Chromaffin vesicle function in intact cells.
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Adenosine Triphosphate↗
Biomedical subjects
B Caughey
Publications and source records attributed to B Caughey.
Equilibrium cation binding selectivity of the carboxylic ionophore narasin A: a comparison with transport selectivities reported in two biological test systems.
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Biological Transport↗
The role of molecular conformation in ion capture by carboxylic ionophores: a circular dichroism study of narasin A in single-phase solvents and liposomes.
Conformational and thermodynamic aspects of cation binding by the carboxylic ionophore narasin A were studied by circular dichroism (CD). In single-phase solvents, dramatic increases in the maximum differential absorption (delta epsilon) of the C-11 carbonyl were observed upon the binding of K+, Na+ and protons to the free anionic form. These changes were associated with major shifts in the conformation equilibrium between extended and pseudocyclic conformers of narasin. Similar CD changes observed upon the binding of K+ to narasin A in dimyristoylphosphatidylcholine vesicles provided evidence that in the membrane environment, comparable conformation changes were associated with ion binding. Variation of the polar and protic properties of single-phase solvents was also found to influence the delta epsilon of the cation bound species of narasin A, supporting previous evidence for polarity-mediated modulation of conformation. Comparison of cation binding affinities indicated that in both single-phase solvents and liposomes, narasin had a marked equilibrium selectivity for K+ over Na+.
Cations↗
Metabolic pools of ATP in cultured bovine adrenal medullary chromaffin cells.
Cultured bovine adrenal chromaffin cells contain a pool of ATP sequestered within the chromaffin vesicles and an extravesicular pool of ATP. In a previous study it was shown that the turnover of ATP in the extravesicular pool was biphasic. One phase occurred with a t1/2 of 3.5-4.5 h whereas the second phase occurred with a t1/2 of several days. The studies described here were undertaken to characterize further the vesicular and extravesicular pools of ATP by examining the effects of metabolic inhibitors, adenosine, and digitonin on ATP utilization and subcellular localization immediately after and 48 h after labeling with [3H]adenosine and 32Pi. Immediately after labeling a combination of cyanide, 2-deoxy-D-glucose, the beta-glucono-1,5-lactone resulted in a 90-95% depletion of the labeled ATP but only a 25% depletion of the endogenous ATP within 30 min. Forty-eight hours after labeling, addition of the inhibitors resulted in a 70% depletion of the [3H]ATP but only a 25% depletion of the [32P]ATP and endogenous ATP. Addition of 10 microM adenosine to the media resulted in a similar loss of [3H]ATP in cells examined immediately after or 48 h after labeling. Adenosine increased the amounts of [32P]ATP when added immediately after labeling but had no effect on the [32P]ATP content when added 48 h after labeling.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine↗
Effects of bromocriptine on pituitary organelle marker enzyme activities in lactating and postlactating rats: selective activation of lysosomal prolactin proteolytic activity.
Effects of the dopamine agonist 2-bromo-alpha-ergocryptine (bromocriptine) on plasma and pituitary PRL and enzyme activities in lactating and postlactating rats have been investigated. Lactating rats which had been suckling their young for 3 days were given a single sc injection of bromocriptine or solvent. The treated and control animals were divided into 2 further groups. One group (lactating rats) was permitted to suckle their pups for a further 12 or 24 h; the young were removed from the other group (postlactating rats). Homogenates were prepared from the anterior pituitaries and assayed for organelle marker enzyme activities. When 0.5-500 micrograms bromocriptine were administered to lactating rats for 24 h, pituitary PRL was increased by all doses, but only the 500-micrograms dose significantly reduced plasma PRL. Total protein was unchanged, lysosomal acid PRL proteolytic activity increased 8-fold, N-acetyl-beta-glucosaminidase and beta-glucuronidase (lysosomes) were unchanged, acid phosphatase (lysosomes and endoplasmic reticulum) was increased by three of four doses, 5'-nucleotidase and alkaline phosphatase (plasma membrane) were increased 4-fold, neutral-alpha-glucosidase (endoplasmic reticulum) and malate dehydrogenase (mitochondria) were unchanged, and catalase (peroxisomes) was significantly increased. Bromocriptine (500 micrograms) administration to lactating and postlactating rats for 12 and 24 h significantly decreased the pituitary DNA but not the total protein content of the pituitaries in all animals. The lysosomal acid PRL proteolytic activity and the lysosomal enzyme activities, N-acetyl-beta-glucosaminidase and beta-glucuronidase, were increased by suckling withdrawal alone. Acid PRL proteolytic activity was further increased (to 18-fold) by coadministration of bromocriptine, whereas the increase in the activities of the other lysosomal marker enzymes was blocked. Malate dehydrogenase activity (mitochondria) was also increased by litter removal and blocked by bromocriptine. The activity of the plasma membrane markers 5'-nucleotidase and alkaline phosphatase were increased by litter removal, and bromocriptine further increased both enzyme activities. The activity of neutral-alpha-glucosidase (endoplasmic reticulum) was unchanged by any treatment. The results demonstrate that bromocriptine produces significant changes in the activities of lysosomal marker enzymes, particularly acid PRL proteolytic activity, as well as marker enzymes of plasma membranes and other organelles in pituitaries of lactating and postlactating rats.
5'-Nucleotidase↗
The solvent polarity dependent conformational equilibrium of the carboxylic ionophore narasin: a proton NMR study.
