Previous work has shown that endogenous chemical mediators, of which histamine is the prototype, increase the permeability of blood vessels by causing gaps to appear between endothelial cells . In the present paper, morphologic and statistical evidence is presented, to suggest that endothelial cells contract under the influence of mediators, and that this contraction causes the formation of intercellular gaps . Histamine, serotonin, and bradykinin were injected subcutaneously into the scrotum of the rat, and the vessels of the underlying cremaster muscle were examined by electron microscopy . To eliminate the vascular collapse induced by routine fixation, in one series of animals (including controls) the root of the cremaster was constricted for 2-4 min prior to sacrifice, and the tissues were fixed under conditions of mild venous congestion . Electron micrographs were taken of 599 nuclei from the endothelium of small blood vessels representing the various experimental situations . Nuclear deformations were classified into four types of increasing tightness (notches, foldsl closing folds, and pinches. In the latter the apposed surfaces of the nuclear membrane are in contact) . It was found that : (1) venous congestion tends to straighten the nuclei in al groups ; (2) mediators cause a highly significant increase in the number of pinches (P < 0 .001), also if the vessels are distended by venous congestion ; (3) fixation without venous congestion causes vascular collapse . The degree of endothelial recoil, as measured by nuclear pinches, is very different from that caused by mediators (P < 0 .001) . (4) Pinched nuclei are more frequent in leaking vessels, and in cells adjacent to gaps (P < 0 .001) ; (5) mediators also induce, in the endothelium, cytoplasmic changes suggestive of contraction, and similar to those of contracted smooth muscle ; (6) there is no evidence of pericyte contraction under the conditions tested . Occasional pericytes appeared to receive fine nerve endings. Various hypotheses to explain nuclear pinching are discussed ; the only satisfactory explanation is that which requires endothelial contraction .

A current hypothesis maintains that the vascular leakage induced by histamine, serotonin, and bradykinin is brought about by a hydrostatic mechanism: The larger veins are said to contract; the venules are therefore submitted to an increase in pressure, which causes their wall to give way. To test this hypothesis, vessels of an exposed striated muscle (rat and rabbit cremaster) were photographed while treated with the above-mentioned substances; the occurrence of vascular leakage was tested by the intravenous injection of carbon black (carbon labeling). All animals were under barbiturate anesthesia, given a neuromuscular blocking agent (gallamine triethiodid), and were maintained by artificial respiration, according to a technique previously described. In 72 rats and 4 rabbits, the first event observed was arteriolar dilatation. Constriction of a small vein was observed in only 1 in 76 experiments (in two other cases it was doubtful). It is concluded that venous spasm cannot account for the leakage induced by the mediators of the histamine type, though such leakage may be enhanced by venous spasm whenever it may occur. An alternative mechanism is proposed and discussed: Histamine-type mediators cause vascular leakage by a direct effect on the venular endothelium, which is induced to contract. FIGURE 2 Effect of bradykinin, 0.01 mg/ml, on rat oremaster. (1) 26 min after a first injection of carbon, a second one was given; 40 sec later, no vessels are labeled. Note caliber of arteriole A. (V = venule; large vessel is a vein.) (2) 20 sec after bradykinin (and 3 min after the second carbon injection). The arteriole has greatly dilated. (3) 50 sec after bradykinin. Arteriole is still dilated; venule V has begun to label. (4) 66 sec after bradykinin. Venule V is strongly labeled. The large vein has not become constricted throughout the sequence. Downloaded from 838 MAJNO, GILMORE, LEVENTHAL FIGURE 3 Sequence illustrating venular labeling after histamine, in the absence of venous contraction, and with minimal oenular dilatation. (1) 6 sec after carbon. Note laminar flow (x) in small vein. The carbon is unevenly distributed. (2) 11 sec after carbon. Laminar flow (x) is now apparent in other vessels. (3) 2 min after carbon. The blood is dark, but no venular labeling has developed. (4) 4 min, 5 sec after carbon and 1 min, 35 sec after Downloaded from 840 MAJNO, GILMORE, LEVENTHAL FIGURE 4 Effect of bradykinin, 0.01 mg/ml, on rat cremaster. (1) 2 min, 8 sec after carbon. Arrow points to a leukocyte "rolling" along the wall of the small vein. V = venule. (2) 5 min after carbon; decreased perfusion of the capillaries during a "resting phase." (3) 23 min after the first injection of carbon a second one was given: 1 min, 45 sec later, there is no vascular labeling at all; circulation is lively. (4) 2 min, 15 sec after the second carbonFIGURE 5 Effect of bradykinin, 0.01 mg/ml, on rat cremaster. (1) Normal circulation. (2) 15 sec after carbon. Note laminar flow (x). (3) 35 sec after bradykinin and 3 min after carbon. No labeling ...

