Endothelial Na-K-Cl cotransport regulation by tonicity and hormones: Phosphorylation of cotransport protein

Martha E O'Donnell, A. Martinez, D. Sun

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Abstract

The Na-K-Cl cotransport system of vascular endothelial cells plays a central role in maintenance and regulation of intracellular volume. Activity of the cotransporter is modulated both by hormones and by extracellular tonicity. Vasopressin and other hormones that stimulate the endothelial cotransporter act via a Ca- and calmodulin-dependent pathway. Little is known, however, about the mechanisms that mediate cell shrinkage-induced stimulation of cotransport activity. In the present study, we evaluated the Ca dependence of cell shrinkage-stimulated Na-K-Cl cotransport activity and cell volume recovery of cultured bovine aortic endothelial cells and also the effects of protein kinase and phosphatase inhibitors on these processes. In addition, to investigate the possibility that hormones and/or hypertonicity regulate endothelial Na-K-Cl cotransport via direct phosphorylation of the cotransporter protein, we employed a monoclonal antibody to the human colonic T84 epithelial cell Na-K-Cl cotransport protein (T4 antibody) for Western blot analysis and immunoprecipitation of phosphoprotein. Our studies revealed that both cell shrinkage-stimulated net K uptake and recovery of intracellular volume were Ca dependent. We also found that hypertonicity- induced stimulation of cotransport activity was blocked by several inhibitors of Ca- and calmodulin-dependent protein kinases. Furthermore, inhibitors of myosin light chain kinase blocked cell shrinkage-stimulated cotransport and recovery of intracellular volume, while having no effect on vasopressin- stimulated cotransport. Western blot analysis of bovine aortic and cerebral microvascular endothelial cell membrane preparations revealed a 170-kDa protein recognized by the T4 antibody. In addition, we found that hypertonicity induced a marked increase in phosphorylation of the endothelial cotransport protein, as did vasopressin, bradykinin, okadaic acid, and calyculin A. Our findings indicate that modulation of endothelial cell Na-K- Cl cotransport activity by hypertonicity and by stimulatory hormones occurs via pathways involving Ca- and calmodulin-dependent protein kinases and direct phosphorylation of the cotransport protein.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume269
Issue number6 38-6
StatePublished - 1995

Fingerprint

Phosphorylation
protein phosphorylation
Endothelial cells
shrinkage
endothelial cells
hormones
vasopressin
Hormones
calmodulin
Endothelial Cells
Vasopressins
protein kinases
Calcium-Calmodulin-Dependent Protein Kinases
L-thyroxine
Proteins
Recovery
cells
Western blotting
Western Blotting
myosin light chain kinase

Keywords

  • bumetanide
  • hypertonicity
  • immunoprecipitation
  • intracellular volume
  • kinases
  • monoclonal antibodies
  • phosphatases
  • vasopressin

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology
  • Agricultural and Biological Sciences(all)

Cite this

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title = "Endothelial Na-K-Cl cotransport regulation by tonicity and hormones: Phosphorylation of cotransport protein",
abstract = "The Na-K-Cl cotransport system of vascular endothelial cells plays a central role in maintenance and regulation of intracellular volume. Activity of the cotransporter is modulated both by hormones and by extracellular tonicity. Vasopressin and other hormones that stimulate the endothelial cotransporter act via a Ca- and calmodulin-dependent pathway. Little is known, however, about the mechanisms that mediate cell shrinkage-induced stimulation of cotransport activity. In the present study, we evaluated the Ca dependence of cell shrinkage-stimulated Na-K-Cl cotransport activity and cell volume recovery of cultured bovine aortic endothelial cells and also the effects of protein kinase and phosphatase inhibitors on these processes. In addition, to investigate the possibility that hormones and/or hypertonicity regulate endothelial Na-K-Cl cotransport via direct phosphorylation of the cotransporter protein, we employed a monoclonal antibody to the human colonic T84 epithelial cell Na-K-Cl cotransport protein (T4 antibody) for Western blot analysis and immunoprecipitation of phosphoprotein. Our studies revealed that both cell shrinkage-stimulated net K uptake and recovery of intracellular volume were Ca dependent. We also found that hypertonicity- induced stimulation of cotransport activity was blocked by several inhibitors of Ca- and calmodulin-dependent protein kinases. Furthermore, inhibitors of myosin light chain kinase blocked cell shrinkage-stimulated cotransport and recovery of intracellular volume, while having no effect on vasopressin- stimulated cotransport. Western blot analysis of bovine aortic and cerebral microvascular endothelial cell membrane preparations revealed a 170-kDa protein recognized by the T4 antibody. In addition, we found that hypertonicity induced a marked increase in phosphorylation of the endothelial cotransport protein, as did vasopressin, bradykinin, okadaic acid, and calyculin A. Our findings indicate that modulation of endothelial cell Na-K- Cl cotransport activity by hypertonicity and by stimulatory hormones occurs via pathways involving Ca- and calmodulin-dependent protein kinases and direct phosphorylation of the cotransport protein.",
keywords = "bumetanide, hypertonicity, immunoprecipitation, intracellular volume, kinases, monoclonal antibodies, phosphatases, vasopressin",
author = "O'Donnell, {Martha E} and A. Martinez and D. Sun",
year = "1995",
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volume = "269",
journal = "American Journal of Physiology - Renal Fluid and Electrolyte Physiology",
issn = "1931-857X",
publisher = "American Physiological Society",
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T1 - Endothelial Na-K-Cl cotransport regulation by tonicity and hormones

T2 - Phosphorylation of cotransport protein

AU - O'Donnell, Martha E

AU - Martinez, A.

