Photoaffinity labeling by [3H]-N5-methyl-N5-isobutylamiloride of proteins which cofractionate with Na+/H+ antiport activity.

N5-Methyl-N5-isobutylamiloride (MIA) is one of a series of 5-N-substituted amiloride analogues which exhibit high affinity and specificity for inhibition of Na+/H+ antiport. Amiloride-sensitive [3H]MIA binding to renal brush border membranes exhibited a Kd of 250 nM and a Bmax of 8.6 pmol/mg of protein.

Chemical inducers of differentiation in a long-term renal cell line.

The long-term renal epithelial cell line LLC-PK1 expresses at confluence several differentiated characteristics of renal proximal tubule including Na/glucose cotransport and several brush border membrane hydrolases. The differentiation-inducing chemical hexamethylene bisacetamide (HMBA) triggers a dramatic induction of Na+/glucose symport, trehalase and maltase, expressed as an increase in the number of cells in the culture that express the differentiated phenotype. Characteristics of the induction response are reviewed in terms of proposed mechanisms of inducer action.

Experimental pancreatic cancer in the Syrian hamster: effect of cholecystectomy.

Because cholecystectomy stimulates hypertrophy and hyperplasia in the hamster pancreas, its effect on experimental pancreatic carcinogenesis was studied in this animal model. Forty female Syrian hamsters underwent cholecystectomy, while 40 others underwent sham operations. Two weeks later, 30 hamsters undergoing cholecystectomy and 30 hamsters undergoing sham operations received 4 weekly subcutaneous injections of N-nitroso-bis (2-oxopropyl) amine (BOP) (10 mg/kg).

Synthesis and in vivo characterization of D-(+)-(N1-[11C]methyl)-2-Br-LSD: a radioligand for positron emission tomographic studies of serotonin 5-HT2 receptors.

D-(+)-N1-Methyl-2-Br-LSD (MBL), which displays high affinity and selectivity for serotonin receptors in vitro, has been labeled with carbon-11 for localization of cerebral serotonin 5-HT2 receptors by positron emission tomography. [11C]MBL was prepared from [11C]iodomethane and D-(+)-2-Br-LSD within 20 min from end of bombardment. The average specific activity of [11C]MBL was 2300 mCi/mumol and the average radiochemical yield was 17%, both at end of synthesis.

Increased trehalase expression after glucose limitation of LLC-PK1 clones.

Expression of the apical membrane disaccharidase trehalase correlates with the development of a proximal tubule-specific differentiated phenotype in the LLC-PK1 cell line derived from renal epithelia. Substitution of galactose for glucose in the culture medium of confluent cultures resulted in a significant trehalase induction; a smaller induction was observed after replacement by fructose, mannose, or the nonmetabolizable sugar alpha-methyl-D-glucose. Induction was specific for trehalase among other microvillar hydrolases, reversible after replacement of glucose, and was increased as a function of decreasing glucose concentration.

Neuropeptide and noradrenaline distributions in rat interscapular brown fat and in its intact and obstructed nerves of supply.

Following demonstration of noradrenergic and peptidergic nerves in rat interscapular brown adipose tissue, neuronal courses were investigated with reference to both the segmentally derived nerve traversing the fat pad and to paravascular projections, employing respectively, sucrose potassium glyoxylic acid and indirect immunofluorescence methodologies. Following ligation of the segmental nerve before its pad entry and distal to the pad, accumulations of noradrenaline, neuropeptide-Y, calcitonin gene-related peptide and substance P were observed immediately proximal to the ligature in all nerve components (T1-5). Two to three days after unilateral nerve resection (proximal to the pad), with or without simultaneous contralateral padectomy, there was, in some sections of the denervated pads either total absence of, or a very sparse distribution of both noradrenergic and peptidergic labelled nerves, thus suggesting minimal paravascular projection of these nerves and a major projection via the defineable segmental nerve route.

Neural and vascular provisions of rat interscapular brown adipose tissue.

