CAPILLARY ENDOTHELIAL CEAIENT IN RELATION TO PERhIEABILITY

ROBERT CHAMBERS AND B. W. ZWEIFACH' Laboratory of Cellular Physiology, Department of Iiiology, Washington Square College,

N e w Y o r k Unzversaty

THREE FIGURES

I n a recent paper on the permeability of blood capillaries Krogh ( ' 3 7 ) emphasized that all solutes which are able to pass through the capillary wall do so without any evidence of a separation of solutes from solvent and that the diffusion is of the fluid in bulk. I n spite of this relatively indiscriminatory type of permeability- Rrogh ascribed the property to the endotlielial cell with the suggestion that the in- creased permeability of a dilated capillary is due to the greater surface presented by the stretched and expanded cells. However, the permea- bility of the capillary wall is in such marked contrast to the highly selective and restricted permeability of cells in general as to throw into question the role of the endothelial cell in the process. Mere thin- ness does not affect protoplasmic permeability. An example of this is the secreting cyst of proximal tubular epithelium of the kidney in tissue culture (Chambers and Kempton, '33). The cyst maintains its selective permeability irrespective of whether the cells of its epithe- lial wall are columnar or are flattened by distension to the thinness of pavement epithelium.

The difference between the permeability of membranes of the endo- thelial type and that of a membrane the permeability of which depends upon its cellular constituents, is strikingly shown in the reaction to NH4C1 of the glomerular tuft of blood capillaries and of the tubular epithelium in the frog's kidney (Chambers and Kempton, '37). When an aqueous solution of NH4C1, an acid salt, is delivered to the glomerulus, by way of the renal artery, the salt passes through as a whole and imparts its acid reaction to the urine. On the other hand, when the salt is delivered directly to the proximal tubule, by way of the renal portal vein, a differential permeability becomes evident. Of the two hydrolytic products, NH, (or NH,OH) readily passes through the cells while the HC1 does not. This results in a pronouncedly alkaline urine.

'This work was made possible through a grant from the Josiah Macy Jr. Foundation, New York City.

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JOURNAL OF C E I J A U L ~ R AND COMPARATlVE YHYSIOLOGY, VOI,. 15, NO. 3 JUNE, 1940

256 ROBERT CllAMBECS AND B. W. ZWEIFACH

The non-cellular constituent of the wall of the capillary, the so-called intercellular cement, was appreciated by Colinheim according to his earlier papers of 1867 and by Arnold in 185'7 who claimed that a loosen- ing of the cement between the endothelial cells formed stigmata to permit diapedesis. The chemical nature of the cement was suggested by Rabl (1893) who pointed out that the black staining of the endothelial lines in the AgNO, technique is due to the formation of a silver pro- teinate, since he found the deposit to be soluble in sodium hyposulfite.

Overton ( '04), in his studies on cellular permeability, stressed the importance of the existence of a cohesive material consisting of a reversible calcium salt of a weak acid for binding together the cells of plant and animal cellular membranes. This he based on the work of Curt Rerbst ('00) who found that the cohesiveness of the blasto- meres of developing sea urchin eggs can be destroyed by exposing the eggs to calcium-free sea water. I t is interesting to note that Ringer (1890) had already discussed the importance of lime salts for main- taining the cohesive nature o€ the intercellular cement. He used the cellular epithelium of tadpoles and of marine plants in his experiments.

These considerations prompted an experimental study on the blood capillaries of the frog with particular reference to tlie iriterendothelial cell-cement substance. The blood vessels of an exposed mesentery, under microscopic observation, were perfused with solutions the pII and calcium-content of which were varied. The effect on the permeability of the #capillaries was noted by the time of appearance of edema in the surrounding tissue and by the behavior of particulate matter in the capillaries.

The results of this investigation indicate that variations in the permeability of the blood capillaries can be readily induced by subject- ing the capillaries to conditions which Herbst and Overton found to affect the physical state of the intercellular cement.

