Vasoactive substances induce cytoskeletal changes in cultured rat glomerular epithelial cells

Ram Sharma, Helen Bergado Lovell, Thomas B. Wiegmann, Virginia J. Savin

Research output: Contribution to journalArticle

72 Citations (Scopus)

Abstract

Angiotensin II (ANG II), atrial natriuretic peptide III (ANP), and sodium nitroprusside (SNP) alter capillary hydraulic conductivity in isolated glomeruli. These agents also affect cyclic nucleotide levels of glomerular epithelial cells (GEC). ANG II increases cAMP, whereas ANP and SNP increase cGMP. The effects of these vasoactive substances on GEC cytoskeleton were tested by incubating cells from primary cultures or an established cell line with each agent. Changes in the cytoskeleton were assessed by staining for F-actin with Bodipy® phallacidin and for tubulin with NBD-colcemid. Control cells exhibited short bundles of F-actin or stress fibers near the base of the cells. These were frequently arranged in parallel and occasionally appeared to radiate from the center of the cell. Microtubules were arranged in a fine network throughout the cell with increased density adjacent to the nucleus and within the nucleolus. Incubation of GEC with 10-7 M ANG II, cholera toxin, or 8Br-cAMP for 2 h at 37°C resulted in rearrangement of F-actin into distinct stellate patterns with a decrease in the relative intensity of the peripheral staining, all concurrent with a fivefold increase in intracellular cAMP. The incubation of GEC with 10-6 M ANP or 10-7 M SNP for 2 h at 37°C resulted in apparent disassembly of stress fibers, sparse and more diffuse fluorescence, and some increase of fluorescence at the periphery of the cells, all concurrent with a 10-fold increase in intracellular cGMP. Cytochalasin D incubation led to complete disassembly of actin filaments. The tubulin-containing microtubule meshwork showed no apparent changes with cAMP- or cGMP-inducing vasoactive agents. Direct measurement of F-actin and G-actin by immunoblotting showed no change in actin levels with vasoactive agents. This study demonstrates the presence of a regulatory system for the cytoskeleton of GEC mediated by vasoconstrictors, which increase cAMP, and vasodilators, which increase cGMP. Changes in F-actin architecture may play an important role in modulating filtration characteristics of GEC in vivo.

Original languageEnglish (US)
Pages (from-to)1131-1138
Number of pages8
JournalJournal of the American Society of Nephrology
Volume3
Issue number5
StatePublished - Nov 1 1992

Fingerprint

Actins
Epithelial Cells
Nitroprusside
Atrial Natriuretic Factor
Cytoskeleton
Angiotensin II
Stress Fibers
Tubulin
Microtubules
Fluorescence
Staining and Labeling
Cytochalasin D
Primary Cell Culture
Cholera Toxin
Cyclic Nucleotides
Vasoconstrictor Agents
Actin Cytoskeleton
Vasodilator Agents
Immunoblotting
Cell Line

Keywords

  • Actin
  • Angtotensin II
  • Atrial natriuretic peptide
  • Sodium nitroprusside
  • cAMP
  • cGMP

ASJC Scopus subject areas

  • Nephrology

Cite this

Vasoactive substances induce cytoskeletal changes in cultured rat glomerular epithelial cells. / Sharma, Ram; Lovell, Helen Bergado; Wiegmann, Thomas B.; Savin, Virginia J.

In: Journal of the American Society of Nephrology, Vol. 3, No. 5, 01.11.1992, p. 1131-1138.

Research output: Contribution to journalArticle

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AU - Lovell, Helen Bergado

AU - Wiegmann, Thomas B.

AU - Savin, Virginia J.

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N2 - Angiotensin II (ANG II), atrial natriuretic peptide III (ANP), and sodium nitroprusside (SNP) alter capillary hydraulic conductivity in isolated glomeruli. These agents also affect cyclic nucleotide levels of glomerular epithelial cells (GEC). ANG II increases cAMP, whereas ANP and SNP increase cGMP. The effects of these vasoactive substances on GEC cytoskeleton were tested by incubating cells from primary cultures or an established cell line with each agent. Changes in the cytoskeleton were assessed by staining for F-actin with Bodipy® phallacidin and for tubulin with NBD-colcemid. Control cells exhibited short bundles of F-actin or stress fibers near the base of the cells. These were frequently arranged in parallel and occasionally appeared to radiate from the center of the cell. Microtubules were arranged in a fine network throughout the cell with increased density adjacent to the nucleus and within the nucleolus. Incubation of GEC with 10-7 M ANG II, cholera toxin, or 8Br-cAMP for 2 h at 37°C resulted in rearrangement of F-actin into distinct stellate patterns with a decrease in the relative intensity of the peripheral staining, all concurrent with a fivefold increase in intracellular cAMP. The incubation of GEC with 10-6 M ANP or 10-7 M SNP for 2 h at 37°C resulted in apparent disassembly of stress fibers, sparse and more diffuse fluorescence, and some increase of fluorescence at the periphery of the cells, all concurrent with a 10-fold increase in intracellular cGMP. Cytochalasin D incubation led to complete disassembly of actin filaments. The tubulin-containing microtubule meshwork showed no apparent changes with cAMP- or cGMP-inducing vasoactive agents. Direct measurement of F-actin and G-actin by immunoblotting showed no change in actin levels with vasoactive agents. This study demonstrates the presence of a regulatory system for the cytoskeleton of GEC mediated by vasoconstrictors, which increase cAMP, and vasodilators, which increase cGMP. Changes in F-actin architecture may play an important role in modulating filtration characteristics of GEC in vivo.

AB - Angiotensin II (ANG II), atrial natriuretic peptide III (ANP), and sodium nitroprusside (SNP) alter capillary hydraulic conductivity in isolated glomeruli. These agents also affect cyclic nucleotide levels of glomerular epithelial cells (GEC). ANG II increases cAMP, whereas ANP and SNP increase cGMP. The effects of these vasoactive substances on GEC cytoskeleton were tested by incubating cells from primary cultures or an established cell line with each agent. Changes in the cytoskeleton were assessed by staining for F-actin with Bodipy® phallacidin and for tubulin with NBD-colcemid. Control cells exhibited short bundles of F-actin or stress fibers near the base of the cells. These were frequently arranged in parallel and occasionally appeared to radiate from the center of the cell. Microtubules were arranged in a fine network throughout the cell with increased density adjacent to the nucleus and within the nucleolus. Incubation of GEC with 10-7 M ANG II, cholera toxin, or 8Br-cAMP for 2 h at 37°C resulted in rearrangement of F-actin into distinct stellate patterns with a decrease in the relative intensity of the peripheral staining, all concurrent with a fivefold increase in intracellular cAMP. The incubation of GEC with 10-6 M ANP or 10-7 M SNP for 2 h at 37°C resulted in apparent disassembly of stress fibers, sparse and more diffuse fluorescence, and some increase of fluorescence at the periphery of the cells, all concurrent with a 10-fold increase in intracellular cGMP. Cytochalasin D incubation led to complete disassembly of actin filaments. The tubulin-containing microtubule meshwork showed no apparent changes with cAMP- or cGMP-inducing vasoactive agents. Direct measurement of F-actin and G-actin by immunoblotting showed no change in actin levels with vasoactive agents. This study demonstrates the presence of a regulatory system for the cytoskeleton of GEC mediated by vasoconstrictors, which increase cAMP, and vasodilators, which increase cGMP. Changes in F-actin architecture may play an important role in modulating filtration characteristics of GEC in vivo.

KW - Actin

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