Charge selectivity of rat intestinal capillaries

GASTROENTEROLOGY 1986;91:1443-6

Charge Selectivity of Rat Intestinal Capillaries Influence of Polycations D. NEIL GRANGER, PETER R. KVIETYS, AUBREY E. TAYLOR Department

of Physiology,

College of Medicine,

The role of fixed anionic sites on the intestinal capillary wall in transvascular protein exchange was assessed by neutralizing the negative charges with polycations. The studies were performed in anesthetized rats with an intestinal lymph cannula. Intestinal lymph flow and lymph and plasma total were measured at regular protein concentrations intervals before and after intravenous infusion of either protamine sulfate, poly-L-Iysine, or polyethyleneimine. Protamine sulfate infusion produced an eightfold increase in lymph flow and a fivefold increase in lymph protein clearance. Lymph flow increased 4.6-fold and lymph protein clearance increased 3.6 times over control in rats receiving the poly-L-Iysine infusion. Polyethyleneimine infusion produced results comparable in magnitude to protamine sulfate; however, the animals were unable to tolerate this agent. The enhanced transcapillary protein fluxes produced by the polycation infusions suggest fhat fixed anionic sites normally impede the egress of proteins from the intestinal vasculature. The permeability of intestinal capillaries to macromolecules is influenced by the size and shape of the permeating solute (1,2). Recknt ultrastructural and physiologic studies indicate that net electrical charge of the solute is also an important determinant df intestinal transcapillary exchange (3,4). The distribution of anionic sites on the blood front of the fenestrated endothelium of intestinal capillaries has been assessed using cationized ferritin (3). A high Received April 3, 1986. Accepted May 20, 1986. Address requests for‘reprints to: D. Neil Granger, PhD., Department of Physiology, MSB 3024, University of South Alabama, Mobile, Alabama 36688. This work was supported by a grant from the National Heart, Lung and Blood Institute (HL26441]. 0 1986 by the American Gastroenterological Association 0016-5085/86/$3.50

MICHAEL A. PERRY, and

University

of South Alabama, Mobile, Alabama

density of anionic sites was demonstrated on the fenestral diaphragms. Binding could not be demonstrated on the membrane of plasmalemmal vesicles and transendothelial channels. These observations suggest that the fenestral diaphragms discriminate against anionic molecules, whereas vesicles and transendothelial channels favor the penetration of anionic molecules and discriminate against cationic molecules. Lymph studies in the small bowel have demonstrated (a) that the steady-state concentration of a positively charged dextran in intestinal lymph is significaqtly less than that of a neutral dextran of equal size and (b) that the capillary osmotic reflection coefficient’ of positive isoenzymes of lactate dehydrogenase is greater than that of neutral and negative isoenzymes (4). These findings suggest that the intestinal capillary wall behaves as a net positively charged barrier. Thus, taken as a whole, the physiologic and ultrastuctural studies indicate that, although there are both fixed anionic and cationic sites on the luminal surface of intestinal capillaries, the cationic sites exert a dominant influence on transcapillary solute exchange. Studies of renal glomerular permeability to macromolecules indicate that this capillary network behaves as a net negatively charged filter (5,6). This contention is supported by reports that systemic infusions of polycations lead to an increased urinary excretion of plasma proteins (T-10). Such polycation-induced alterations in urinary protein excretion have been attributed to neutralization of fixed anionic sites on the glomerular capillary wall. Although similar fixed anionic sites have been demonstrated in intestinal capillaries (3), the importance of these anionic sites in intestinal transcapillary protein exchange remains unclear. Thus, the objective of the present study was to assess the role of fixed anionic sites in intestinal transvascular protein exchange by neutralizing the negative charges with

1444

GASTROENTEROLOGY Vol. 91,No.6

GRANGERETAL.

polycations. Our results indicate that fixed anionic sites play an important role in preventing excessive protein leakage across intestinal capillaries.

