Kevin Strange

Maintenance of cell volume in the central nervous system

Maintenance of the ionic and osmotic composition and volume of intra- and extracellular fluids in the brain is crucial for normal functioning of the central nervous system (CNS). Osmoregulation in the CNS is mediated by solute and water transport across the blood-brain barrier, choroid plexus and plasma membrane of glial cells and neurons. Despite its clinical and physiological significance, however, little is known about the underlying cellular and molecular mechanisms by which CNS osmotic and ionic balance is maintained. In this review, I will discuss our current understanding of cell volume regulation in the CNS and how it relates to various disease processes, such as hyponatremia, renal failure and hypernatremia. A detailed understanding of brain osmoregulatory processes represents a fundamental physiological problem and is required for the treatment of numerous disease states, particularly those encountered in the practice of nephrology.

CHARACTERIZATION OF PICLN PHOSPHORYLATION STATE AND A PICLN-ASSOCIATED PROTEIN KINASE

pICln is a ubiquitous cellular protein that has been proposed to be a volume-sensitive Cl- channel or a channel regulator. Detailed biochemical, cellular and molecular characterization of pICln is required to understand its function. Our goal in the present investigation was to define further the biochemical properties of pICln and the proteins that associate with it. Immunoprecipitation of pICln from 32P-orthophosphoric acid-labeled C6 glioma cells revealed that the protein is phosphorylated constitutively, primarily on serine residues. Protein kinase activity was detected in pICln immunoprecipitates, revealing that a constitutively active protein kinase co-precipitates with pICln. A specific association between pICln and a protein kinase was also observed in affinity assays using a recombinant GST-pICln fusion protein. The pICln-associated kinase displayed broad substrate specificity and was inhibited in a concentration-dependent manner by heparin, zinc and 5,6-dichloro-1-beta-D-ribofuranosylbenose (DRB). These characteristics resembled those of casein kinase I and II. The pICln-associated kinase was not recognized, however, by antibodies against these two enzymes. Association of the kinase with pICln was disrupted by increasing concentrations of NaCl in the washing buffer, suggesting that electrostatic interactions are involved in kinase binding. Mutagenesis experiments corroborated this observation. Truncation of pICln demonstrated that two highly charged clusters of acidic amino acid residues are both necessary and sufficient for kinase binding. Phosphopeptide mapping demonstrated that pICln contains at least two phosphorylated serine residues that are located on trypsin cleavage fragments rich in acidic amino acid residues. We propose that the kinase or a kinase binding protein binds to acidic amino acids located between D101 and Y156 and phosphorylates nearby serine residues.

Volume Regulation in the Collecting Duct and Related Epithelia

Under normal physiological conditions, cells of tight, Na+-reabsorbing urinary epithelia, such as the collecting tubule, are exposed to both anisosmotic and isosmotic volume stress. A common but poorly understood response of these cells to volume perturbation is the activation of mechanisms that restore volume to its original resting value. The purpose of this article is to review briefly what is currently known about volume regulatory processes in the collecting tubule, amphibian and mammalian bladder, and frog skin. Specifically, the role of solute loss and accumulation pathways, transcellular cross-talk mechanisms, and volume sensor/transducer systems will be discussed. Key questions in need of future research will be emphasized.

Optical Methods for Quantifying Cell Volume Changes in Isolated Nephron Segments

Animal cell volume is determined by extracellular and intracellular solute content. Measurements of cell volume changes induced by osmotic, ionic, electrophysiological and/or pharmacological manipulations can therefore provide important and often unique insights into membrane solute and water transport properties. In addition, volume measurements allow the elucidation of cell volume regulatory mechanism. The purpose of this brief review article is to describe the requirements for measuring volume changes in isolated nephron segments and to illustrate the types of questions that can be addressed using this powerful approach.