Tissue fixation and osmium black formation with nonvolatile octavalent osmium compounds

Summary Several compounds of osmium^VIII, including potassium osmiamate and coordination complexes of OsO₄ with ammonia and various heterocyclic nitrogen compounds, have been synthesized and characterized. They have also been evaluated as substitutes for OsO₄ in postfixation of biological specimens and in light and electron microscopic cytochemical methods resulting in osmium black formation.The most useful of these osmic compounds, a molecular addition complex of hexamethylenetetramine (methenamine) with OsO₄, has a negligible vapor pressure of OsO₄. It has the molecular formula C₆H₁₂N₄.2OsO₄ and has been designated osmeth. Although it has only limited solubility, aqueous solutions of the compound (or of OsO₄) can be rapidly prepared by dissolution in a minimal amount of dimethylformamide and subsequent dilution with distilled water or buffer. Although stable in the solid state, the complex in solution undergoes partial dissociation releasing OsO₄, and the odor of OsO₄ becomes apparent.Such solutions of osmeth are (0.25%) considerably less concentrated with respect to OsO₄ than solutions (1–2%) ordinarily employed for ultrastructural preservation or in cytochemical studies. Osmeth has limited value for postosmication after glutaraldehyde fixation because the generation (release) of OsO₄ appears to be slow. Adequate osmication of tissue blocks exists only at the surface, but effective osmication can be achieved throughout tissue sections. In cytochemical reactions resulting in the formation of osmium blacks, the osmeth solutions are as effective as OsO₄ solutions of equivalent concentrations. Our findings indicate that OsO₄ solutions of less than 1% may be satisfactorily utilized in many cytochemical studies.Osmeth is safer and more convenient to handle than OsO₄ because small amounts may be solubilized as needed. It should be considered as a substitute for OsO₄ in ultrastructural cytochemistry.This investigation was supported by NIH research grant number DE 02668 from the National Institute of Dental Research and by NIH grant number RR 05333 from the Division of Research Facilities and ResourcesVisiting Professor, Dental Research Center, University of North Carolina at Chapel Hill, Jan.-May, 1975. Supported in part by USPHS Grant HD 09209     

Osmiophile Substanz in Blattzellen der Brombeere (Rubus fruticosus L. s. 1.)

Zusammenfassung Nach Fixierung der Brombeerblätter (Rubus fruticosus L. s. l.) mit Aldehyden (Formaldehyd, Glutaraldehyd sowie Acrolein) und Postfixierung mit OsO₄ erscheint im Cytoplasma gewisser Zellen und im Stroma gewisser Chloroplasten solcher Zellen eine elektronenoptisch dichte (schwarze) Substanz. Diese Substanz hat einen ausgesprochen osmiophilen Charakter, der ebenso wie das häufige Vorkommen von Myelinfiguren auf die Anwesenheit von Lipoiden hinweisen dürfte. Die Darstellung dieser osmiophilen Substanz nur durch Aldehydfixierungen mit nachfolgender OsO₄-Fixierung könnte als Indiz für das Vorliegen von Fixierungsartefakten gewertet werden, der gute Erhaltungszustand aller anderen Zellelemente spricht dagegen für das Vorhandensein eines realen Zellbestandteils, den lediglich (und nur teilweise) die Aldehyd/OsO₄-Fixierung darzustellen imstande ist. Eine endgültige Entscheidung zwischen diesen beiden Möglichkeiten auf Grund eines stichhaltigen Beweises ist zur Zeit jedoch nicht möglich.

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Osmiophilic substance in leaf cells of blackberry

Summary After fixation of leaves of blackberry (Rubus fruticosus L. s. l.) with aldehydes (formaldehyde, glutaraldehyde, acrolein) and postfixation with OsO₄ an electron-optically dense (black) substance appears in certain cells and in the stroma of certain plastids of such cells. This substance has a pronounced osmiophilic character which indicates—just as the occurence of numerous myelin figures—the presence of lipids. The only occurence of this osmiophilic substance after aldehyde/OsO₄ fixation procedures indicates the presence of fixation artifacts, the perfect preservation of other cell elements on the contrary the existance of a true formation which can be shown only by aldehyde/OsO₄ fixation, and even this only to some extent. A definite decision between these two possibilities is at present not yet possible.