METHOD

The solutions were perfused under a given constant air pressure, recorded by a mercury manometer and maintained by the usual device of two intercommunicating bottles partially filled with water and placed a t different levels. The perfusate was introduced by cannulating the aorta, the outflow on its return escaping through a cut in the tip of the ventricle. While the animal was being perfused, the observations were made on the capillary circulation in the mesentery spread over a specially constructed moist chamber (Zweifach, '37) on the stage of a microscope. A coverslip was mounted on the mesentery the under-

CAPILLARY CEMENT AN11 PlCKMEABlLITY 257

surface of which was free for micro-manipulation. A perfusion pressure of 30 mm. Hg was always maintained unless otherwise indicated.

Three types of solutions were used : Amphibian Ringer, Ca-free Ringer and Ringer containing twice the noriiial concentration of calcium. Each of these were prepared at two p H levels, viz., 6.6 to 6.8 and 7.4 to 7.6, with phosphate buffers. The pH of the solutions, colorimetrically tested before and after each experiment, was found to remain constant. All the solutions contained 0.5% asli-free gelatin (Eastman Kodak Co.). The use of gelatin has been described in a review article of Amberson ( ’37). Frog’s Ringer, containing 0.5% gelatin and buffered to p H 7.4 to 7.6, was used as control. This was found to maintain the conditions of normal circulation with no visible maiiifestations of edema for a period of well over 2 hours.

To the calcium-free solutions 2 cc. of 0.05 niol Na oxalate were added for every 100 cc. of the perfusate. This amount of Na oxalate is sufficient to neutralize the amount of calcium nornially present in frog’s Binger and was used to couutei*nct the effect of calcium which might enter from the tissues.

All the solutions contained a suspension either of carbon or of red blood cells. The carbon suspension was prepared in warmed gelatin- Ringer solution and immediately filtered. The cell suspension was prepared with washed rooster blood cells. The filtered carbon suspen- sion flowed readily through the capillaries. The avian cell suspension in frog’s Ringer gave a strikingly natural appearance to the circulation since the cells maintained their normal plasticity and non-adhesiveness as they coursed through the blood vessels of the frog.

The mesentery was kept moist throughout the course of an experiment with a solution similar, except for the lack of the gelatin, to the perfusate being used.

Several criteria were used to ascertain the ef‘fcct of varying the concen- tration of calcium in the perfusate. Macroscopic evidence was obtained by noting the time of development of edema in the exposed intestinal loop and in the spleen. The latter, attached by a short stalk to the root of the intestinal mesentery, is almost spherical and about 5 mm. in diameter in the average sized frog. Whenever marked edema ap- peared, it did so rather suddenly, the spleen attaining well over one- third and the intestine fully twice the normal dimensions.

The microscopic evidence depended upon the type of particulate material suspended in the perfusate. In the case of the avian red cells, evidence for a marked leakiness was a spreading out of the cells from the central channel of the stream in the capillaries, often culminating

258 ROBERT CHAMBERS AND B. W. ZWEIFACH

in extravasation of many of the cells and in general stasis. This is in contrast to the normal condition in which the cells course rapidly along a central channel in the sfream the cells keeping away from the walls except at occasional sharp bends of the capillary. When the control solution (normal Ringer-gelatin, pH 7.5) was used, no capillary stasis was observed up to 2 hours of perfusion. I n the case of a carbon suspension, the larger particles behaved in a similar way to the red cells. A marked leakiness of the wall was detected by the dispersal of all the particles throughout the capillary lumen and in a pronounced adhesion of the carbon, often to tlie extent. of occluding the lurnen. Extrusion of the carbon particles tlirongh openings in the capillary wall was noted in extreme cases only.

EXPERIMEKTAL

A. E x p e r i m e n t s O ~ L frogs with ioztact circulation

The various vessels in the capillary bed are given the terminology introduced by Zweifach ( ’37). The arteriole ends in the arterio-venular capillary which maintains a deficient muscularity throughout until it merges into a venule. I n the carbon perfusions the arterial end of the a-v capillary can be readily distinguished from the venous end which develops marked adhesive properties much earlier. The true capillaries with no muscle cells are side-branches of the a-v capillaries.