Analysis of the cell surface movements of membrane moieties of lymphocytes induced by ligands is of interest both in regard to yielding information on the structural organization of the membrane, and also in terms of biological and immunological functional significance. The purpose of this paper is to present data on the fine structural aspects of these events and the topography of cell surface markers before, during, and after such movements. The observations made are consistent with and support several theses concerning the nature of such intramembranous movements, and the prerequisites necessary for such events to occur, and have marked bearing on current theories concerning the structure and behavior of cell membranes. Materials and MethodsReagents.--Antisera were prepared as in the previous paper. These antisera, together with concanavalin A (Con A)1 and the labels used for their ultrastructural detection, are shown in Table I. Immunoglobulin (Ig) was isolated from the three antisera by the preparative methods outlined previously (1). Rabbit anti-mouse Ig (RAMG) was chemically coupled to ferritin using difluorodinitrodiphenyl sulfone (FNPS) as conjugating reagent (2). 40 g.g of globulin and 116/zg of ferritin (Pentex 6 times crystallized, lot No. 7-1, Pentex Biochemical, Kankakee, IlL) were dissolved in a total of about 5 ml of phosphate-buffered saline (PBS), pH 7.0. About 4.5 ml 2% Na2CO~ was added and then, with stirring at 4°C, 0.5 ml of FNPS (0.5% in acetone) was added dropwise. Stirring was continued for 24 hr at 4°C. The solution was then dialyzed for 5 days against PBS in the cold, and spun for 30 min at 7500 g. Free Ig and ferritin were separated from the conjugated Ig by electrophoresis. The solution was electrophoresed for 20 hr on a Pevikon block (500 v, 56 amp) in barbital buffer, pH 8.6, 0.05 ~, with sodium phosphate buffer, pH 7.45, 0.2 ~, in the outer troughs. The fraction containing the ferritin-Ig

Histamine, administered to dogs by various routes, produced peribronchial interstitial edema. Using colloidal carbon as an electron-dense tracer, it was shown that bronchial venules had become leaky at the same time that the minute blood vessels of the pulmonary circulation remained unaffected by the histamine. The effect of histamine was rapid and brief in duration: wide interendothelial gaps appeared in the walls of bronchial venules through which blood and tracer escaped into the interstitium. This action of histamine seemed to be related to the presence in the bronchial venular endothelium of abundant cytoplasmic fibrils that presumably have contractile capacity. Water content of the lungs was consistent with morphologic evidence that histamine promoted the accumulation of fluid in the lungs. The same pattern of response was observed after bradykinin or compound 48/80, a mast cell degranulator. In contrast to these three agents, serotonin did not affect bronchial venular permeability to colloidal carbon. Consideration of these observations in the light of the large extent of the bronchial venular plexus, raises the possibility that the bronchial venular system may play an important reabsorptive role under normal circumstances and that it may also be involved in the genesis of certain types of interstitial pulmonary edema. KEY WORDSserotonin bradykinin 48/80 permeability bronchial circulation endothelial contraction electron microscopy colloidal carbon • In the systemic circulation, histamine, serotonin, and other vasoactive substances selectively affect the venular end of the microcirculatory bed (1-4). Whether the consecutive segments of the microcirculation the lung react differently to such agents, is unknown. Also unknown is whether the minute blood vessels of the bronchial and pulmonary circulations differ with respect to ultrastructure and permeability. These uncertainties merit clarification since the lung in many species is rich in histamine and other humoral substances that are capable of altering vascular permeability (5). Attempts by others to determine the effects of a variety of humoral agents on the permeability of small pulmonary blood vessels (6-8) have been inconclusive because the agents were administered in such quantities, and by such routes, that they exerted systemic as well as local effects.

Stroma-free hemoglobin is an electron-opaque molecule useful as a tracer for the ultrastructural stuty of pulmonary capillary permeability. After this tracer was infused into the isolated pulmonary lobe of the dog, the endothelial junctions of the capillaries, as revealed by electron microscopy, act like distensible pores, thus allowing the tracer to escape when the pulmonary artery pressure was raised above 50 millimeters of mercury.