Correlation of open cell-attached and excised patch clamp techniques

The excised patch clamp configuration provides a unique technique for some types of single channel analyses, but maintenance of stable, long-lasting preparations may be confounded by rundown and/or rapid loss of seal. Studies were performed on the amiloride-sensitive Na⁺ channel, located on the apical surface of A₆ cells, to determine whether the nystatininduced open cell-attached patch could serve as an alternative configuration.Compared to excised inside-out patches, stable preparations were achieved more readily with the open cell-attached patch (9% vs. 56% of attempts). In both preparations, the current voltage (I-V) relation was linear, current amplitudes were equal at opposite equivalent clamped voltages, and E _rev was zero in symmetrical Na⁺ solutions, indicating similar Na⁺ activities on the cytosolic and external surfaces of the patch. Moreover, there was no evidence that nystatin altered channel activity in the patch because slope conductance (3–4pS) and E _rev (75 mV), when the bath was perfused with a high K:low Na solution (E _Na=80 mV), were nearly equal in both patch configurations.Our results therefore indicate that the nystatininduced open cell-attached patch can serve as an alternative approach to the excised inside-out patch when experiments require modulation of univalent ions in the cytosol.We thank Dr. Olaf S. Andersen for his suggestions in the development of the open cell-attached recording technique. This work was supported by a National Institutes of Health grant (DK-18061)     

The duct system of the avian salt gland as a transporting epithelium: structure and morphometry in the duck Anas platyrhynchos

Summary The duct system of the nasal salt gland of the duck comprises central canals, secondary ducts and main ducts. The secondary and main ducts consist of a layer of columnar cells overlying a layer of small cuboidal cells. The columnar cells have complex intercellular spaces showing evidence of Na⁺ K⁺ -ATPase at the apical regions. Approximately 70% of surface area of the duct system is external to the gland. During adaptation to salt water the duct system increases in size as does the gland. Although the components of the gland of adapted ducks, including the duct system within the gland, increase in size compared with normal ducks, the percentage volume densities of the components remain similar in both categories of ducks, i.e. the duct system increases in size in proportion to the glandular tissue. The volume of the duct system external to the gland is six to seven times larger than the volume within the gland. Thus, if ductal modification of secreted fluid occurs, it will be most likely to take place in the ducts external to the gland.Total surface areas of the duct system were measured from serial sections of glands and ducts from one normal and one adapted duck. These were used to calculate possible flux rates of water and sodium across the duct epithelium, assuming the occurrence of either water reabsorption or sodium secretion. Although these flux rates are high it is shown that they are similar to calculated flux rates across the luminal surface of the secretory tubules.     

Thymic nurse cells: morphological study during their isolation from murine thymus

Summary Thymic nurse cells (TNC), which are multicellular complexes composed of epithelial cells and thymocytes, were obtained from C3H-mice thymuses. They were described by means of light and electron microscopy. The morphology of epithelial cells forming isolated TNC compared to that of small tissue fragments obtained by enzymatic digestion revealed that TNC could be derived from all parts of the thymus: cortex, corticomedullary junction and medulla, the cortex being their principal source. This variety of origin, the presence of several epithelial cells inside a single TNC, the presence of non-lymphoid cells, and the various locations of eleaved desmosomes confirmed that their aspect in vitro as round and sealed structures can be considered to be an artifact due to the isolation technique used. Indeed, during this procedure, they are formed by a process of wrapping of the epithelial cytoplasm around the tightly associated thymocytes. All three epithelial cell types: cortical reticular cells, medullary reticular cells, and medullary globular cells can form TNC.A portion of this work was presented at the first Thymus Workshop. Rolduc, Netherlands, April, 1988     

The axolotl thymus: cell types of the microenvironment

Summary The three-dimensional structure of the axolotl (urodele amphibian) thymus was studied by combined scanning and transmission electron microscopy. The epithelial cell is the major component of the microenvironment forming the meshwork where thymocytes differentiate. Three different types of epithelial cells could be defined by their intracytoplasmic organelles and their localization in the subcapsular or deeper part of the organ. These epithelial cells participate in various types of lymphostromal interactions. Other stromal elements, such as interdigitating reticular cells, macrophages, eosinophil granulocytes and epithelial cysts were also defined. The absence of a true cortico-medullary differentiation in the axolotl thymus, the presence of different stromal elements and the physiological significance of these various microenvironments are discussed.     

Recombinant human granulocyte-colony stimulating factor and recombinant human macrophage-colony stimulating factor synergize in vivo to enhance proliferation of granulocyte-macrophage, erythroid, and multipotential progenitor cells in mice

Combinations of low dosages of purified recombinant human (rh) macrophage-colony stimulating factor (M-CSF; also termed CSF-1) and rh granulocyte-colony stimulating factor (G-CSF) were compared alone and in combination for their influence on the cycling rates and numbers of bone marrow and splenic granulocyte-macrophage, erythroid, and multipotential progenitor cells in vivo in mice pretreated with iron-saturated human lactoferrin (LF). LF was used to enhance detection of the stimulating effects of exogenously added CSFs. Concentrations of each CSF that were not active in vivo when given alone were active when given together, with the other CSF. The concentrations of rhM-CSF and rhG-CSF needed to increase progenitor cell cycling in the marrow and spleen were reduced by factors of 40-200 when these CSFs were administered in combination with low dosages of the other CSF. At the concentrations of rhM-CSF and rhG-CSF tested, synergism was not noted on absolute numbers of progenitor cells or total nucleated cell counts per organ or circulating in the blood. These findings may have potential relevance when considered in a clinical setting where the CSFs might be used in combination with other biotherapy and/or chemotherapy.     