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Herrn Professor Dr. Z.Devidé und Frau Dr. M.Wrischer danke ich bestens für die während der Durchführung der Untersuchungen und der Abfassung des Manuskriptes erwiesene Hilfe.     

An improved procedure for the X-ray microanalysis of acid phosphatase activity in lysosomes

Summary The useful detection of acid phosphatase activity with cerium as a capturing agent is confirmed. By introducing a freeze step in combination with a preincubation, reliably localized, lysosomal precipitates are obtained and aspecific ones prevented.Short (t<1 h) postfixation with either OsO₄ plus K₄Fe (CN)₆ or OsO₄ plus aminotriazole, added to lysosomal cerium localization a high membrane contrast.The detection of cerium by X-ray microanalysis is improved by a better spectral separation of the osmium (M ) and cerium (L ) peaks.     

Electron staining of the cell surface coat by osmium-low ferrocyanide

Summary In aldehyde-fixed liver and renal cortex of rat and mouse several variations of postfixation with osmium tetroxide plus potassium ferrocyanide (Fe^II) were tried. Depending on the ferrocyanide concentration different staining patterns were observed in TEM.-Osmium-High Ferrocyanide [40 mM (1%) OsO₄+36 mM (1.5%) Fe^II, pH 10.4], stains membranes and glycogen. Cytoplasmic ground substance, mitochondrial matrices and chromatin are partially extracted, cell surface coats remain unstained. Membrane contrast, but extraction too, are higher with solutions containing cacodylate- than phosphate-buffer.-Osmium-Low Ferrocyanide [40 mM (1%) OsO₄+2 mM (0.08%) Fe^II, pH 7.4], stains cell surface coats and basal laminae, but not glycogen, except for special cases. The trilaminar structure of membranes is poorly delineated. Signs of cytoplasmic extraction are not visible. The surface coat staining is stronger and more widespread with solutions containing phosphate- instead of cacodylate-buffer; it is enhanced by section staining with lead citrate. The cell surface coat stain does not traverse tight junctions nor permeate membranes.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

A technique for the electron microscopic autoradiography of Al adenosine receptors in brain tissue

Summary A procedure for the electron microscopic autoradiography of Al adenosine receptors is described. Fresh tissue slices from rat hippocampus were incubated with the radioactive adenosine analogs: Cyclohexyl[³H]adenosine, 5-N-ethylcarboxamido[³H]adenosine or [¹²⁵I]-iodohydroxyphenylisopropyladenosine. Various fixation agents were tested with respect to the retention of these ligands by the tissue. While most of the ligands were lost in aldehyde fixation they were retained by osmium tetroxide probably via a crosslinking reaction. The final method of choice was an aldehyde prefixation (in the case of [¹²⁵I]-iodohydroxyphenylisopropyladenosine with 4% buffered paraformaldehyde) during which more than 90% of the nonspecifically bound ligands were washed out while 40% of the specifically bound ligands remained. Subsequent fixation with osmium tetroxide (1%) allowed a standard protocoll for dehydration and embedding to be used with only minimal (less than 5%) further loss of the ligands. Electron microscopic autoradiography provided evidence for a specific distribution of the binding sites for [¹²⁵I]-iodohydroxyphenylisopropyladenosine.Abbreviations GA Glutardialdehyde - PFA Paraformaldehyde - OsO₄ Osmiumtetroxide - CHA Cyclohexyladenosine - NECA N-ethyl-carboxamidoadenosine - PIA Phenylisopropyladenosine - I-HPIA Iodohydroxyphenylisopropyladenosine - HPIA Hydroxyphenylisopropyladenosine     

