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63 results on '"Gilmont, Robert R"'

62. Isolation and Characterization of Sertoli Cell Plasma Membranes and Associated Plasminogen Activator Activity1

Author

Marzowski, Joanne, Sylvester, Steven R., Gilmont, Robert R., and Griswold, Michael D.

Abstract

Plasma membranes were isolated from the cultured Sertoli cells of 20-day-old rat testes by differential centrifugation and sucrose density fractionation. The distribution and purity of subcellular components was determined by marker enzyme analysis of gradient fractions. The plasma membrane fraction showed an enrichment in two plasma membrane marker enzymes, 5′-nucleotidase and ouabain-sensitive Na+/K+-ATPase-specific activities, of 9- and 23-fold, respectively. Forty-two percent and 52% of the total cellular 5′-nucleotidase and ouabain-sensitive Na+/K+-ATPase activities, respectively, were found in the membrane fraction. The protein yield of plasma membrane was approximately 6% of the total cellular protein. Two-dimensional polyacrylamide gel electrophoresis was used to compare [35S] methionine- and [3H] glucosamine-labeled membrane proteins. The incorporation of [35S] methionine and [3H] glucosamine was increased in several proteins when the cultured Sertoli cells were treated with follicle-stimulating hormone, insulin, retinol, and testosterone.Isolated Sertoli cell membranes contained a membrane-associated form of plasminogen activator. Analysis of this plasminogen activator demonstrated that the membrane-associated enzyme existed primarily as a single 38,000–40,000-Mrform.

Published
1985
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63. Real-time dynamic movement of caveolin-1 during smooth muscle contraction of human colon and aged rat colon transfected with caveolin-1 cDNA.

Author

Somara S, Bashllari D, Gilmont RR, and Bitar KN

Subjects
Acetylcholine pharmacology, Age Factors, Animals, Caveolin 1 genetics, Cells, Cultured, Colon drug effects, Fluorescence Recovery After Photobleaching, Humans, Membrane Microdomains metabolism, Microscopy, Confocal, Muscle Strength, Muscle, Smooth drug effects, Mutation, Myocytes, Smooth Muscle drug effects, Protein Transport, Rats, Recombinant Fusion Proteins metabolism, Time Factors, Tissue Engineering, Transfection, Aging, Caveolin 1 metabolism, Colon metabolism, Gastrointestinal Motility drug effects, Muscle Contraction drug effects, Muscle, Smooth metabolism, Myocytes, Smooth Muscle metabolism
Abstract

Caveolin-1 (cav-1) plays a key role in PKC-α and RhoA signaling pathways during acetylcholine (ACh)-induced contraction of colonic smooth muscle cells (CSMC). Aged rat CSMC showed sluggish contractility, concomitant with reduced expression of cav-1 with an associated reduction in activation of PKC-α and RhoA signaling pathway. Real-time monitoring of live human CSMC transfected with yellow fluorescent protein-tagged wild-type caveolin 1 cDNA (YFP-wt-cav-1) cDNA in the present study suggests that cav-1 cycles within and along the membrane in a synchronized, highly organized cytoskeletal path. These studies provide, for the first time, the advantages of real-time monitoring of the dynamic movement of caveolin in living cells. Rapid movement of cav-1 in response to ACh suggests its dynamic role in CSMC contraction. Human CSMC transfected with YFP-ΔTFT-cav-1 dominant negative cDNA show fluorescence in the cytosol of the CSMC and no movement of fluorescent cav-1 in response to ACh mimicking the response shown by aged rat CSMC. Transfection of CSMC from aged rat with YFP-wt-cav-1 cDNA restored the physiological contractile response to ACh as well as the dynamic movement of cav-1 along the organized cytoskeletal path observed in normal adult CSMC. To study the force generation by CSMC, three-dimensional colonic rings were bioengineered. Colonic bioengineered rings from aged CSMC showed reduced force generation compared with colonic bioengineered rings from adult CSMC. Colonic bioengineered rings from aged CSMC transfected with wt-cav-1 cDNA showed force generation similar to colonic bioengineered rings from adult rat CSMC. The data suggest that contraction in CSMC is dependent on cav-1 reorganization dynamics, which restores the physiological contractile response in aged CSMC. We hypothesize that dynamic movement of cav-1 is essential for physiological contractile response of colonic smooth muscle.

Published
2011
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