The innervation of rat interscapular brown adipose tissue has been studied by light and fluorescence microscopy and electron microscopy after treatment with "false" adrenergic neurotransmitters 5- and 6-hydroxydopamine. The vascular markers neoprene latex and thioflavin S were used to define the blood vascular arrangements within the around the tissue. Catecholaminergic innervation was revealed by fluorescence microscopy at both parenchymal and vasomotor sites.

Neuropeptides in interscapular and perirenal brown adipose tissue in the rat: a plurality of innervation.

The occurrence of neuropeptides in rat brown adipose tissue has been investigated. Immunohistochemical studies on interscapular and perirenal brown fat have demonstrated unequivocally the presence of substance-P (SP)-like, neuropeptide-Y (NPY)-like and calcitonin gene related-peptide (CGRP)-like immunoreactive elements (putative nerves) in adventitial distribution on inter- and intralobular supply arteries and in accompanying nerve bundles. At a more peripheral level, some NPY-like immunoreactive elements and a greater number of CGRP-like immunoreactive elements were observed in the parenchymal field.

Purification and reconstitution of a 75-kilodalton protein identified as a component of the renal Na+/glucose symporter.

A 75-kilodalton (kDa) protein was purified from solubilized renal brush border membranes by using high-pressure liquid chromatography (HPLC) and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Functional and immunological properties identified the 75-kDa protein as a component of the Na+/glucose symport system. The purified protein was specifically recognized by a monoclonal antibody that functionally interacts with the Na+/glucose symporter.

Monoclonal antibodies that bind the renal Na+/glucose symport system. 1. Identification.

Phlorizin is a specific, high-affinity ligand that binds the active site of the Na+/glucose symporter by a Na+-dependent mechanism but is not itself transported across the membrane. We have isolated a panel of monoclonal antibodies that influence high-affinity, Na+-dependent phlorizin binding to pig renal brush border membranes. Antibodies were derived after immunization of mice either with highly purified renal brush border membranes or with apical membranes purified from LLC-PK1, a cell line of pig renal proximal tubule origin.

Monoclonal antibodies that bind the renal Na+/glucose symport system. 2. Stabilization of an active conformation.

Conformation-dependent fluorescein isothiocyanate (FITC) labeling of the pig renal Na+/glucose symporter was investigated with specific monoclonal antibodies (MAb's). When renal brush border membranes were pretreated with phenyl isothiocyanate (PITC), washed, and then treated at neutral pH with FITC in the presence of transporter substrates Na+ and glucose, most of the incorporated fluorescence was associated with a single peak after resolution by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular mass of the FITC-labeled species ranged from 79 to 92 kDa.

Apical trehalase expression associated with cell patterning after inducer treatment of LLC-PK1 monolayers.

Trehalase, a differentiation-specific marker of renal proximal tubule brush border membrane, is expressed in confluent long-term cultures of the renal epithelial cell line LLC-PK1. The level of trehalase is greatly increased after treatment of cultures with differentiation inducers such as hexamethylene bisacetamide (HMBA), accompanied by increases in other apical membrane-associated differentiated functions (Yoneyama and Lever: J. Cell.

N1-methyl-2-125I-lysergic acid diethylamide, a preferred ligand for in vitro and in vivo characterization of serotonin receptors.

Methylation of 2-125I-lysergic acid diethylamide (125I-LSD) at the N1 position produces a new derivative, N1-methyl-2-125I-lysergic acid diethylamide (125I-MIL), with improved selectivity and higher affinity for serotonin 5-HT2 receptors. In rat frontal cortex homogenates, specific binding of 125I-MIL represents 80-90% of total binding, and the apparent dissociation constant (KD) for serotonin 5-HT2 receptors is 0.14 nM (using 2 mg of tissue/ml).

Localization of serotonin 5-HT2 receptors in living human brain by positron emission tomography using N1-([11C]-methyl)-2-Br-LSD.

N1-([11C]-Methyl)-2-Br-LSD ([11C]-MBL) has been developed as a positron emission tomography (PET) imaging agent for serotonin 5-HT2 receptors. In vitro receptor binding assays with nonradioactive MBL show high-affinity binding to serotonin 5-HT2 receptors (Ki = 0.5 nM), a secondary interaction of 8-fold lower affinity with dopamine D2 receptors, and low-affinity interactions with alpha 1-adrenergic as well as serotonin 5-HT1 receptors.