Separation of proteins by ion-exchange chromatography using gradient elution

Chromatographic separation of proteins by the gradient elution method using DEAE Toyopearl 650^® was carried through. The concentration gradient was effected by changing the ionic strength of NaCl in the carrier buffer solution. Bovine serum albumin and hemoglobin were used as model proteins for separation. The experimental chromatogram was compared with theoretical results of Yamamoto et al. [1, 2]. Adsorption equilibria of the proteins onto the carrier were measured and expressed by a function of the ionic strength. The retention volume and peak width of the resulting chromatogram can be calculated from the equilibrium data using the Yamamoto theory. The calculated results agreed well with the experimental data.The method presented in this paper will be useful to predict the viability of ion-exchange chromatography in protein separation.List of Symbols c kg m^–3 concentration in the liquid phase - c _s kg m^–3 concentration in the solid phase - D _s m² s^–1 intraparticle diffusivity - d _p m particle diameter - E _z m² s^–1 longitudinal diffusivity of the protein - E _{z I} m² s^–1 longitudinal diffusivity of ionic strength - H /(1 – ) - I kmol m^–3 ionic strength - I _O kmol m^–3 initial ionic strength - I _p kmol m^–3 ionic strength at the peak - I _s kmol m^–3 ionic strength in the solid phase - I/V mol (dm³)^–2 slope of the ionic gradient elution - m distribution coefficient - m distribution coefficient at I - m _I distribution coefficient for ionic strength - Q cm³s^–1 flow rate - R m particle radius - R _s degree of separation - r m radial position inside particles - t s time - u m s^–1 linear velocity - V cm³ eluted volume of liquid - V _p cm³ eluted volume of liquid at the peak - V _T cm³ volume of the packed bed - W cm³ peak width - Z m bed height - z m vertical position in the bed - z _p m peak position from the inlet of the bed - (t) delta input at time - void fraction - ₁ s first moment - ₂ s² second central moment - s superficial space time     

Surface Topography and Cytomatrix Elements in Protoplasts from Plant Cells

We studied the surface topography of protoplasts from callus of Daucus sativus(Hofm.) Roehl. and from mesophyll cells of Nicotiana tabacumL. We also followed the distribution of actin elements of the cytomatrix in the subcortical cytoplasm layer. Protoplasts were prepared for scanning electron microscopy by a modified method without drying in the critical point apparatus. After postfixation with OsO₄, carrot and tobacco protoplasts had a similar topography of the surface: it was rough and had few pores. When carrot protoplasts were not postfixed with OsO₄, their surface looked different: it was folded and had 1.5-m pores, which sometimes were bordered with globules 0.3 m in diameter, or it consisted of conical cells varying in depth and size of their bases. We believe that, when protoplasts were not fixed with OsO₄, they lost lipid-containing structures from their surface, and what remained was the protein carcass of the plasmalemma and the underlying layer of the cytoplasm. The inner surface of opened carrot protoplasts had elaborate topography, apparently produced by the elements of the cytomatrix, that is, a relatively thick layer of the cortical cytoplasm, where, using phalloidin–colloidal gold and transmission electron microscopy, we could observe a dense network of actin filaments.     

Ultrahistochemical study on the ruthenium red surface staining

Summary The cell coat picture effect which is usually obtained with the conventional RR method, that is with the RR/OsO₄ coupled reaction, is investigated. In this first paper, each of conceivable events which might take place between RR, OsO₄ and cell surface membrane is discussed or studied. Various tests are carried out on ascites Ehrlich carcinoma cells and Zajdela ascites hepatoma cells treated with numerous chemical reagents, as also on a few pure proteins. The set of data supports the concept that the staining pattern is due to the combination in surface membranes of RR with a colloidal-like form of OsO₂. The latter might occur during the formation of stable cyclic osmic acid diesters between OsO₄ and membrane unsaturated lipids. A possibility by which the resulting marker is though also to be in a colloidal-like state is put forward. A next report will deal with this problem.     

Alterations in the morphology of glycoconjugate molecules caused by histochemical procedures: Comparison of renal glomeruli and articular cartilage

Summary The fine structure of the glycoconjugate molecules was investigated in the glomerular capillary wall of the rat kidney fixed by vascular perfusion, and in the human and rat articular cartilage fixed by immersion. Kidney and cartilage were either prefixed in aldehyde alone (group a), or with the addition of Alcian Blue 8 GX (group b), or Alcian Blue and 0.3m MgCl₂ (group c), or Acridine Orange at a low (0.01%) and high (0.1%) concentration (group d). The specimens were postfixed either in OsO₄ phosphate or cacodylate, with the exception of some of the samples in group a, for which a solution of potassium ferrocyanide-reduced OsO₄ was used (group e). All samples were conventionally dehydrated and embedded in Epon. In addition, some of the tissue samples in group c were cryoprotected, frozen in liquid Freon (–150° C) or in nitrogen slush (–210° C), both postfixed and dehydrated by cryosubstitution, and embedded in Epon (group f).The present investigations demonstrate that some well known extracellular structures such as the laminae rarae of the glomerular basement membrane or the interfibrillar matrix of the articular cartilage can be considerably altered in their morphology by the histological procedures applied. Whereas the precipitated glycoconjugates, as seen after staining with cationic dyes or reduced OsO₄ and conventional dehydration, can easily be recognized, the superposition of the extended molecules, as preserved by freezing and substitution, prevents their demonstration in native conformation.Preliminary reports on some of these findings were given at the Symposium on Morphological Sciences, Rome 1988 (Realeet al., 1989), at the XI Meeting of the Federation of European Connective Tissue Societies, Amsterdam 1988 (Brandes & Reale, 1988) and at the Symposium Recent Advances in Hereditary Nephritis, Milan, July 1989 (Reale & Luciano, 1990).     