Materials and Methods Fifteen male Sprague-Dawley rats (Charles River Laboratories, Kingston, N.Y.), weighing between 314 and 450 g,were used in this study. All rats were housed in an environmentally controlled vivarium with a 12-h light/ dark cycle. The rats were allowed free access to a standard pellet diet and water. Access to food, but not water, was discontinued 18-24 h before experimental use. All animals were anesthetized with sodium pentobarbital (60 mgkg, i.p.). The animals were placed on an electric heating pad in a supine position to maintain body temperature at 87°C. A tracheostomy was performed to ensure a patent airway. A cannula was inserted into the carotid artery to monitor systemic arterial pressure. Arterial pressure was continuously recorded on a Grass physiologic recorder (Grass Instrument Company, Quincy, Mass.). The jugular vein was also cannulated for administration of saline at a rate of 0.5 ml/l66 g body wt . h to compensate for evaporative water loss. The venous catheter was also used to administer the polycations and for collection of blood samples. The abdomen was opened along the midline, and the large intestine, duodenum, and pancreas were surgically removed. The remainder of the small intestine (jejunumileum) was covered with saline-soaked gauze and wrapped with plastic wrap to minimize evaporation and tissue dehydration. Intestinal temperature was maintained at 87°C with a thermistor-controlled infrared lamp. A cannula (PE-10-50, 0.28-0.58 mm ID) was inserted into the main lymphatic vessel emerging from the mesenteric pedicle. Lymph flow was determined by observing lymph movement in a calibrated pipette (56-166 ~1, full scale), which was connected to the lymphatic cannula. The pipette was positioned horizontally at the level of the intestinal segment to eliminate any hydrostatic effects on lymph flow. Lymph and plasma total protein concentration were measured with an American Optical refractometer (American Optical’ Scientific Instruments, Buffalo, N.Y.). Transcapiilary protein clearance was calculated as the product of steady-state lymph flow and the lymph-toplasma protein concentration ratio. The intestinal segment was weighed at the end of each experiment, allowing for expression of both lymph flow and transcapillary protein clearance as milliliters per minute X 100 g gut weight. The polycations used in the study were protamine sulfate, poly+lysine, and polyethyleneimine (Sigma Chemical Co., St. Louis, MO.). Each polycation was dissolved in Tyrode’s solution and the pH was adjusted to 7.4. Lymph flow and lymph-to-plasma protein concentration ratio were allowed to reach a steady state at the onset of each experiment. After obtaining control measurements, one of the polycation solutions was infused into the jugular vein at a rate of 0.1-0.2ml/min for 10 min to deliver a total dose of 20 mg/kg body wt (7,8). Lymph flow and lymph and plasma protein concentrations were mea-

sured immediately after termination of the polycation infusion and at lo-min intervals thereafter for a period of i h. All values are reported as mean 2 SE. A paired Student’s t-test was used to determine whether the polycation infusions significantly altered intestinal transcapillary protein clearance and lymph flow. A probability value of <0.05 was considered statistically significant.

Results The basal values obtained in all animals (n = 15) were 91 + 2 mmHg for mean arterial pressure, 0.22 + 0.04 ml/pin. 100 g for lymph flow, 3.1 + 0.2 g% for lymph protein concentration, 4.9 ? 0.1 g% for plasma protein ccincentration, 0.62 2 0.03 for lymph-to-plasma protein concentration ratio, and 0.14 + 0.02 ml/min. ZOO g for franscapillary protein clearance. These values are similar to results previously reported for the rat (11,12). There was no significant difference in the basal values between experimental groups. The results of a single poly-t-lysine infusion ex-

2.500 poly-I-lysine r I 8~ 1.675 x f g 1.250 B ii f 0.625 3

1.260-

0

-10 0 10 20'30 40 50 60 70' Time (@I

Figure

1. Time-course of effects of intravenous @oly-L-lysine on intestinal lymph flow and transcapillary p?otein clearance in a single experiment.