As a preliminary to the perfusion experiments, observations were made on the exposed mesentery of the frog with an intact blood circula- tion. 1. Treutmemt with AgNO,. A micropipette, filled with a solution of

loo/; AgNO,, was inserted through the mesothelium and the solution gently sprayed on the outer surface of the wall of a capillary in the microscopic field. Within 2 minutes the blackened outlines of the endothelial cells appeared with no interruption of blood flow in the capillary. Twenty to 30 minutes later, small black globules were seen to come off the endothelial lines and were swept away by the blood stream. This process continued until all trace of the stain disappeared without any change indicating leakiness or rupture of the capillary wall. This suggested that the stained intercellular material was being replaced by fresh material.

2. Carbon suspemion in the circulati*%tg blood. A heavy suspension of carbon black was prepared in frog’s Ringer and allowed to stand f o r several days in a tall glass cylinder. The supernatant fluid, containing the finer carbon particles, was decanted off and used for

ChPILLAUY CEMENT A N D PERBIEABILITY 259

the iiijections. A frog was prepared for ohserving tlie meseiiteric blood vessels and about $ cc. of the suspension was injected into the heart. The amount gave a pale greyish tinge to the circulating blood. About 4 hour after injection, the walls of the capillaries in the mesentery showed delicate, black lines which progressively coarsened with the iiicreasing accumulation of the adhering carbon particles. TYitliin 1 to I + hours the deposition of the carbon outlined most of the endothelial cells iii the same way as with the AgNO, treatment. Figure 1 presents tlirce photographs of segments of actively streaming capillaries showing carbon deposits along the endothelial lines.

Ari iiiimediate response of locally prodding the wall of a capillary was tlie plastering of red cells against the irritated portion of the wall

Flg. 1 Three photographs showing carlion deposited along the eiidothelial lines of living c~pi l la r ies in the mesentcry of t h e frog. a. A n a-v capillary with rapid circulation which accentuated the deposition of carbon in longitudinal striae. b and c. True capillaries with cuibon tending to outline the endothclial cclls especially in c.

followed by a marked depositioii of carbon in the saiiie region. The continued deposition of carbon occurred primarily along the endothelial lines, but later spread over the entire inner surface of the endothelium of the irritated area. The capillary was then compressed with the shaft of a microneedle just above the irritated region. This temporarily interrupted the flow of blood and the consequent drop in internal pressure caused the attached red cells and some of the accuniulated carbon to fall way from the wall. Most of the carbon, however, remained attached. The needle was now removed to restore the flow of blood. Gradually, more and more of the carbon on the inner surface of the capillary fell away except for tlie carbon adhering to the endothelial lines.

260 ROBERT CHAMBERS A N U B. I\-. ZWEIFBCH

B. Perfusion with artificial f lu ids contaimiitg carbon ilt suspeizsioii

I n the experiments with the various perfusates a pronounced effect observable is the difference in the sticking of carbon to the capillary walls. When calcium-free perfusates are used the sticking tends to occur very early and is transitory. This is due to the gradual dissolution of the intereridothelial cement, there being a preliminary swelling with an accompanying stickiness which disappears as the swollcn cement goes into solution and is washed away. On the other hand, when the perfusate contains excess free calcium, the developing stickiness is due to the excessive production of cement which progressively spreads ovw the entire inner surface of the vessels.

Some of the experimental data are presented in the form of proto- cols of typical experiments. In a number of the protocols mention is iiiade of occasional leucocytes which are left behind after the blood had been replaced by the perfusate.

Perfusates containing excess calcium tended to clot the blood and clog the ressels unless the blood was first washed out with a perfusate containing the normal amount. of calcium. Normally, the replacement of the blood is complete within the first minute of the perfusion. With normal amphibian Ringer-gelatin + carbon the perfusion can be carried on for about 2 hours before conditions appear which differ from those ohserved with the intact blood circulation. During the first hour or so of perfusion with normal Ringer solutions, an increase in the perfusion pressure to 60 mm. Hg simply distends the capillaries without other visible effects. The data of a typical experiment with normal Ringer arc compared in protocol I with those of a calciurri-free perfusate. 1. Perf irsions with, alkaline ( p H 7.4) a i d acid ( p H 6.8) calcium-free

Ringrr-geZatii/i. With the alkaline perfusate the capi1lai.y walls became excessively leaky within 35 to 40 minutes. This resulted in an increased sluggishness of the flow, an early effect of which was the scattering of the suspended particles throughout the lumen of the capillary instead of their being conceritrated in the central channel of the stream. The piling up of the carbon in irregular clumps along the walls is a result of an outward diffusion of fluid through a leaky membrane which holds back the suspended particles as by a filter. This type of carbon accumulation was characteristic for calcium-free perfusions.