Multiple amounts of DNA related to the size of chloroplasts

Summary Plants containing genetically small or large plastids appear in some euploid and trisomic types of Beta vulgaris.Small tissue samples of the first 6–10 leaves of nearly 30 euploid plants from 7 different generations were incubated in a solution containing ³H-thymidine in dark/light cycles, for up to 72 hr. For semiquantitative autoradiography the chloroplasts were then prepared on the slides with various kinds of isolation media, and fixed with OsO₄, glutaraldehyde, formaldehyde, or ethanol. The specifity of incorporation was tested by observing the extractability of label after differential treatment with acid or nucleases.Chloroplasts in leaves 2–11 cm long preferentially incorporate ³H-thymidine. The silver grains over plastids appear to be in clusters (centres). A relationship between the number of grains and also between the number of centres on the one hand, and the chloroplast size on the other could be found.It is concluded that chloroplasts occur in various degrees of polyenergide organization, as has been described, for example, for blue-green algae. Regarding the presence and degree of polyploidy—the other form of genetic polyvalency —the experiments provided no information. A remarkable variation in chloroplast size (and number of labelled centres) was observed, not only between different plants or between different leaves of a plants, but also within small tissue samples.     

Physiological characteristics of various species of strains of carboxydobacteria

Seven strains of aerobic carbon monoxide-oxidizing bacteria (carboxydebacteria) when growing on CO as sole source of carbon and energy had doubling times which ranged from 12–42 h. The activity profiles obtained after discontinuous sucrose density gradient centrifugation indicated that the CO-oxidizing enzymes are soluble and the hydrogenases are membrane-bound in all strains examined. The CO-oxidizing enzymes of Pseudomonas carboxydohydrogena, Pseudomonas carboxydoflava, Comamonas compransoris, and the so far unidentified strains OM2, OM3, and OM4 had a molecular weight of 230,000; that of Achromobacter carboxydus amounted to 170,000. The molecular weights of the CO-oxidizing and H₂-oxidizing enzymes turned out to be identical. The cell sonicates were shown to catalyze the oxidation of both CO and H₂ with methylene blue, thionine, phenazine methosulfate, toluylene blue, dichlorophenolindophenol, cytochrome c or ferricyanide as electron acceptors. Methyl viologen, benzyl viologen, FAD⁺, FMN⁺, and NAD(P)⁺ were not reduced. The spectrum of electron acceptors was identical for all strains tested. Neither free formate, hydrogen nor oxygen gas were involved in the CO-oxidation reaction. Methylene blue was reduced by CO at a 1:1 molar ratio. The results indicate that CO-oxidation by carboxydobacteria is catalyzed by identical or similar enzymes and that the reaction obeys the equation CO+H₂OCO₂+2H⁺+2e^- as previously shown for Pseudomonas carboxydovorans.Dedicated to Otto Kandler remembering almost three decades of enjoyable cooperation     

Electron microscopic characterization of calcium-binding physodes in the green alga Mougeotia scalaris

Summary Effect of the covalently cross-linking agents glutardialdehyde and osmium tetroxide, and of adsorption of the vital dye, neutral red, to the matrix of the calcium-binding vesicles from the green alga Mougeotia scalaris has been analysed in situ, both in terms of structural preservation and of the calcium-binding capacity of the vesicles. Upon cell fixation in glutardialdehyde without OsO₄, the vesicles appear to dissolve, but upon simultaneous fixation in glutardialdehyde with OsO₄ (1% w/v), the vesicles retain a globular form, are evenly stained by osmium and appear to be surrounded by a membrane-like structure. This structure was also observed around the vesicles in cells preincubated for 10 min in 0.1 mM neutral red and then fixed in glutardialdehyde/OsO₄ for 1 h. More detailed information of the matrix structure is obtained when simultaneous fixation of the Mougeotia cells was shortened to 15 min: a membrane-like structure was no longer observed around the vesicles. After cell treatment in the presence of neutral red, no calcium at all was found inside the vesicles. A small amount of calcium remained, when cells were fixed simultaneously and extensively in the absence of neutral red. However, calcium was found, to a considerable extent, inside the vesicles after short simultaneous fixation of the cells in the absence of neutral red. Based on the ultrastructural and elemental features presented here, the calcium-binding vesicles in Mougeotia appear to represent a member of the large family of (calcium-binding) physodes in lower plants (CaBP).     