AU - Sun, D.

PY - 1995

Y1 - 1995

N2 - The Na-K-Cl cotransport system of vascular endothelial cells plays a central role in maintenance and regulation of intracellular volume. Activity of the cotransporter is modulated both by hormones and by extracellular tonicity. Vasopressin and other hormones that stimulate the endothelial cotransporter act via a Ca- and calmodulin-dependent pathway. Little is known, however, about the mechanisms that mediate cell shrinkage-induced stimulation of cotransport activity. In the present study, we evaluated the Ca dependence of cell shrinkage-stimulated Na-K-Cl cotransport activity and cell volume recovery of cultured bovine aortic endothelial cells and also the effects of protein kinase and phosphatase inhibitors on these processes. In addition, to investigate the possibility that hormones and/or hypertonicity regulate endothelial Na-K-Cl cotransport via direct phosphorylation of the cotransporter protein, we employed a monoclonal antibody to the human colonic T84 epithelial cell Na-K-Cl cotransport protein (T4 antibody) for Western blot analysis and immunoprecipitation of phosphoprotein. Our studies revealed that both cell shrinkage-stimulated net K uptake and recovery of intracellular volume were Ca dependent. We also found that hypertonicity- induced stimulation of cotransport activity was blocked by several inhibitors of Ca- and calmodulin-dependent protein kinases. Furthermore, inhibitors of myosin light chain kinase blocked cell shrinkage-stimulated cotransport and recovery of intracellular volume, while having no effect on vasopressin- stimulated cotransport. Western blot analysis of bovine aortic and cerebral microvascular endothelial cell membrane preparations revealed a 170-kDa protein recognized by the T4 antibody. In addition, we found that hypertonicity induced a marked increase in phosphorylation of the endothelial cotransport protein, as did vasopressin, bradykinin, okadaic acid, and calyculin A. Our findings indicate that modulation of endothelial cell Na-K- Cl cotransport activity by hypertonicity and by stimulatory hormones occurs via pathways involving Ca- and calmodulin-dependent protein kinases and direct phosphorylation of the cotransport protein.

AB - The Na-K-Cl cotransport system of vascular endothelial cells plays a central role in maintenance and regulation of intracellular volume. Activity of the cotransporter is modulated both by hormones and by extracellular tonicity. Vasopressin and other hormones that stimulate the endothelial cotransporter act via a Ca- and calmodulin-dependent pathway. Little is known, however, about the mechanisms that mediate cell shrinkage-induced stimulation of cotransport activity. In the present study, we evaluated the Ca dependence of cell shrinkage-stimulated Na-K-Cl cotransport activity and cell volume recovery of cultured bovine aortic endothelial cells and also the effects of protein kinase and phosphatase inhibitors on these processes. In addition, to investigate the possibility that hormones and/or hypertonicity regulate endothelial Na-K-Cl cotransport via direct phosphorylation of the cotransporter protein, we employed a monoclonal antibody to the human colonic T84 epithelial cell Na-K-Cl cotransport protein (T4 antibody) for Western blot analysis and immunoprecipitation of phosphoprotein. Our studies revealed that both cell shrinkage-stimulated net K uptake and recovery of intracellular volume were Ca dependent. We also found that hypertonicity- induced stimulation of cotransport activity was blocked by several inhibitors of Ca- and calmodulin-dependent protein kinases. Furthermore, inhibitors of myosin light chain kinase blocked cell shrinkage-stimulated cotransport and recovery of intracellular volume, while having no effect on vasopressin- stimulated cotransport. Western blot analysis of bovine aortic and cerebral microvascular endothelial cell membrane preparations revealed a 170-kDa protein recognized by the T4 antibody. In addition, we found that hypertonicity induced a marked increase in phosphorylation of the endothelial cotransport protein, as did vasopressin, bradykinin, okadaic acid, and calyculin A. Our findings indicate that modulation of endothelial cell Na-K- Cl cotransport activity by hypertonicity and by stimulatory hormones occurs via pathways involving Ca- and calmodulin-dependent protein kinases and direct phosphorylation of the cotransport protein.

KW - bumetanide

KW - hypertonicity

KW - immunoprecipitation

KW - intracellular volume

KW - kinases

KW - monoclonal antibodies

KW - phosphatases

KW - vasopressin

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