When the perfusate was acidified to pI3 6.5; the effects noted mere even more striking. A comparison of the results with the calcium containing and calcium-free perfusates in protocol I shows strikingly the effect of the lack of calcium especially when the perfusate was more acid than normal.

CAPILLARY CEMENT AND PERMEABILITY 261

Protocol I . Acidified, ca.lcium-free compared with alkaline, calcium- containing Ringer-gelat,in + carbon.

Calcium-free perfusate, pH 6.6 to 6.8

0 min. Perfusion started, pres- sure at 30 mm. Hg.

10 min. Occasional, slight, linear deposits of carbon on capillary wall.

15min. Caybon washed off wall but tending to be pe- riplicral in stream.

20mjn. Carbon sucked in sudden spurts from central stream toward wall at various points.

25min. Rate of flow slowed. Edema developing.

30 min. Lencocytes which are on wall have flattened out ; many undergoing rapid diapedesis.

35min. Eddies appear piling up carbon in spots along wall.

40 min. Extrusion of carbon through wall.

50 min. C’logging of capillaries by large clumps of carbon.

60 min. Flow considerably slowed and abnormal. Experiment ended.

80 min. . . . . . . .

110 min. , . . , , . . . . . . . . . , . . . . . , . .

C alcinm-cont aining perf us ate, pH 7.4 to 7.6

Carbon confined to central channel of capillary. No deposition of carbon.

l l any leucocytes adhering t.o inner wall of venous end of a-v capil- laries. A few undergoing dia- pedesis.

Finer carbon particles beginning to stick to wall of venous a-v capillaries.

Occasional leucocyte attached to wall.

Longitudinal striae of carbon on cement lines accentuated in di- rection of flow.

Carbon accumulated only along cndot,helial lines.

Flow rapid and normal.

Carbon lines evident in most capillaries.

Sticking still confined to endothe- lial lines. Axial flow persisting. Slight edema developing.

Experiment ended

In the acidified calcium-free perfusate the wall of the capillary became sticky very quickly. Eddies of swirling carbon particles ap- peared a s early as 20 to 25 minutes after starting the perfusion and the extrusion of the carbon through the walls became evident within 40 minutes. As in previous experiments, tliese alterations in the vessel

262 ROBERT CHAMBERS AND B. W. Z W E I F A C H

wall were especially noticeable in the venous capillary segments. An increased perfusion pressure (up to 60 mm. Hg.) resulted in the distension and rupture of the capillary at numerous spots evidenced by a spurting out of carbon masses.

In the perfusions with calcium-f ree solutions many of the adhering leucocytes were soon dislodged and carried away. Others become flattened against the wall and underwent active diapedesis. In the acidified calcium-free perfusate (pH 6.6 to 6.8) an extensive and rapid emigration of the leucocytes was accompanied by other evidences of leakiness.

2. Perfusions with acid, normal Ringer-gelat in , p H 6.6 t o 6.8. The adhesion of the carbon to the endothelial lines developed earlier than with the more alkaline normal perfusate. Edema, as shown by the swelling of the intestine and of the spleen, occurred within 50 to 60 minutes after the perfusion had been started. When the perfusion was carried on fo r longer than 1 hour, carbon became deposited over the entire inner surface of the venous a-v capillaries. Occasionally, during the preparation of the mesentery f o r the experiment, excessive handling produced a slight injury to some of the capillaries. During the perfu- sion these vessels acquired an extra heavy coating of carbon.

3. Perfusions with double calcium Riizger-gelatin, p H 68. The excess calcium in the perfusion fluid increases the adhesiveness of the carbon to the capillary wall so that after a period of time long heavy lines of carbon become prominent in the venous, a-v capillaries. With an alkaline perfusate (pH 7.5), a slight edema began to develop only after 100 to 110 minutes.

With the acidified perfusate (protocol 11) the carbon adhered, not only to the endothelial lines, but also to the inner surfaces of the endothelial cells. The perfusate encountered increasing resistance dur- ing its passage through the vessel and many of the capillaries became clogged within 15 minutes. It is significant that an increase in the perfusion pressure had no effect on the diameter or the relative im- permeability of the capillaries a t this stage.

