Ultrastructural localization of Na+,K+-ATPase in rat and rabbit kidney medulla.

Na+,K+-ATPase was localized at the ultrastructural level in rat and rabbit kidney medulla. The cytochemical method for the K+-dependent phosphatase component of the enzyme, using p-nitrophenylphosphate (NPP) as substrate, was employed to demonstrate the distribution of Na+, K+-ATPase in tissue-chopped sections from kidneys perfusion-fixed with 1% paraformaldehyde-0.25% glutaraldehyde. In other outer medulla of rat kidney, ascending thick limbs (MATL) were sites of intense K+-dependent NPPase (K+-NPPase) activity, whereas descending thick limbs and collecting tubules were barely reactive. Although descending thin limbs (DTL) of short loop nephrons were unstained, DTL from long loop nephrons in outer medulla were sites of moderate K+-NPPase activity. In rat inner medulla, DTL and ascending thin limbs (ATL) were unreactive for K+-NPPase. In rabbit medulla, only MATL were sites of significant K+-NPPase activity. The specificity of the cytochemical localization of Na+,K+-ATPase at reactive sites in rat and rabbit kidney medulla was demonstrated by K+-dependence of reaction product deposition, localization of reaction product (precipitated phosphate hydrolyzed from NPP) to the cytoplasmic side of basolateral plasma membranes, insensitivity of the reaction to inhibitors of nonspecific alkaline phosphatase, and, in the glycoside-sensitive rabbit kidney, substantial inhibition of staining by ouabain. The observed pattern of distribution of the sodium transport enzyme in kidney medulla is particularly relevant to current models for urine concentration. The presence of substantial Na+,K+-ATPase in MATL is consistent with the putative role of this segment as the driving force for the countercurrent multiplication system in the outer medulla. The absence of significant activity in inner medullary ATL and DTL, however, implies that interstitial solute accumulation in this region probably occurs by passive processes. The localization of significant Na+,K+-ATPase in outer medullary DTL of long loop nephrons in the rat suggests that solute addition in this segment may occur in part by an active salt secretory mechanism that could ultimately contribute to the generation of inner medullary interstitial hypertonicity and urine concentration.

Recombinant retroviruses containing novel reporter genes.

Two histochemical marker genes, Drosophila alcohol dehydrogenase (ADH) and human placental alkaline phosphatase (ALP), were cloned into the recombinant retroviral vectors pLJ and pgag beta-actin. The resulting vectors were transfected into retroviral producer cell lines, psi CRE and psi CRIP, and stable recombinant retrovirus producers were isolated. Recombinant virus was harvested and used to transduce genes into several cell lines, singly or in conjunction with lacZ (Escherichia coli beta-galactosidase)-containing retrovirus. Cell lines were then stained using standard histochemical methods for recombinant gene expression. We found that multiple gene products could be identified in the same cell populations and in the case of ALP and beta-galactosidase, in the same cells. The resulting reagents should be useful for a variety of cell-marking studies including those involving multiple clonal analysis and developmental studies for gene therapy.

Purification of mouse brain (Na+ + K+)-ATPase catalytic unit, characterization of antiserum, and immunocytochemical localization in cerebellum, choroid plexus, and kidney.

The denatured catalytic polypeptide of mouse brain (Na+ + K+)-adenosine triphosphatase(ATPase) was separated from microsomal membranes on polyacrylamide gels and used as an immunogen. The antiserum, characterized by immunoblots, recognizes the polypeptide corresponding to the catalytic unit in various fractions of mouse brain and cross-reacts with the catalytic unit from lamb kidney, duck salt gland, and electroplax. The same polypeptide in brain and salt gland is recognized by antiserum raised against purified lamb kidney enzyme. Light microscopy was performed with the peroxidase-conjugated second antibody method. In mouse cerebellum, immunochemical staining outlines Purkinje cell and granule cell perikarya. Intense activity is associated with regions of high synaptic content including the pericellular basket meshes and preaxonal regions of Purkinje cells and the glomeruli in the granular layer. In the molecular layer, the neuropil is diffusely reactive with distinct vertically oriented processes evident. White matter exhibits light stain deposition. Choroid plexus presents abundant reaction product only at ependymal apical surfaces, while the ependymal lining of the fourth ventricle displays little or no immunoreactivity. Specificity of the antiserum was demonstrated further in mouse kidney where staining conforms to the well-characterized localization of the enzyme along basolateral surfaces of cortical and medullary tubules. The biochemical and immunocytochemical data show the efficacy of generating antisera to brain (Na+ + K+)-ATPase using catalytic polypeptide as an immunogen.

Phagocytosis and surface morphology in cultured retinal pigment epithelial cells.