Alterations in cytokeratin expression precede histological changes in epithelia of vitamin A-deficient rats

Summary Normal epithelial cell differentiation is charactezied by the production of distinct cytokeratin proteins. It is well known that epithelia of several organs show squamous metaplasia in a vitamin A-deficient status. It is not yet known whether these histological changes are concomitant with a change in cytokeratin expression. Therefore, 3-week-old female rats (BN/BiRij) were fed a vitamin A-deficient diet for 8 weeks. The cytokcratin expression in epithelia of various organs was monitored immunohistochemically during the induction of vitamin A deficiency. Therefore, monoclonal antibodies specific for human cytokeratin 4, 5, 5+8, 7, 10, 14, 18 and 19 were used. In a normal vitamin A status, the distributional pattern for the different cytokeratins in rats was similar to that reported for human tissue. No change in cytokeratin expression was seen in trachea, skin, liver and colon at any time point studied. Squamous metaplasia in urinary bladder and salivary glands was observed after six weeks on the vitamin A-deficient diet. This was concomitant with a substitution of cytokeratins 4, 5+8, 7, 18 and 19 by cytokeratin 10. The latter cytokeratin is specific for keratinzed squamous epithelium. A change in cytokeratin expression was observed in bladder, ureter, kidney, salivary glands, uterus and conjunctiva before histological alterations appeared. In conclusion, the changes in cytokeratin expression observed under vitamin A deficiency in epithelia in vivo are in agreement with those described in other studies for epithelial cells in vitro. The changes in cytokeratin expression and the subsequent differentiation into squamous cells occurs in basal cells of the bladder but not in transitional cells. Furthermore, histological alterations are preceded by changes in cytokeratin expression indicating that vitamin A status controls cytokeratin expression in vivo.     

Occurrence of unusual heterogeneous lipid-containing granule-storing cells in the main excretory duct epithelium of the male mouse submandibular gland

Summary The fine structure of the main excretory duct epithelium of the male mouse submandibular glands was investigated by scanning and transmission electron microscopy. Three principal cell-types were observed: type I and II, and basal cells. This epithelium was characterized by the presence of intercellular canaliculi. Type-I cells were the most numerous. They had an abundance of mitochondria, well-developed Golgi apparatus, a few electron-lucent lipid-containing granules and poorly developed basal infoldings. These cells were also characterized by many glycogen granules throughout the cytoplasm and abundant smooth endoplasmic reticulum in the apical cytoplasm. Type-II cells were the second most numerous. Their most characteristic feature was the presence of abundant heterogeneous lipid-containing granules having acid phosphatase activity at the periphery. They were concentrated in the infra- and supranuclear cytoplasm. The granules may be derived from mitochondrial transformation and seem to be a special kind of secondary autolysosome. Type-II cells also contained abundant mitochondria throughout the cytoplasm, much smooth endoplasmic reticulum in the apical cytoplasm, a well developed Golgi apparatus adjacent to the heterogeneous lipid-containing granules and no basal infoldings. Basal cells were situated adjacent to the basal lamina. They had a large nucleus and the cytoplasm was filled with glycogen granules.     

Morphometric investigations of the colon mucosa in chronicTrypanosoma cruzi infected rats

The objective of this investigation was to study the morphometry of the epithelial mucosa in the chronic phase ofT. cruzi infection. Nine young female Wistar rats were inoculated withT. cruzi. Ten months after inoculation the animals were sacrificed and the proximal colon was collected for morphometric measurements of the thickness of the muscle layers, the number of neurons in the myenteric plexus, the crypt cell population (CCP), crypt cell production per crypt (CCPC) and turnover time (TT) of the epithelium. There was no muscle layer hypertrophy but there was significant denervation in the group inoculated withT. cruzi, which also showed hyperplasia of the epithelium. The data suggest that denervation of the myenteric plexus did not induce hypertrophy of the propria muscle layer itself but altered the morphometry of the colonic epithelium inT. crwzi-infected animals, with increased development of CCP and TT. It is possible that this epithelial hyperplasia, as a consequence of a longer crypt cell TT, increased the absorption and secretion activities of the colon, which in turn may participate in the genesis of the enteromegalies observed in the chronic phase of Chagas’ Disease.     

General method for the derivation and numerical solution of epithelial transport models

Summary A general method is presented for the formulation and numerical evaluation of mathematical models describing epithelial transport. The method is based on the principles of conservation of mass, and maintenance of electroneutrality within the cells and bathing solutions. It is therefore independent of the specific membrane transport mechanisms, and can be used to evaluate different models describing arbitrary transport processes (including passive, active and cotransport processes). Detailed numerical methods are presented that allow computation of steady-state and transient responses under open-circuit, current-clamp and voltage-clamp conditions, using a general-purpose laboratory minicomputer. To evaluate the utility of this approach, a specific model is presented that is consistent with the Koefoed-Johnson and Ussing hypothesis of sodium transport in tight epithelia (Acta Physiol. Scand. 42:298–308, 1958). This model considers passive transport of an arbitrary number of permeant solutes, active transport of sodium and potassium, and osmotically induced water transport across the apical and basolateral membranes. Results of the model are compared to published experimental measurements in rabbit urinary bladder epithelium.