Protocol 11: Double-calcium Ringer-gelatin, carbon, pH 6.8. The blood was first washed out with the normal perfusate.

0 min. Perfusion started, pressure at 30 mm. Hg. 7min. Sticking of carbon appearing early in venous a-v capillaries.

15 min. Thick ridges of carbon deposited along endothelial lines. Many true capillaries becoming clogged at their venous ends.

25 min. Leucocytes rounded and sticky, clinging to wall. 30min. Carbon adhering to inner surface of venous, a-v capillaries.

CAPILLARY CEMENT AND PERMEABILITY 263

40 min. Adhering leucocytes coated with sticky material to which carbon

60 min. Heavy deposit of carbon over entire inner surface of all capil-

75 min. A-v capillaries, hitherto open, are becoming clogged with

particles adhere.

lary walls.

carbon. Edema developing.

C. Revers ib i l i t y of ef fects with double perf usions These experiments demonstrate the reversibility of the effects ob-

tained by the presence or absence of calcium in the perfusates. Protocol 111 shows that these extreme co~~ditions are readily reversible, provided the perfusion is not carried on too long. Protocol 111: Acid (pH 6.8) double-calcium followed by acid, calcium- free Ringer-gelatin + carbon. Pressure at 30 mm. Hg.

The blood was first washed out of the vessels by perfusing with normal Ringer-gelatin solution f o r 3 minutes. The experimental solu- tion was then introduced.

20 niin. Rapid accumulation of carbon along the endothelial lines. 25 min. Carbon deposited in thick ridges. 30 min. Circulation still normal except for increasing sluggishness.

40 min. (10’ after change). Washing away of carbon becoming evident. 50 min. (20’ after change). Carbon deposits found only occasionally. 60 min. (30’ after change). Peripheral eddies in the stream and char-

acteristic clumps of carbon begin to form. 65 min. (35’ after change). Edema is evident.

Leakiness and non-sticky condition of the capillary wall, resulting from perfusion with a Ca-free solution, can be completely reversed by changing the perfusate to one containing calcium. When the perfusate is a normal calcium Ringer-gelatin solution at pH 7.4, the reversion reaches a steady state in which there is not sufficient leakage to induce edema and in which the stickiness of the wall remains restricted to the intercellular lines.

0 min. Changed to double-calcium perfusate.

Changed to calcium-free perfusate.

D. P e r f u s i o n with suspensioms containirzg avian red cells A remarkably close resemblance to normal circulating blood was

obtained by perfusing a suspension of rooster red cells in normal frog- Ringer-gelah. To accentuate the effect of the lack of calcium a calcium- free perfusate acidified to pH 6.8 was used. The avian cells suspended in this perfusate had been washed several times in calcium-free Ringer before preparing the suspension.

264 ROBERT CHAMBERS AND B. W. ZWEIFACH

The red cells became plastered against the capillary wall in increasing numbers within 30 to 35 minutes. This was not a result of adhesiveness. Lateral currents, set up by the leaky condition of the endothelial membrane, displaced the cells from the central channel and drove them against the wall. When a change occurred in the direction or rate of flow, a red cell, lying against the wall, could be seen to fall away. A similar dislodging of the cells could be induced by gently pressing on the capillary with a microneedle.

Cells remaining on the walls began to be extruded within a few minutes through weak spots in the wall, a s if they were being squeezed through minute pores. The extruded portion of the cell bulged on the outside of the wall. A t this stage the portion of the cell on the inside could be lifted momentarily off the wall by prodding with a microneedle. Within about 75 minutes owing to the excessive loss of fluid the cells became concentrated especially in the venous a-v capil- laries and small venules. Stasis ensued in about 90 minutes.

Perfusions of suspension of red cells were also made with double calcium Ringer-gelatin at p H 7.5. The perfusion maintained an adequate circulation for 30 to 40 minutes and no extravasation was observed. A marked diminution in the rate of flow occurred about 45 minutes after beginning the perfusion, the vessels becoming crowded with red cells.