Phagocytosis of latex spheres was studied in explant cultures of retinal pigment epithelium from rabbit, calf, and rat (normal and dystrophic). Calf and rabbit pigment epithelial cells showed a "latent period" of about 12 and 17 hr, respectively, during which latex spheres were not ingested even when the cultures were pre-incubated for up to 24 hr prior to exposure. Scanning electron microscopy demonstrated a profusion of apical processes on the surface of cells from both species that was present throughout the latent period. In contrast, rat pigment epithelial cells showed no latent period and phagocytosed latex spheres after a pulse of 1 hr. It was concluded that the latent period is not common to all species and, when present, is not due to an absence of apical processes. The surfaces of normal and dystrophic pigment epithelial cells showed abundant apical processes that did not differ from each other in morphology, number, or distribution. In cells from pigmented species (calf and rat), frequent fusions were observed between latex phagosomes and melanosomes or melanin granules.

Hospital diabetes education: classroom and self-instruction.

This study was conducted to assess the effectiveness of two approaches to instructing hospitalized patients with diabetes mellitus on the food exchange system. Both groups were given the same pretest and post-test, but one group received self-instruction and the other group received classroom instruction. The pretest scores of the two groups indicated differences in their initial understanding of the food exchange system. The mean score on the pretest for the classroom instruction group was 1.95 points higher than for the self-instruction group on the 18-point test. The self-instruction group gained 3.45 more points than the classroom instruction group on their post-test scores. An analysis of gain between pretest and post-test for both groups indicated that both treatments could be considered effective. However, when the pretest was analyzed as an independent variable, it was shown to have an influence on the post-test results. In controlling for the pretest in the analysis of covariance, the LAP treatment was found to be more effective than the classroom instruction for teaching patients with diabetes mellitus about the food exchange system. As diabetes affects persons from all demographic groups, a variety of programs are needed to meet individual needs (15). Demands for education programs in institutions are being met as patients' needs change. The two instructional approaches assessed in this research are recommended as viable options to use in educating the increasing number of persons with diabetes mellitus.

Results of outpatient laparoscopic appendectomy in women.

The results of laparoscopic appendectomy under outpatient conditions are reported here from a retrospective analysis of the data for 78 women (aged 17-55) selected as having clinically acute or subacute appendicitis. Patients with severe disease presenting with sepsis or peritonitis were excluded, since they were referred to hospital. The duration of the procedure in the 78 women was 30-120 minutes (mean: 45), and only one minor intraoperative complication (a slight lesion to the uterus from the working trocar) was encountered. Follow-up was carried out by daily telephone interviews and a physical examination on the third or fourth postoperative day. Five postoperative complications (four cases of peritonitis and one stump insufficiency) were found two to seven days after the laparoscopic appendectomy, and these had to be treated by laparotomy. No severe sequelae or mortality were encountered. The calculated costs of the laparoscopic approach (DM 1,000.00 in total for anesthesia and operation) compared favorably with a conventional inpatient regimen covering seven days (DM 3,000.00-5000.00). We conclude that laparoscopic appendectomy under outpatient conditions is a safe and cost-effective modality for treating acute and subacute appendicitis in selected patients.

Early experience with laparoscopic appendectomy in women.

Experience and the surgical technique of laparoscopic appendectomy in 70 female patients over a period of more than three years is described. Three women were pregnant. Complications occurred in one case.

Laparoscopic appendectomy in pregnancy.

Since 1982 we have operated on more than 150 patients using the laparoscopic appendectomy technique. Our complication rate was 0.75% and the patients included six pregnant women in all stages of pregnancy. There were no complications in this group of six women.

[Endoscopic surgery in a gynecologic outpatient clinic]. / Endoskopische Chirurgie in einer gynäkologischen Tagesklinik.

From 1988 to 1989, we operated on 784 women in our daily ambulant clinic. In 462 of these cases, we performed laparoscopic surgery with 679 therapeutical interventions. Sterilisation is the most frequent operation in our cases of Fallopian tube surgery, but we have also had tubal pregnancy in 5 cases. In ovarian surgery, we found a great number of benign ovarian cysts followed by a high rate of ovarian adhesions, which, counted together with the Fallopian tube and intestinal adhesions, amounted to 201 cases. In 9 women, the extirpation of a myoma was necessary and the largest one was 5.5 cm in diameter. In comparison with the total number of our surgical interventions, we had a high rate of appendectomies. Each surgeon is aware of the various locations of the appendix, of its close relation to the adnexae and the difficulty of clinical diagnosis of appendicitis in women, especially in pregnant women. We removed the appendix in two pregnant women (4th and 18th week of pregnancy). A pathological substrate was found in more than 80%. Because of a good preliminary selection of our patients, we found no carcinoma in the abdominal cavity, except from an early metastasis on the appendix of an ovarian carcinoma. This patient came for staging re-laparoscopy after treatment of the ovarian carcinoma years ago. Since the beginning of 1989, we have used a Neodym: Yag laser in endoscopic operations. The good cutting effect loses its benefits of precision and time saving through bleeding as soon as a large vessel is involved.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasoactive intestinal peptide stimulates ion transport in avian salt gland.

