 52, 64-95. 7. WAGNER, R. C., ANDREWS, S. B., AND MATTHEWS, M. A. (1977). A fluorescence assay for micropinocytosis in isolated capillary endothelium. Microvasc. Res. 14, 67-80. 8. SIMIONESCU, M., AND SIMIONESCU, N. (1978). Constitutive endocytosis of the endothelial cell. J. Cell Biol. 79, 381a. 9. SIMIONESCU, M., AND SIMIONESCU , N. (1978). Isolation and characterization of endothelial cells from the heart microvasculature. Microvnsc. Res. 16, 426-452. 10. WEIGELE, M., DEBERNARDO, W., LEIMGRUBER, R., CLEELAND, R., AND GELJNBERG, E. (1973). Fluorescent labelling of proteins, a new methodology. Biochem. Biophys. Res. Commun. 54, 899-906. 11. CARTER, R. D., JOYNER, W. L., AND RENKIN, E. M. (1974). Effects of histamine and some other substances on molecular selectivity of the capillary wall to plasma proteins and dextrans. Microvasc. Res. 7, 31-48. 12. JOYNER, W. L., CARTER, R. D., RAZES, G. S., AND RENKIN, E. M. (1974). Influence of histamine and some other substances on blood-lymph transport of plasma protein and dextran in the dog paw. Microvasc. Res. 7, 19-30. 1251-labelled albumin into blood 13. JOHANSSON, B. R. (1978). Movement of interstitially microinjected
capillaries Microvasc.
of rat skeletal muscle demonstrated Res.
with electron
Statementof ownership,managementand circulationrequiredby United
microscopic
autoradiography.
16, 354-361.
the Act of October
23. 1%2, Section 4369, Title
39,
States Code: of MICROVASCULAR
RESEARCH
Published bimonthly by Academic Press, Inc., III Fifth Avenue, New York, N.Y. 10003. Number of issues published annually: 6. Editors: David Shepro, Boston University, Boston, Massachusetts and Benjamin Zweifach, University of California, San Diego, La Jolla, California 92037. Owned by Academic Press, Inc., 111 Fifth Avenue, New York, N.Y. 10003. Know” bondholders. mortgagees, and other security holders owning or holding percent or more of total amount of bonds, mortgages or other securities: None. Paragraphs 2 and 3 include, in cases where the stockholder or security holder appears upon the bwks of the company as trust.% or in any other fiduciary relation, the name of the person or corporation for whom such “ustee is acting, also the statements in the hvo paragraphs show the aBiant’s Ml knowledge and belief as to the circumstances and conditions under which stockholders and security holders who do not appear upon the books of the company as trustees. hold stock and securities in a caI.ucity other than that of a bona fide owner. Names and addresses of individuals who are stockholders of a corporation which itself is a stockholder or holder of bonds, mortgages or other securities of the publishing corporation have bee” included in paragraphs 2 and 3 when the interests of such individuals are equivalent to of the total amount of the stock or securities of the publishing corporation. Total no. copies printed: average no. copies each issue during preceding 12 months: 1048; single issue nearest to filing date: 1006.Paid circulation (a) to term subscribers by mail, carrier delivery or by other means: average no. copies each issue during preceding 12 months: 643; single issue nearest to tiling date: 644; (b) Sales through agents, news dealers, or otherwise: average no. copies each issue during preceding 12 months: 0; single issue nearest to tiling date: 0. Free distribution by mail, carrier delivery, or by other means: average no. copies each issue during preceding 12 months: 58; single issuenearest to tiling date: 58. Total no. of copies distributed: average no. copies each issue during preceding 12 months: 701; single issue nearest to tilii date: 702. (Signed) Roselle Coviello, Senior Vice President
I
I percent ormore