When the double-calcium solution was acidified to pI-1 6.6 to 6.8, the circulation rapidly became abnormal within 10 to 15 minutes, being unusually slow and sluggish throughout. The true capillaries became quickly clogged and, within 30 minutes, the flow was confined to the a-v capillaries. Ten to 15 minutes later the flow was intermittent and uncertain. No definite symptoms of edema appeared. This is in marked contrast to the slowing of the flow and the marked edema observed after perfusing with calcium-free solutions.

E. T h e e f f e c t o f variations in pH on the appearamce o f stickiness in the capillaries and of edema

A series of perfusion experiments were made in which the pH of the perfusion fluid was varied from 6.6 to 8.0 using phosphate mixtures as buffers. Three kinds of Ringer-gelatin mixtures were used, one with the normal amount of calcium, the second with double the normal amount of calcium, and the third was free of calcium.

The results, given in figures 2 and 3, were taken from fifty-nine perfusions, each point on the curves representing an average from several separate perfusions, viz., 4 t o 5 for the points in the p H range between 7.0 and 7.6, and 2 to 3 at the two extreme ends of the curves.

CAPILLARY C E M E N T A N D P E R M E A B I L I T Y 265

The results from perfusates containing the normal amount of calcium arc plotted in figure 2 A those from perfusates in which the calcium content was doubled are in figure 2 B. The curves in both figures are similar. It is to be noted that the two curves of each figure tend to parallel one another especially where the slope is steep along the inter- mediate pH ranges. With increasing alkalinity and acidity, respec- tively, of the medium, the permeability of the capillary wall and the

Fig. 2 (A) Normal Ringer-gelatin perfusate. (B) Calcium-free Ringer-gelatin perfusate. Graphs showing time of appearance of carbon-sticking and of edema in relation t o variations in pH of the perfusate. Normal pH range of frog’s blood indicated between vertical broken lines.

stickiness to carbon tend to reach a comparatdvely constant value as shown by the flattening of the curves. It is also to be noted that, in both charts, sticking of the carbon occurs before the appearance of edema by a fairly constant interval of time along the entire curve. With the normal calcium perfusate the interval is about 20 to 25 minutes. Wit.h the double calcium perfusate the interval is longer; viz., 35 to 50 minutes. The appearance of edema, with the normal perfusate at the

266 ROBERT C H A M B E R S AND B. W. ZWEIFACH

70-

' 60- w

Z 5 50- - 2 40-

- 220- 230- w

I- I O -

pH of normal frog blood (pH 7.5), figure 2 A, did not occur until about 110 minutes after commencing the perfusion. Figure 2 8 shows that the appearance of edema is delayed with the increase in alkalinity of the perfusate. An increase in acidity causes an early onset of edema.

The reactions are best explained by assuming that the component which conditions the diffusion of fluid through the capillary wall has the nature of a calcium salt. The sigmoid shape of the curve suggests a dissociation phenomenon. The component is increasingly stabilized on the alkaline side where calcium ionization is less and is rapidly

0 - CARBON

A- EDEMA CLUMPING

l l

weakened and dissipated on the acid side where the calcium ionization is greater.

The steepness of the curve through the pH range of pH 7.4 to 6.8 in figure 2 A indicates a pronounced susceptibility of tlie wall within this range. The time of appearance of edema was fairly uniform and in accord with the protocols described in this paper. In the case of perfusates containing an excess of calcium the variation was much greater between the average values represented in figure 2 B and those in the individual protocols. With double calcium the analysis of the causes of edema and of sticking is complicated by the developing back-pressure resulting from a progressive clogging of the venous vessels.

CAPILLARY C E M E X T A N D PERMEABILITY 267

Figure 3 gives curves showing the effect of calcium-free perfusates. I n contrast to the effect of the calcium-containing perfusates it is to be noted that edema occurred very early. The curve denoting the time of carbon clumping is not to be compared with those in figure 3 which represent carbon-sticking. In the 'calcium-free perfusions the appearance of carbon on the walls occurred later than the edema and there was no evidence of sticking. The material responsible for the stickiness was washed away. The deposition of the carbon in clumps against the wall was caused by the force of the fluid diffusing out through excessively porous portions of the vessel wall. The consequent weakened condition of the wall was demonstrated by raising the perfusion pressure to 60 mm. Hg whereupon the vessel became sli<ghtly distended and suddenly the carbon particles spurted singly and in clumps at several points through the wall into the surrounding tissue.