Two dimensional homonuclear (1H-1H) chemical shift correlation, double resonance and nuclear Overhauser effect difference spectroscopy were used to determine spectral parameters of narasin acid in different solvents approximating the range of polarities encountered within a biological membrane. The observed chemical shift and coupling constant changes were consistent with a polarity mediated shift between two conformational states, with the major conformational adjustments occurring in two specific backbone regions of the molecule previously described as "hinges" (1,2). Evidence suggests that the conformational equilibrium is not only mediated by solvent polarity but may in part be determined by the intrinsic propensity of narasin to form inclusion complexes with H+.
Chemical Phenomena↗
Analytical subcellular fractionation of rat pituitary homogenates with special reference to the subcellular localization and properties of alkaline phosphatases.
Alkaline phosphatase activities of the virgin rat anterior pituitary were studied with a highly sensitive fluorometric assay. Tissue whole homogenates were fractionated on sucrose density gradients in a Beaufay automatic zonal rotor and the gradient fractions assayed for alkaline phosphatase, prolactin and various organelle marker enzymes. Alkaline phosphatase was distributed between two peaks on the gradient. The low-density (1.10-1.15 g . cm-3) alkaline phosphatase component co-sedimented with the plasma membrane marker, 5'-nucleotidase, had an apparent Km for 4-methylumbelliferyl phosphate of approx. 59 microM, and was inhibited by levamisole. The high-density (1.20-1.25 g . cm-3) peak was resistant to levamisole-inhibition, had an apparent Km of approx. 30 microM and its distribution was distinct from plasma membrane, Golgi, lysosome, endoplasmic reticulum, mitochondria and prolactin granule markers on the isopycnic gradients.
Alkaline Phosphatase↗
Growth of infective forms of Trypanosoma rhodesiense in vitro, the causative agent of African trypanosomiasis.
A new approach to the culture of African trypanosomes led to the growth of the infective forms of the causative agent of human African trypanosomiasis. Infective cultures of Trypanosoma rhodesiense were initiated and maintained in vitro on Chinese hamster lung cells. By changing daily one-third of the Hepes-buffered RPMI 1640 medium containing 20 percent fetal bovine serum, the trypanosome numbers increased to 3 X 10(6) to 5 X 10(6) cells per milliliter. After 80 days in vitro at 37 degrees C, the cultured trypomastigotes are infective for mice and rats and morphologically similar to bloodstream trypomastigotes in having a subterminal kinetoplast and a surface coat. In addition, they possess L-alpha-glycerophosphate oxidase, the predominant steady-state terminal oxidase of bloodstream trypomastigotes.
Animals↗
Growth of infective forms of Trypanosoma (T.) brucei on buffalo lung and Chinese hamster lung tissue culture cells.
Infective cultures of Trypanosoma (T.) brucei (strain 427) have been initiated and maintained on Chinese hamster lung tissue culture cells and buffalo lung tissue culture cells. By changing daily one-third of the RPMI-1640 plus 20% fetal bovine serum medium, the cell numbers can be maintained at 2--4 X 10(6) cells/ml. The cultured trypanosomes on these two tissue culture cell types were infective to mice and morphologically similar to bloodstream slender trypomastigotes in having a subterminal kinetoplast and a surface coat. In addition, they possessed the L-alpha-glycerophosphate oxidase, the predominant steady state terminal oxidase identified in bloodstream trypomastigotes.
Animals↗
Inhibition of scrapie-associated PrP accumulation. Probing the role of glycosaminoglycans in amyloidogenesis.
Accumulation of an abnormal, protease-resistant form of an endogenous protein, PrP, is a characteristic feature of scrapie and related transmissible spongiform encephalopathies. This abnormal isoform is also present in the amyloid plaques that are often observed in these diseases. In mouse neuroblastoma cells persistently infected with scrapie, the abnormal protease-resistant isoform of PrP is derived from an operationally normal protease-sensitive precursor. Conversion of PrP to the protease-resistant state occurs either on the plasma membrane or along an endocytic pathway by an unknown mechanism. Inhibitors of protease-resistant PrP accumulation have been identified, and these include the amyloid-binding dye Congo red and certain sulfated glycans. The similarity of these compounds to sulfated glycosaminoglycans, which are components of all natural amyloids, has led to the hypothesis that the inhibitors act by competitively blocking an interaction between endogenous glycosaminoglycan(s) and PrP that is critical for amyloidogenic PrP accumulation. The proven prophylactic effect of these sulfated glycans in animal models of scrapie suggests that they represent a group of compounds that might interfere with the pathogenic formation of amyloid in a variety of diseases, such as Alzheimer's disease.
Amyloid↗
Prion protein and the scrapie agent: in vitro studies in infected neuroblastoma cells.
The mouse neuroblastoma cell line N2a was persistently infected with the Chandler strain of the mouse scrapie agent. Although the infection did not spread to infect > 1% of the cells, clones were established that had from 50 to 100% infected cells. These clones expressed the abnormal protease-resistant form of prion protein (PrP), which is believed to mediate brain degeneration in animals with scrapie and bovine spongiform encephalopathy and in humans with kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome. With this in vitro system, Congo red and several sulfated polysaccharides, including heparin and pentosan polysulfate, were found to inhibit accumulation of protease-resistant PrP. These results and additional data confirming PrP binding to heparin suggested a possible role for sulfated glycosaminoglycans in the generation of protease-resistant PrP during scrapie infection. Accumulation of protease-resistant PrP was also blocked in vitro by expression of foreign PrP molecules, indicating that PrP from different species might compete for common substrates in this process. These results using scrapie-infected cell lines provide new opportunities for development of drugs capable of blocking the brain degeneration caused by scrapie and other transmissible spongiform encephalopathies.
Animals↗