POLYCATIONS AND CAPILLARY EXCHANGE

December 1986

periment are presented in Figure 1. Intestinal lymph flow and transcapillary protein clearance were dramatically altered by the IO-min intravenous infusion of poly-L-lysine. Both parameters increased to peak levels immediately after termination of the polycation infusion. Lymph flow increased more than sixfold and transcapillary protein clearance increased 5.2-fold during the initial postinfusion period. Both parameters declined subsequently but remained two to three times the preinfusion values at the end of the experiment. Figure 2 summarizes the peak responses obtained from 7 rats that received poly+lysine. Intravenous infusion of the polycation produced a 4.6-fold (range, 3.0- to Il.@fold) increase in lymph flow and a 3.6-fold (range, 1.5-to 5.2-fold) increase in transcapillary protein clearance. Intravenous infusion of protamine sulfate produced changes in lymph flow and transcapillary protein clearance comparable to those elicited by poly+lysine. Protamine sulfate consistently produced an abrupt increase in both parameters, followed by a gradual decline toward basal values (similar to the pattern depicted in Figure 1). Figure 3 summarizes the peak responses obtained from 6 rats that received protamine sulfate. This polycation produced an eightfold (range, 2.5-to 15.3-fold) increase in intestinal lymph flow and a 5.1-fold (range, 1.8-to 9.8-fold) increase in transcapillary protein clearance. Polyethyleneimine was infused intravenously in 2 rats. Although polyethyleneimine also produced an increase in both lymph flow (5.8- and 8.2-fold) and transcapillary protein clearance (2.9- and 4.4-fold), the animals were unable to tolerate this agent. Thirty to forty minutes after termination of the polyethyleneimine infusion the animals died of severe hypotension. Thus, additional experiments were not performed using this agent.

I-J Control 0

Poly-I-lysine *

Lymph Flow

Transw&ary Clearance

Figure 2. Summary of effects of intravenous poly+lysine on intestinal lymph flow and transcapillary protein clearance (n = 7). Asterisk denotes p i 0.05 relative to corresponding control.

1445

2.00 0 * 1.50

1

Control

?? Protamine Sulfate

I

0”

s

x 1.00

.c E 5 E

* 0.50

,, ~ Lymph Flow

Transcapillary Protein Clearance

Figure 3. Summary of effects of intravenous protamine sulfate on intestinal lymph flow and transcapillary protein clearance (n = 6). Asterisk denotes p < 0.05 relative to corresponding control.

Discussion A number of physical factors are known to influence the rate of escape of macromolecules from the microcirculation (1,2]. These include size, configuration, deformability, and net electrical charge of the solute. Most studies have focused on the effect of solute size on microvascular exchange, with relatively little attention devoted to the influence of other physical properties of the solute. The kidney is a notable exception to this generalization inasmuch as the role of net solute charge has been extensively studied (5-10). Three approaches have been used to define the charge-selective properties of the glomerular capillary wall: (a) microscopic assessment of the degree of penetration and binding of charged electron-dense tracers, (b) comparison of urinary clearances of charged and neutral solutes, and (c) urinary protein excretion measurements after neutralization of fixed anionic sites with polycations. The results obtained from all three approaches are consistent with the concept that renal glomerular capillaries behave like a negatively charged filter. There is less information available regarding the charge-selective properties of intestinal capillaries. Data derived from studies using the tracer penetration and binding approach indicate a nonuniform distribution of fixed charge sites on the intestinal capillary wall such that fenestral diaphragms are expected to discriminate against anionic molecules, whereas stomata1 diaphragms (which cover vesicle openings], plasmalemmal vesicles, and transendothelial channels may favor the penetration of cationic molecules (3). Comparisons of lymph clearances of charged and neutral macromolecules reveal that the intestinal capillary wall behaves as a net positively charged filter that impedes the movement

1446

GRANGER ET AL.