A further difference in the nature of the reaction between the calcium- containing and calcium-free perfusates can be seen by comparing the shape of the different curves. In figure 3 the curves tend to be linear. This suggests a reaction with a graded increase in intensity at the successive pH ranges from 8.0 down to 6.6.

DISCUSSION

The experiments described in this paper indicate the important role of the intercellular cement in the permeability of the capillary endo- thelium. The indications are that the permeability of the endothelial membrane may be explained without taking into account the permea- bility of the individual cells.

A histological study by Volterra ('25) is significant because of the attention it focusses on the non-cellular constituent of the blood capil- laries. Volterra described a clearly defined, argentophilic, amorphous, pericapillary sheath which, in inflamed tissues, was swollen and unstainable with silver. He interpreted the increased permeability of the capillaries to the observed changes in the sheath and generalized that the permeability of the capillary is a property of the amorphous, non-cellular component.

The role of calcium in controlling the permeability of blood vessels has already been considered by several investigators. Chiari and Januschke ( '10) induced a reduction of edematous blisters by injecting the neighboring tissue with CaCl,. They ascribed the reduction to a decrease in the permeability of the blood vessels. Hirschfelder ( '24) repeated this but was unable to obtain consistent results. A claim for a similar action of CaC1, was made by Hamburger ('22) from his

268 ROBERT CHAMBERS AND €3. W. ZWEIFACH

perfusion experiments on the hind legs of frogs. The evidence presented by Haniburger was somewhat fragmentary and has been regarded by Krogh ( '29) as inconclusive.

Landis, in a recent review article ( '37), remarked upon the clificulty of interpreting the recorded effects of calcium on capillary permea- bility. He referred to Hamburger (loc. cit.) but also to others who used indirect methods and secured uncertain or confusing results.

The experiments on the effects of calciuni described in this paper are all with the use of artificial perfusates. They have the disadvantage of not dealing with possible permeability changes in the presence of the normal, circulating blood. However, they have the advantage of affording controlled variations in the calcium content, essential as a preliminary for further work under more normal conditions. I n so far as they go, the experiments clearly indicate that the physical state of the capillary wall depends upon the amount of calcium present in the perfusate. Moreover the conditions produced are reversible. They appear to be closely analogous to those of epithelial membranes in which the intercellular cement reacts to inorganic salts as if it were itself a salt. It is also significant that a relatively slight change in pH of the perfusion fluid, as shown in figure 2, is sufficient to increase markedly the permeability of the capillaries. This is explicable on the basis of regarding the cement substance as an organic salt the ioniza- tion of which is increased with an increase in acidity. With increasing ionization the cement would become more soluble and more apt to he dissipated. The absence of calcium from the medium would still further increase the instability through the elimination of the rela- tively stable but reversible calcium salt and its replacement by a sodium or potassium salt.

It is interesting to note that Ringer (loc. cit.) appreciated the stabilizing effect of the alkalinity of the medium for he states that NaHCO, and tribasic phosphates tend to prevent disintegration of the intercellular cement even in the absence of calcium. In this relation is the finding by Volterra (loc. cit.) that the argentophilic nature of the non-cellular, pericapillary component becomes appreciably di- minished in inflamed tissues. This would be explicable on the ground that the greater acidity of the inflamed regions inhibits the formation of a stable silver salt with the non-cellular component. The fact that Landis ('28, '34) found no effect of pH change on the permeability of the mesenteric capillaries of the frog might be due, as he himself has pointed out, t o the method he employed. He exposed the mesentery to acidified Ringer's solution without, however, determining whether

CAPILLdRY CEMENT A S D PERMEABILITY 269

this caused an actual change of pH in the tissue and around the capillaries.

Ellinger and Heyman ('21) stressed the importance of serum in inhibiting edema. Drinker ( '33) repeated the experiments and found that the use of salt solutions with gun1 acacia alone was insufficient to prevent edema, whereas the addition of 20% horse serum was effective. He mentions Krogh's suggestion of a hormone in controlling the capillary permeability but leaves the question open. Gum acacia contains a considerable amount of bound calcium and it is possihle that the availability of this calcium is enhanced by something in the added serum. It is also possible that the proteins added with the horse-serum coat the inner surface of the capillaries. This would act to decrease the permeability of the wall.