Primary malignant lymphoma of the heart. Antemortem cytologic diagnosis.

A case of primary malignant lymphoma of the heart diagnosed cytologically is reported. The patient presented with pericardial tamponade and ventricular arrhythmias and developed rapidly progressive and intractable cardiac failure. Two-dimensional echo-cardiography, which demonstrated the pericardial effusion, showed progressive impairment of the left ventricular contraction.

Demonstration of a catecholaminergic innervation in human perirenal brown adipose tissue at various ages in the adult.

From light and electron microscopic studies on perirenal fat from human donors 27, 39 and 47 years old, unequivocal evidence is found for the presence of islands of multilocular adipocytes. After glyoxylic acid condensation for visualisation of biogenic amines, catecholaminergic nerve plexuses were demonstrated around the arteries of supply to these islands and within the intercellular spaces between their constituent adipocytes. Transmission electron microscopy revealed the cytology of these adipocytes to be similar to that of brown adipocytes in rodents.

Microsomal chemiluminescence of benzo[a]pyrene-7,8-dihydrodiol and its synthetic analogues trans- and cis-1-methoxyvinylpyrene.

The cis- and trans-methoxyvinylpyrene (MVP) analogues of benzo[a]pyrene-7,8-dihydrodiol (7,8-diol) produce specific microsomal chemiluminescence comparable to that produced from 7,8-diol in Aroclor-induced rat liver microsome preparations. The chemiluminescence quantum yields, emission spectra, and the concentration and the temporal kinetics of these three substrates have been examined. Radiolabelled 7,8-diol and t-MVP exhibit significant covalent binding (more than 14%) to microsomal protein when metabolized enzymatically.

Proliferative and metastatic potential of exfoliated colorectal cancer cells.

Exfoliated colorectal carcinoma cells were obtained by lavage of 27 freshly resected tumor-bearing segments of human bowel with the use of either Hartmann's solution or medium 199. The tumor cells were isolated from the lavage fluid on Nycodenz (Nyegaard, Oslo, Norway) columns. Their proliferative and metastatic potentials were investigated by their ability to incorporate tritiated thymidine ([3H]dThd) and by their ability to form experimental pulmonary tumors following iv injection into immune-deprived stain A mice.

Expression of differentiated functions in kidney epithelial cell lines.

The expression of differentiated functions in long-term cell lines MDCK and LLC-PK1 derived from renal epithelia is briefly summarized. Topics include differentiated transport properties and enzymatic functions expressed by these cell lines, modulation of a differentiated phenotype by exogenous inducers, substratum and cell-cell contact, and possible mechanisms of inducer action.

Transport by the (Na+,K+) ATPase: modulation by differentiation inducers and inhibition of protein synthesis in the MDCK kidney epithelial cell line.

MDCK kidney epithelial cell cultures exposed to the differentiation inducer hexamethylene bisacetamide (HMBA) for 24 hours exhibited a 50% decrease in transport activity per (Na+,K+)-ATPase molecule (turnover number) but an unchanged number of pump sites (Kennedy and Lever, 1984). Inhibition of protein synthesis by either 10 microM cycloheximide or 2 microM emetine blocked the inhibitory effects of HMBA on Na+/K+ pump efficiency assessed by measurements of [3H]-ouabain binding to intact cells, (Na+,K+) ATPase activity of detergent-activated cell extracts, and ouabain-sensitive Rb+ uptake. In the absence of inducer treatment, inhibition of protein synthesis increased Na+/K+ pump turnover number by twofold while maintaining Na+/K+ pump activity per cell at a constant level.

The pre- and postnatal development and ageing of interscapular brown adipose tissue in the rat.

Development and ageing changes in interscapular brown adipose tissue have been studied in white Wistar rats by light, fluorescence and electron microscopy. Cellular aggregation was noted in the fetal interscapular area on the 15th day of gestation and vascularised primitive lobules of brown adipose tissue became unequivocally identifiable on the 17th day in utero. Brown adipocyte precursors appeared to be derived directly from mesenchymal cells and were uniquely characterised by larger (0.