Flagellar surface morphology ofChlamydomonas eugametos

Summary Flagellar surfaces ofChlamydomonas eugametos fixed in OsO₄ were compared to those fixed in glutaraldehyde by scanning and transmission electron microscopical techniques. They appeared to be quite different. Flagella fixed in OsO₄ had a pleated membrane that vesiculated when Tris was present during fixation. Flagella fixed in glutaraldehyde of various concentrations and buffer strengths, appeared to have disc-bearing appendages which were continuous with the membrane. On growing flagella the appendages occurred after 2 hours and they increased in size and complexity during the next 3 to 4 hours. No structural differences were observed between flagella of vegetative cells and those of gametes. Mating competence of gametes appeared to parallel the development of mature appendage-bearing flagella. The possibility that membrane vesicles, found in the cell culture medium, are derived from the flagellar appendages is discussed.     

Aspects of nitrogen fixation in Chlorobium

Four strains of the green sulfur bacterium Chlorobium were studied in respect to nitrogen nutrition and nitrogen fixation. All strains grew on ammonia, N₂, or glutamine as sole nitrogen sources; certain strains also grew on other amino acids. Acetylene-reducing activity was detectable in all strains grown on N₂ or on amino acids (except for glutamine). In N₂ grown Chlorobium thiosulfatophilum strain 8327 1 mM ammonia served to switch-off nitrogenase activity, but the effect of ammonia was much less dramatic in glutamate or limiting ammonia grown cells. The glutamine synthetase inhibitor methionine sulfoximine inhibited ammonia switch-off in all but one strain. Cell extracts of glutamate grown strain 8327 reduced acetylene and required Mg²⁺ and dithionite, but not Mn²⁺, for activity. Partially purified preparations of Rhodospirillum rubrum nitrogenase reductase (iron protein) activating enzyme slightly stimulated acetylene reduction in extracts of strain 8327, but no evidence for an indigenous Chlorobium activating enzyme was obtained. The results suggest that certain Chlorobium strains are fairly versatile in their nitrogen nutrition and that at least in vivo, nitrogenase activity in green bacteria is controlled by ammonia in a fashion similar to that described in nonsulfur purple bacteria and in Chromatium.Non-common abbreviations MSX Methionine sulfoximine - MOPS 3-(N-morpholino) propane sulfonic acid This paper is dedicated to Professor Norbert Pfennig on the occasion of his 60th birthday     

Bicarbonate uptake in the marine macroalga Ulva sp. is inhibited by classical probes of anion exchange by red blood cells

We demonstrate in this work that HCO _inf3 ^sup– uptake in the marine macroalga Ulva sp. features functional resemblances to anion transport mediated by anion exchangers of mammalian cell membranes. The evidence is based on (i) competitive inhibition of photosynthesis by the classical red-blood-cell anion-exchange blockers 4,4-dinitrostilbene-2,2-disulfonate and 4-nitro-4-isothiocyanostilbene-2,2-disulfonate under conditions where HCO _inf3 ^sup– , but not CO₂, was the inorganic carbon form taken up; (ii) inhibition of HCO _inf3 ^– uptake by pyridoxal phospate, indicating the involvement of lysine residues in the binding/translocation of HCO _inf3 ^sup– ; and (iii) inhibition of HCO _inf3 ^sup– (but not of CO₂) uptake by exofacial trypsin treatments, indicating the functional involvement of a plasmalemma protein. It is suggested that HCO _inf3 ^sup– uptake mediated by such a putative anion transporter can be a fundamental step in providing inorganic carbon for the CO₂-concentrating system of marine marcoalgae in an environment where the HCO _inf3 ^sup– concentration is high, but the CO₂ concentration and rates of uncatalyzed HCO _inf3 ^sup– dehydration are low.Abbreviations CI ionorganic carbon - DIDS 4,4-diisothiocyanostilbene-2,2-disulfonate - DNDS 4,4-dinitrostilbene-2,2-disulfonate - NIDS 4-nitro-4-isothiocyanostilbene-2,2-disulfonate - PLP pyridoxal phosphate - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase     