Vitamin C has been shown, from separate sources in the literature, to be concerned both with calcium metabolism and with the deposition of intercellular cement substance. The experiments described in this paper indicate a relation between these two factors, since the experi- ments show a dependence of the intercellular cement of capillaries upon the presence of calcium. -4 similar relationship is indicated in the work of TVolbach and Howe ('26) who found that vitamin C deficiency is responsible for the failure of the forinatioii of intercellular substance in the healing of wounds. The lack of vitamin C is also known to be responsible for capillary hemorrhages. This suggests a connection between the weakness of the capillary wall and a deficiency of the intercellular cement which may be due to disturbed calcium availability.

The cement appears to be a product of the endothelial cells. This is not unusual. Wolbach and Howe (Ioc. cit.) have indicated a similar production by various connective tissue cells. The secretion of material to form an extraneous hyaline layer on the protoplasmic surface of developing echinoderm eggs has been the object of a detailed study (R. Chambers and M. J. Kopac, in press).

Concerning the endothelial cell, the observations of Volterra (loc. cit.) indicate the presence of an extraneous material on the outer surface of the endothelium. The evidence presented in this paper shows that the cement may be secreted not only between the endothelial cells but also over the surfaces facing the capillary lumen. This was demonstrated by increasing the stabilization of the cement with the use of perfusates containing excess calcium. When normal Ringer's solution was used, the cement was less stiff and, hence, whatever was present on the exposed inner surface of the capillary, tended to be carried away by the stream- ing fluid in the capillaries. Therefore under normal conditions the

270 BOBERT CHAMBERS AND B. W. ZWEIFACH

stickiness is restricted, at least as concerns the inner surface of the capillary wall, to the contiguous borders of the endothelial cells.

In our experiments the two indicated factors which enhance the stability of the cement are tlie alkalinity and the calcium content of tlie perfusing fluid. An important role of the endotlielial cell may thus be the continual secretion of an intercellular cement, the chemical stability of which controls the permeability of the blood capillary.

SUMMARY

I. The inter-endothelial cell cement during normal blood circulation.

1. The endothelial lines can be stained, under viable conditions, by spraying 10% AgNO, on the outer surface of the capillaries with a micropipette. The blackened substance of the cement is gradually washed away by the blood stream and replaced by fresh cement.

2. Carbon, suspended in the blood stream, adheres to the sticky cement without adhering to the exposed surfaces of the endothelial cells. In this way the carbon-deposit outlines the endothelial cells.

3. When the capillary is prodded with a microneedle, there occurs an immediate flattening of red cells against the irritated wall and a local accumulation of carbon. This indicates an excessive leakage of fluid through that region of the capillary.

11. The effect of perfusion of physiological salt solutions containing ash- free gelatin (0.5%) on the inter-endothelial cell-cement. (All the per- fusates contained particulate matter, either carbon or rooster red cells, in suspension.)

A. By varying the calcium content of the perfusate. 4. The capillary bed caii be perfused with a normal Ringer-gelatin

solution fo r over 100 minutes with no visible abnormalities. 5. With solutions lacking calcium, a softening of the cement is

indicated by its increased stickiness and its being washed away. 6. With solutions containing double the amount of calcium, the

stickiness of the endothelial lines becomes intensified and later the entire inner surface of the endothelial wall becomes sticky.

B. By varying the pH. 7. An increase in acidity of the medium induces early and intense

stickiness followed by pronounced leakiness of the capillary wall.

CAPILLARY CEMENT A N D PEBMEABILITY 271

111. The relation between the induced changes in the endothelial cement and the extrusion of particulate matter and the appearance of edema.

8. The lack of calcium and increase in acidity soften the cement (evidenced by increased stickiness to carbon) and enhance its dissipa- tion. These conditions favor the extrusion of formed elements of the perfusate (e.g., carbon, leucocytes, red cells) and accelerate the outward diffusion of the perfusate.

CONCLUSION

An important role of the endothelial cell is the continual secretion of an intercellular cement, the chemical stability of which controls the permeability of the blood capillary.

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