of cationic molecules and enhances the egress of anionic molecules (4). Thus, the data derived from the two approaches indicate that, although there are both fixed anionic and cationic sites on the luminal surface of intestinal capillaries, the cationic sites exert a dominant influence on microvascular solute exchange. The objective of the present study was to determine whether fixed anionic sites in intestinal capillaries exert a significant influence on microvascular protein exchange. Neutralization of fixed anionic sites was achieved by systemic administration of polycations. All polycations studied produced a rapid increase in net capillary fluid filtration (lymph flow) and capillary protein leakage (lymph protein clearance). The time-course and the magnitude of the enhanced protein leakage induced by the polycations were similar to previously reported effects of intravenous protamine sulfate and poly-L-lysine on urinary protein excretion (7). Our observation that polycations enhance capillary protein leakage indicates that the fixed anionic sites on fenestral diaphragms play an important role in preventing excessive filtration of circulating albumin and other negatively charged plasma proteins across intestinal capillaries. Although the enhancement of capillary protein leakage induced by polycations is generally attributed to neutralization of fixed anionic sites on the capillary wall, there is some evidence to indicate that polycations may alter transcapillary protein efflux through other mechanisms. Studies on the kidney suggest that polycations not only neutralize anionic sites but distort glomerular basement membrane structure, thereby altering porosity (8). Another possible explanation for the effects of the polycations on intestinal capillary protein flux is capillary recruitment, i.e., an increase in the number of perfused or filtering capillaries. Recruitment of all intestinal capillaries could account for up to a fourfold increase in net capillary protein efflux (1). Such large increments in perfused capillary surface area only occur under conditions of severe ischemia or tissue hypoxia (13). Inasmuch as transcapillary protein clearance increased up to lo-fold after protamine sulfate administration, it seems unlikely that capillary recruitment alone can account for the polycation-induced protein leakage. Our finding that polycations increase protein leakage across intestinal capillaries may be of significance regarding the mechanism by which activated neutrophils and eosinophils increase capillary per-

GASTROENTEROLOGY Vol. 91. No. 6

meability. When activated, neutrophils and eosinophils release, along with a variety of other substances, cationic proteins that have been shown to cause enhanced capillary protein leakage (14). Although the mode of action of cationic proteins on tE.e microcirculation remains undefined, the results of our study suggest that the basic proteins may increase vascular permeability by neutralizing fixed anionic sites in the capillary wall. Thus, modulation of the negative charge density of the microvasculature may be an important component of the inflammatory response.

References 1. Granger DN, Barrowman

JA. Microcirculation of the alimentary tract. I. Physiology of transcapillary fluid and solute exchange. Gastroenterology 1983;84:846-68. across 2. Taylor AE, Granger DN. Exchange of macromolecules the microcirculation. In: Renkin EM, Michel CC, eds. Handbook of physiology, microcirculation. Washington, D.C.: American Physiological Society, 1984:467-520. N, Simionescu M, Palade GE. Differentiated 3. Simionescu microdomains on the luminal surface of the capillary endothelium. Preferential distribution of anionic sites. J Cell Biol 1981;90:605-13. 4. Perry MA, Benoit JN, Kvietys PR, Granger DN. Restricted transport of cationic macromolecules across intestinal capillaries. Am J Physiol 1983;245:G568-72. 5. Brenner BM, Hostetter TH, Humes HD. Glomerular permselectivity: barrier function based on discrimination of molecular size and charge. Am J Physiol 1978;34:F455-60. 6. Karnovsky MJ. The structural basis for glomerular filtration. In: Kidney disease-present status. IAP Monograph No. 20. Baltimore: Williams & Wilkins, 1979:1-41. 7. Vehaskari VM, Root ER, Germuth FG, Robson AM. Glomerular charge and urinary protein excretion: effects of systemic and intrarenal polycation infusion in the rat. Kidney Int 1982;22:127-35. 8. Barnes JL, Radnik RA, Gilchrist EP, Venkatachalam MA. Size and charge selective permeability defects induced in glomerular basement membrane by a polycation. Kidney Int 1984;25:11-9. 9. Bertolatus JA, Hunsicker LG. Glomerular sieving of anionic and neutral bovine albumins in proteinuric rats. Kidney Int 1985;28:467-76 10. Lambert PP, Doriaux M, Sennesael J, Vanholder R, LammensVerslijpe M. Pathogenicity of cationized albumin in the dog: renal and extrarenal effects. Clin Sci 1984;67:19-33. 11. Barrowman JA, Granger DN. Effects of experimental cirrhosis on splanchnic microvascular fluid and solute exchange in the rat. Gastroenterology 1984;87:165-72. 12. Anzueto L, Benoit JN, Granger DN. A rat model for studying the intestinal circulation, Am J Physiol 1984;246:G56-61, 13. Shepherd AP. Role of capillary recruitment in the regulation of intestinal oxygenation. Am J Physiol 1982;242:G435-41. 14. Klebanoff SJ, Clark RA, eds. The neutrophil: function and clinical disorders. Amsterdam: North Holland Publishing Company, 1978.