Gallotannin, consisting mainly of low molecular weight esters such as penta- and hexagalloylglucoses (commercially available as tannic acid produced from Turkish nutgall), can be used for increasing and diversifying tissue contrast in electron microscopy. When applied on tissue specimens previously fixed by conventional methods (aldehydes and OsO4), the low molecular weight galloylglucoses (LMGG) penetrate satisfactorily the cells and induce general high contrast with fine delineation of extra- and intracellular structures, especially membranes. In some features, additional details of their intimate configuration are revealed. Various experimental conditions tested indicate that the LMGG display a complex effect on fixed tissues: they act primarily as a mordant between osmium-treated structures and lead, and concomitantly stabilize some tissue components against extraction incurred during dehydration and subsequent processing. Experiments with aldehyde blocking reagents (sodium borohydride and glycine) suggested that the LMGG mordanting effect is not dependent on residual aldehydes groups in tissues.

Interstitial cells of heart atrioventricular and sigmoid valves were examined in several laboratory animals (rabbit, hamster, rat, and mouse) and in humans. These cells constitute a large fraction of the total cell population of the valve; in mouse atrioventricular valves, they amount to approximately 30% of the volumetric density. By their ultrastructural features and functional properties, valvular interstitial cells are intermediate between fibroblasts and vascular smooth muscle cells. Like fibroblasts, valvular interstitial cells lack a basal lamina establishing direct and extensive contacts with collagen fibers, elastin microfibrils, and proteoglycans of the matrix. The cells have numerous slender and long processes, connected to one another, forming a complex cellular framework spanning the entire valve. Similar to smooth muscle cells, valvular interstitial cells are extensively coupled by communicating junctions as shown by thin sections, freeze-fracture, lanthanum staining, and carboxyfluorescein microinjection. The cells contain numerous bundles of actin filaments, which are decorated by the S1 fragment of heavy meromyosin. Valvular interstitial cells also express cyclic guanosine-monophosphate-dependent protein kinase, as detected by immunofluorescence and immunoperoxidase histochemistry. Motor nerve endings are located closely apposed to valvular interstitial cells: structurally most of them appear to be of the adrenergic type. Valvular interstitial cells contract on epinephrine or angiotensin II stimulation as shown both in culture and in situ (valvular strips). Taken together these observations suggest that VIC may have contractile properties, which can account for a controlled tonus, actively correlated with the cyclically changing forces acting on valves during diastole and systole.

Local differentiations within the endothelium of both muscular (diaphragm, myocardium) and visceral (pancreas, jejunal villi) capillaries have been studied in rats on sectioned and freeze-cleaved preparations . Four distinct parts have been recognized in the endothelial cells of all these vessels on the basis of subcellular components present in each part and on the basis of variations in the local frequency of plasmalemmal vesicles : (a) the parajunctional zone, (b) the peripheral zone, (c) the organelle region, and (d) the nuclear region . Our data indicate that -16, -7 .0, and 8 .5% of the endothelial cytoplasmic volume (in the peripheral zone) is accounted for by vesicles, their content, and their membranes, respectively . The average density of vesicular openings per µm 2 is 78 in diaphragm, 89 in myocardium, 25 in pancreas, and 10 in jejunal mucosa capillaries . The frequency of fenestrae is 1 .7 times as high in jejunal (26/µm 2) as in pancreatic capillaries (15/µm2), the corresponding fractional areas being -9 .5 and -6 0/,, respectively, of the endothelial surface . Intercellular spaces occupy a relatively small area (-0 .08 to 0 .2%) of the inner endothelial surface .

The pathway followed by macromolecules across the wall of visceral capillaries has been studied by using a set of tracers of graded sizes, ranging in diameter from 100 A (ferritin) to 300 A (glycogen) . Polysaccharide particles, i .e . dextran 75 (mol wt -75,000 ; diam -125 A), dextran 250 (mol wt 250,000 ; diam -225 A), shellfish glycogen (diam -200 A) and rabbit liver glycogen (diam -300 A), are well tolerated by Wistar-Furth rats and give no vascular reactions ascribable to histamine release . Good definition and high contrast of the tracer particles were obtained in a one-step fixation-in block staining of the tissues by a mixture containing aldehydes, Os04 and lead citrate in phosphate or arsenate buffer, pH 7 .4, followed by lead staining of sections . The glycogens and dextrans used move out of the plasma through the fenestrae and channels of the endothelium relatively fast (3-7 min) and create in the pericapillary spaces transient (2-5 min) concentration gradients centered on the fenestrated sectors of the capillary walls . The tracers also gained access to the plasmalemmal vesicles, first on the blood front and subsequently on the tissue front of the endothelium . The particles are temporarily retained by the basement membrane . No probe moved through the intercellular junctions . It is concluded that, in visceral capillaries, the fenestrae, channels, and plasmalemmal vesicles, viewed as related parts in a system of dynamic structures, are the structural equivalent of the large pore system .

