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Notes: Transmission electron microscopy and ultracytochemistry were employed in an attempt to localize the enzyme calcium adenosine triphosphatase (Ca-ATPase) in the rod outer segments (ROS) of the toad retina. Utilizing a one-step incubation procedure, Ca-ATPase was identified as an electron-dense precipitate in the intradiskal spaces of the rod disks (vesicles) of the ROS. Analytical microscopy identified the reaction product as lead phosphate. The formation of the reaction product was dependent on the presence of ATP (the substrate) and calcium ions. However, calcium ions could be substituted for by magnesium ions. In addition, the reaction was vanadate sensitive. The latter is known to inhibit Ca-ATPase activity. Such data appear to indicate the presence of a Ca-Mg-ATPase in association with the rod disks. Since cyclic guanosine monophosphate (cyclic GMP), rather than calcium ions, is currently believed to be the primary intracellular messenger associated with phototransduction, the presence of an ROS Ca-ATPase may indicate other functions for this cation in the physiology and biochemistry of the visual process. Ca-ATPase might play a role in directional calcium fluxes between intracellular compartments.

Notes: A cytochemical technique for the electron microscopic localization of calcium adenosine triphosphatase (Ca-ATPase) was utilized to localize this enzyme in the enterocytes of rachitic and vitamin D-replete chicks. In animals treated with cholecalciferol (CC, vitamin D3), an electron-dense reaction product was located along the basolateral membranes of the absorptive cells within 72 hr after injection. Similarly, a reaction product was identified in association with the basolateral membranes within 24 hr after injection of 1,25-dihydroxycholecalciferol, the active metabolite of vitamin D. A microvillar reaction product was not seen in either of these two groups. Electron-dense reaction products were also seen in association with mitochondria and scattered throughout the cytoplasm of these enterocytes. The Ca-ATPase reaction product was dependent upon the presence of medium calcium and substrate (ATP), was inhibited by vanadate, and was heat labile. In the rachitic animals, a reaction product indicative of Ca-ATPase activity was not seen in association with either the basolateral membranes or the mitochondria. These data appear to indicate that an energy-requiring calcium-activated membrane pump plays a role in the flux of calcium across the enterocytes of the small intestine.

Notes: New methods of tissue preparation were developed to study the morphology and distribution of calcium ions in duodenal enterocytes from normal, rachitic, and vitamin D-replete (either cholecalciferol [CC] or 1,25-dihydroxycholecalciferol [1,25-DHCC] treated) chicks. Frozen hydrated sections were prepared from cryofixed tissues by ultracryomicrotomy at - 125° C. Sections were subsequently freeze-dried by increasing the temperature to - 100°C. The latter temperature was maintained throughout both the structural and elemental analyses. In cells from normal, rachitic, and vitamin D-treated [CC] animals the brush border from lanthanum-infused tissues was electron dense and calcium-lanthanum positive by x-ray analysis. In the absence of lanthanum, i.e., sucrose-infused duodena, the microvilli were still calcium positive. In the terminal web region of normal and CC-treated enterocytes, numerous, apparently interconnected, tubules and vesicles were seen. Vacuole-like structures were also seen. Such structures were especially prominent in the enterocytes from the vitamin-treated [CC] animals. Except for the vacuoles, the tubules and vesicles were electron dense in the lanthanum-infused duodena, and clear in sucrose-infused tissues. In both instances, the structures were calcium positive. Similar, but even larger structures were seen below the terminal web. Here however, the tubules and vesicles seemed to be organized into multiple complex interconnecting networks, i.e., tubulo-vesicular complexes. Both the tubules and the vesicles seemed to be interconnected via smaller channel-like entities. The extensiveness of this structure was better appreciated in the enterocytes from lanthanum-infused tissues, where it appeared similar in structure and complexity to an en face view of the sarcoplasmic reticulum of skeletal muscle. These intestinal complexes were less well developed, decreased in number, and quite often absent, in the apical cytoplasm of absorptive cells from rachitic chicks.In the enterocytes from animals treated for 24 hours with 1,25-DHCC, the same highly developed tubulo-vesicular networks were again seen in the enterocyte apical cytoplasm. They were even more developed in the 1,25-DHCC-treated animals. All structures were intensely calcium positive in enterocytes from both the lanthanum- and the sucrose-infused preparations. Numerous endocytotic (pinocytotic) vesicles were seen at the lumenal plasmalemma. Similar structures were also apparent in the terminal web region of the 1,25-DHCC-treated enterocytes. Exocytotic vesicles were seen at the apical aspect of the lateral cell membrane, below the level of the junctional complex. All components of this unique system contained high concentrations of calcium. A similar, apically located, tubulovesicular complex has not been described in the enterocytes of conventionally prepared tissues obtained from either normal or vitamin D-replete duodena from rats, mice, chicks, etc. Thus it appears that this system, which could play a role in intestinal calcium transport, is extremely labile and apparently not preserved (maintained) by ordinary biological fixatives and/or by routine methods used for the preparation of tissues for electron microscopy. Therefore, cryofixation, ultracryomicrotomy, etc., may prove to be essential for the identification of transiently induced states of cell activation associated with hormones, growth factors, nerve impulses, etc.Since the dilated components of the tubulo-vesicular system described herein resemble (size, shape, density, etc.) the so-called calcium lysosomes previously described by our group in normal and vitamin D-replete chick enterocytes, the effect of 1,25-DHCC on certain lysosomal acid hydrolases was determined. After 24 hours treatment with 1,25-DHCC, there was a three to fourfold increase in the release of lysosomal enzymes such as acid phosphatase, cathepsin B, and B-N-acetyl-D-glucosaminidase into the medium of isolated enterocytes in comparison to untreated control cells. This would seem to indicate that the tubulo-vesicular network described here is comprised, at least in part, of lysosomes.