Avian salt glands are considered to be under the control of cholinergic nerve fibers. Here we report evidence that vasoactive intestinal peptide (VIP) also regulates ion transport. Nerve fibers stained immunocytochemically with anti-VIP were distributed throughout the tissue within the peritubular connective tissue and were in close proximity to the secretory tubules. VIP applied to primary cultures of the secretory cells elicited active ion transport as assayed by short-circuit current (Isc) analysis. The mucosal-to-serosal positive Isc was produced in a dose-dependent fashion [(EC50) = 3.1 X 10(-9) M], was potentiated by theophylline, and was inhibited by either ouabain or furosemide. This Isc was independent of activation by cholinergic agonists. VIP also increased ouabain-sensitive respiration 14-18% in acutely isolated cells from salt-stressed and unstressed animals. These data demonstrate for the first time that VIP is present in the avian salt gland and can act as a secretagogue by directly affecting the secretory cells. In addition, the results provide evidence for direct control of ion transport by an adenosine 3',5'-cyclic monophosphate-linked neurohormone in both adult unstressed and fully salt-stressed animals.

Role of CFTR in lysosome acidification.

The role of CFTR in lysosome acidification was examined in CFPAC-1 pancreatic adenocarcinoma cells with the delta F508 mutation that were transduced with a retroviral vector (PLJ-CFPAC) or with the normal CFTR gene (CFTR-CFPAC). Steady-state lysosomal pHi in intact cells was lower in PLJ-CFPAC cells than CFTR-CFPAC cells (3.55 vs 3.80) and was not affected by cAMP or forskolin. Initial rates of ATP-dependent acidification of isolated lysosomes and steady-state ATP-dependent pHi were similar in both cell lines over a range of chloride concentrations and were not altered when cells were exposed to cAMP or to forskolin prior to preparation of lysosomes. These observations suggest that CFTR plays no role in acidification of lysosomes, possibly due to limited permeability of lysosomal membranes to chloride.

Freeze-fracture and morphometric analysis of occluding junctions in rectal glands of elasmobranch fish.

The structure of occluding junctions in secretory and ductal epithelium of salt-secreting rectal glands from two species of elasmobranch fish, the spiny dogfish Squalus acanthias and the stingray Dasyatis sabina, was examined by thin-section and freeze-fracture electron microscopy. In both species, occluding junctions between secretory cells are shallow in their apical to basal extent and are characterized by closely juxtaposed parallel strands. Average strand number in the dogfish was 3.5 +/- 0.2. with a mean depth of 56 +/- 5 nm; in the stingray a mean of 2.0 +/- 0.2 strands encompassed an average depth of 18 +/- 3 nm. In contrast, the linear extent of these junctions was remarkably large due to the intermeshing of the narrow apices of the secretory cells to form the tubular lumen. Morphometric analysis gave values of 66. 8 +/- 2.5 and 74.9 +/- 4.6 m/cm2 for the length of junction per unit of luminal surface area in the dogfish and stingray, respectively. This junctional morphology is similar to that generally described for "leaky" epithelia. In comparison, the stratified ductal epithelium which carries the NaCl-rich secretion to the intestine is characterized by extensive occluding junctions which extend 0.6-0.8 mum in depth and consist of a mean of 12 strands arranged in an anastomosing network, an architectural pattern typical of "tight" epithelia. The length density of these junctions in the dogfish rectal gland was 7.6 +/- 0.1 m/cm2. The junctional architecture of the rectal gland secretory epithelium (few strands, large junctional length densities) is similar to that described for several other hypertonic secretory epithelia [20, 34] and is compatible with the recent model for salt secretion in rectal glands [39] and in other C1- secretory epithelia which posits a conductive paracellular pathway for trans-epithelial Na+ secretion from intercellular space to the lumen to form the NaCl-rich secretory product.

Primary culture of duck salt gland. I. Morphology of confluent cell layers.

Dissociated avian salt gland secretory cells were maintained in primary culture after plating on hydrated collagen gels. When seeded at 3 X 10(6) cells/cm2, confluent cell sheets formed within 2-3 days, whereas cultures seeded at lower densities formed a complex reticulum of cell aggregates, which remained nonconfluent even after 7 days. Scanning electron microscopy showed that the free surface of 3-day confluent cultures consisted of intermixed convex and flattened cell membranes with prominent junctional boundaries and abundant microvilli. Transmission electron microscopy indicated that these cultures were multilayers of 1-4 cells in thickness. The plasma membranes of the superficial cells were polarized into apical and basolateral regions displaying, respectively, microvilli and interdigitating lateral membrane folds. These membrane domains were separated by shallow occluding junctions, which consisted of both single strands and simple net-like arrays in freeze-fracture images. Underlying epithelial cells retained lateral membrane folds and formed desmosomal contacts with superficial and neighboring cells. These cultures, unlike the intact tissue, allow direct access to the apical and basolateral cell surfaces for electrophysiological analysis of transmural active ion transport.