Whale skeletal muscle myoglobin (mol wt 17,800 ; molecular dimensions 25 X 34 X 42A) was used as a probe molecule for the pore systems of muscle capillaries . Diaphragms of WistarFurth rats were fixed in situ at intervals up to 4 h after the intravenous injection of the tracer, and myoglobin was localized in the tissue by a peroxidase reaction . Gel filtration of plasma samples proved that myoglobin molecules remained in circulation in native monomeric form . At 30-35 s postinjection, the tracer marked '75% of the plasmalemmal vesicles on the blood front of the endothelium, 157 of those located inside and none of those on the tissue front . At 45 s, the labeling of vesicles in the inner group reached 6070 but remained nil for those on the tissue front . Marked vesicles appeared on the latter past 45 s and their frequency increased to -80% by 60-75 s, concomitantly with the appearance of myoglobin in the pericapillary spaces. Significant regional heterogeneity in initial labeling was found in the different segments of the endothelium (i .e., perinuclear cytoplasm, organelle region, cell periphery, and parajunctional zone) . Up to 60 s, the intercellular junctions and spaces of the endothelium were free of myoglobin reaction product ; thereafter, the latter was detected in the distal part of the intercellular spaces in concentration generally equal to or lower than that prevailing in the adjacent pericapillary space . The findings indicate that myoglobin molecules cross the endothelium of muscle capillaries primarily via plasmalemmal vesicles . Since a molecule of this size is supposed to exit through both pore systems, our results confirm the earlier conclusion that the plasmalemmal vesicles represent the large pore system ; in addition, they suggest that the same structures are, at least in part, the structural equivalent of the small pore system of this type of capillaries . late tracer (1), but the structural identity of the small pore system in the same type of vessels is still in doubt . Using horseradish peroxidase (HRP) (mol wt e-40,000, and -40-50 A) as a histochemically detectable tracer, Karnovsky (2) and Cctran and Karnovsky (3) arrived at the conclusion that it reaches the pericapillary spaces mainly through the intercellular junctions of the 4 2 4

We investigated the luminal surface of the continuous endothelium of the microvasculature of the murine heart and diaphragm to find out whether it has differentiated microdomains. The probes were ferritin molecules, cationized to pl 's 6.8, 7.15, 7.6, 8.0 and 8.4, which were introduced by retrograde or anterograde perfusion through the aorta or vena cava after the blood was removed from the vasculature. The pattern of labeling was analyzed by electron microscopy and assessed quantitatively by morphometry in arterioles, capillaries, and venules identified in bipolar microvascular fields in the diaphragm. The results showed that the plasmalemma proper was heavily but discontinuously labeled by all cationized ferritins (CF) used, the labeling being less extensive on the venular endothelium. CF had access as individual molecules to a fraction of the vesicular population opened on the luminal front of the endothelium. Plasmalemmal vesicle labeling increased from ~10 to ~25% as the pl decreased from 8.4 to 6.8. Vesicle labeling also increased with CF concentration in the perfusate. All CF binding sites were removed by pronase and papain. Heparinase and heparitinase caused only a slight reduction in CF labeling. Neuraminidase decreased the extent and density of labeling, especially on the plasmalemma proper of the venular endothelium; this decrease was particularly pronounced in old animals.In work already published we demonstrated that the luminal surface of the endothelium of routine fenestrated capillaries has biochemically differentiated microdomains generated by a preferential distribution of anionic sites (26, 28) and glycoconjugates (29). These microdomains are characteristically associated with structures involved in microvascular permeability, i.e, plasmalemmal vesicles, transendothelial channels, fenestrae, and their corresponding diaphragms (18, 23). (Differentiated microdomains were also found on the abluminal plasmalemma of the endothelium of fenestrated capillaries [30].) The chemistry of the anionic sites was partially defined in experiments in which a cationic probe, cationized ferritin (CF),~ was applied after the microvascular beds were perfused with proteases and glycosaminoglycan-degrading enzymes. The results showed that the sites are provided by proteogly1Abbreviations used in this paper: CF, cationized ferritin; DPBS, Dulbecco's phosphate-buffered saline.cans and sialoglycoproteins on the plasmalemma proper, and by heparan sulfate proteoglycans on fenestral diaphragms (32). In subsequent experiments, a comparable approach was used to map anionic sites on the continuous endothelium of the pulmonary microvasculature (22). Differentiated microdomains, comparable to those detected in fenestrated capillaries, were found associated with plasmalemmal vesicles and the plasmalemma proper. In addition, CF-detectable anionic sites were absent or present only at low density over large areas of highly attenuated, vesicle-free endothelium directly apposed to the alveolar wall.In this paper we present...

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Synthetic pentamonogalloylglucose applied to fixed tissues acts as a mordant, inducing high and diversified contrast similar to that obtained with natural gallotannins of low molecular weight (LMGG). By the separate use of each of the two moieties of the galloylglucose molecule, it was found that gallic acid is the mordanting agent. Glucose may contribute, however, to the effect by increasing the solubility and cross-linking potential of the compound, since the mordanting induced by gallic acid alone is weaker than that produced by its hexose esters. As suggested by results obtained with various phenolics and benzoic acid derivatives, the functional groups required for the mordanting effect of such agents are the carboxyl group, and at least one hydroxyl group concomitantly present on the benzene ring. In the case of galloylglucoses, it is assumed that the effect is due to hydrolysis products (gallic, digallic, or trigallic acids) or to the multiple hydroxyl groups of the intact molecule. Esters of gallic acid (propyl-and methylgallate), as well as pyrogallol, produce a "reversed staining" of all membranes, except for those of communicating (gap) junctions.In the previous paper (21), we showed that natural gallotannins of low molecular weight (LMGG) commercially available as tannic acid AR, usually extracted from Turkish nutgalls, can be used for increasing and diversifying contrast in electron microscopy, The effect was obtained on tissues previously fixed in one step by Os04 or in two steps by aldehydes and Os04. We demonstrated that the gallotannins act as mordants and ascribed this effect to their major components, namely, the penta-and hexagalloylglucoses, the relative low molecular weight of which may explain their satisfactory penetration into the cells.To exclude the possibility that other minor components or contaminants occurring in these natural gallotannins might be responsible for the mordanting effect, we have carried out experiments with synthetic pentagalloylglucose (i.e. synthetic ester of gallic acid with glucose [5,6] which has been found to be chemically identical to one of the main components of natural gallotannins [1,6,15, 181).In additional experiments we tried to find out which part of the galloylglucose molecule accounts for the mordanting effect and which functional groups are involved in this process. 622