H2: heterodisulfide oxidoreductase,a second energy-conserving system in the methanogenic strain Gö1

Washed everted vesicles of the methanogenic bacterium strain Gö1 catalyzed an H₂-dependent reduction of the heterodisulfide of HS-CoM (2-mercaptoethanesulfonate) and HS-HTP (7-mercaptoheptanoylthreonine phosphate) (CoM-S-S-HTP). This process was independent of coenzyme F₄₂₀ and was coupled to proton translocation across the cytoplasmic membrane into the lumen of the everted vesicles. The maximal H⁺/CoM-S-S-HTP ratio was 2. The tranmembrane electrochemical gradient thereby generated was shown to induce ATP synthesis from ADP+P_i, exhibiting a stoichiometry of 1 ATP synthesized per 2 CoM-S-S-HTP reduced (H⁺/ATP=4). ATP formation was inhibited by the uncoupler 3,5-di-tert-butyl-4-hydroxy-benzylidene-malononitrile (SF 6847) and by the ATP synthase inhibitor N,N-dicyclohexylcarbodiimide (DCCD). This energy-conserving system showed a stringent coupling. The addition of HS-CoM and HS-HTP at 1 mM each decreased the heterodisulfide reductase activity to 50% of the control. Membranes from Methanolobus tindarius showed F₄₂₀H₂-dependent but no H₂-dependent heterodisulfide oxidoreductase activity. Neither of these activities was detectable in membranes of Methanococcus thermolithotrophicus.Abbreviations _H+ transmembrane electrochemical gradient of H⁺ - CoM-SH 2-mercaptoethanesulfonate - F₄₂₀ (N-l-lactyl--l-glutamyl)-l-glutamic acid phosphodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin-5-phosphate - F₄₂₀H₂ reduced F₄₂₀ - HTP-SH 7-mercaptoheptanoylthreonine phosphate - DCCD N,N-dicyclohexylcarbodiimide - SF 6847 3,5-di-ert-butyl-4-hydroxybenzylidenemalononitrile - Mb. Methanobacterium - Ml. Methanolobus - Mc. Methanococcus - MV methylviologen - BV benzylviologen - MTZ metronidazole     

Membrane transport ofp-nitrophenyl-α-galactoside by the melibiose carrier ofEscherichia coli

Summary p-Nitrophenyl--galactoside (-pNPG) was found to be a substrate for the melibiose transport system ofEscherichia coli. This sugar enters induced cells via the carrier and is split by -galactosidase to galactose andp-nitrophenol. In mutant cells lacking the -galactosidase [³H]--pNPG accumulated to concentrations 15 times higher than the external medium. The transport of -pNPG is inhibited by both Na⁺ and Li⁺. Na⁺ (10mm) reduced the K_m for -pNPG from 0.45 to 0.18mm and reduced theV _max from 6.7 nmoles/min/mg dry wt to a value of 3.0.     

Luminal alkalinization in the intestine of the goby

Summary The rate of luminal alkalinization in vitro byGillichthys mirabilis posterior intestine as measured by a manual pH stat technique was 0.70±0.05 Equiv/cm² h; acidification of the mucosal medium was never observed. The rate of HCO ₃ ^– secretion (J ^HCO ₃) was reduced by ouabain, serosally-applied DIDS, removal of serosal HCO ₃ ^– and replacement of media Cl^– with gluconate. HCO ₃ ^– secretion was enhanced replacement of Cl^– with isethionate and unaffected by mucosal DIDS, furosemide or acetazolamide.J ^HCO ₃ was reduced at mucosal pH above or below 7.5. These results support active HCO ₃ ^– secretion via a Cl^–/HCO ₃ ^– exchange mechanism on the basolateral membrane and a conductive exit pathway for HCO ₃ ^– , H⁺ or OH^– on the apical membrane.Abbreviations DIDS diisothiocyanostilbene-2,2-disulfonic acid - TEP transepithelial potential - GBR Gillichthyts bicarbonate Ringer - GUR Gillichthys unbuffered, bicarbonate-free Ringer - GER Gillichthys EPPS-buffered, bicarbonate-free Ringer - EPPS N-(2-hydroxyethyl)piperazine-N-3-propanesulfonic acid     

Sulfate transport in rabbit ileum: Characterization of the serosal border anion exchange process

Summary The preceding paper [30] shows that transepithelial ileal SO₄ transport involves Na-dependent uptake across the ileal brush border, and Cl-dependent efflux across the serosal border. The present study examines more closely the serosal efflux process. Transepithelial mucosa (m)-to-serosa (s) ands-to-m fluxes (J _ms,J _sm) across rabbit ileal mucosa were determined under short-circuit conditions. SO₄ was present at 0.22mm. In standard Cl, HCO₃ Ringer's,J _ms ^SO4 was 81.3±5.3 (1se) andJ _ms ^SO4 was 2.5±0.2 nmol cm^–2 hr^–1 (n=20). Serosal addition of 4-acetamido-4-isothiocyanostilbene-22-disulfonate (SITS), 44-diisothiocyanostilbene-22-disulfonate (DIDS) or 1-anilino-8-naphthalene-sulfonate (ANS) inhibited SO₄ transport, SITS being the most potent. Several other inhibitors of anion exchange in erythrocytes and other cells had no effect on ileal SO₄ fluxes. In contrast to its effect on SO₄ transport, SITS (500 m) did not detectably alter Cl transport.Replacement of all Cl, HCO₃ and PO₄ with gluconate reducedJ _ms ^SO4 by 70% and increasedJ _ms ^SO4 by 400%. A small but significantJ _net ^SO4 remained.J _ms ^SO4 could be increased by addition to the serosal side of Cl, Br, I, NO₃ or SO₄. The stimulatory effect of all these anions was saturable and SITS-inhibitable. The maximalJ _ms ^SO4 in the presence of Cl was considerably higher than in the presence of SO₄ (73.1 and 42.2 nmol. cm^–2 hr^–1, respectively;p<0.001). TheK _1/2 value for Cl was 7.4mm, 10-fold higher than that for SO₄ (0.7mm). Omitting HCO₃ and PO₄ had no measurable effects on SO₄ fluxes.This study shows that (i) SO₄ crosses the serosal border of rabbit ileal mucosa by anion exchange; (ii) the exchange process is inhibited by SITS, DIDS and ANS, but not by several other inhibitors of anion exchange in other systems; (iii) SO₄ may exchange for Cl, Br, I, NO₃ and SO₄ itself, but probably not for HCO₃ or PO₄; (iv) kinetics of the exchange system suggest there is a greater affinity for SO₄ than for Cl, although the maximal rate of exchange is higher in the presence of Cl; and, finally (v) SITS has little or no effect on net Cl transport.     

Rhabdomeric membrane and smooth endoplasmic reticulum in photoreceptors of Manduca sexta: modulations associated with the diurnal light/dark cycle and effects of chromophore deprivation

Summary Aberrations of photoreceptor ultrastructure resulting from carotenoid/retinoid (vitamin A) deprivation were studied in the retina of Manduca sexta. The syndrome of chromophore deficiency included hypertrophy of smooth endoplasmic reticulum, variable dilation of rhabdomeric microvilli, the insertion of endomembrane fingers into such enlarged microvilli, and the formation of rhabdomeric vacuoles, intracellular compartments containing microvilli similar to those of the rhabdomere. Retinas were processed either with conventional procedures employing preliminary aldehyde fixation followed by heavy metal postfixation, or by fixation and incubation in unbuffered OsO₄. The latter method deposits osmium throughout the endomembrane system, within the rhabdomeric vacuoles, and in the extracellular space of the rhabdom. However, the intravillous fingers were rarely impregnated with osmium, despite their continuity with densely stained cisternae of the smooth endoplasmic reticulum. We suggest that the insertion of endomembrane fingers into dilated microvilli results from a cytoskeleton-mediated link between cisternae of the smooth endoplasmic reticulum and the rhabdomeric membrane, an association that may be important in the turnover of photoreceptor membrane. We interpret endomembrane hypertrophy and development of rhabdomeric vacuoles as symptoms of disturbance in the pathway leading to the assembly of the rhabdomere resulting from reduced synthesis of visual pigment.