Dietmar Kültz

Mitogen-activated protein kinases are in vivo transducers of osmosensory signals in fish gill cells.

The abundance and activity of three subgroups of mitogen-activated protein (MAP) kinases, the extracellular signal regulated kinase 1 (ERK1), stress-activated protein kinase 1/ Jun N-terminal kinase (SAPK1), and stress-activated protein kinase 2/ p38 (SAPK2), were measured in gill epithelium of the euryhaline teleost Fundulus heteroclitus exposed for 1 h to 4 weeks to hyper- and hyposmotic stress. The abundance of ERK1, SAPK1 and SAPK2 was analyzed by standard Western immunodetection. MAP kinase activity is a function of phosphorylation and was measured using phospho-specific and MAP kinase subgroup-specific antibodies. The abundance of the 63 kDa fish isoform of SAPK2 increases significantly during hyper- but not hyposmotic stress while ERK1 and SAPK1 protein levels remain unchanged during both types of osmotic stress. In contrast to this small effect of osmotic stress on MAP kinase abundance, the activity of all MAP kinases decreases significantly in response to hyperosmotic stress and increases significantly during hyposmotic stress. These results demonstrate for the first time that the activity of all major MAP kinases is osmoregulated in gill epithelium of euryhaline fish. Based on these results we conclude that MAP kinases are important components of salinity adaptation and participate in osmosensory signaling pathways in gill epithelium of euryhaline fishes.

Mitochondria-rich (MR) cells and the activities of the Na+/K+-ATPase and carbonic anhydrase in the gill and opercular epithelium of Oreochromis mossambicus adapted to various salinities

1. 1. The activities of the Na+K+-ATPase and carbonic anhydrase and the number and size of mitochondria rich cells were determined in the gills and opercular epithelia of tilapias adapted to freshwater, brackish water, seawater, 130% seawater and 170% seawater. 2. 2. In the gill all parameters examined increased significantly in correlation to an enhanced environmental salinity, whereas in the opercular epithelium the activities of the enzymes remained constant. 3. 3. On the basis of size-frequency analysis two classes of mitochondria rich cells were found.

Cellular and epithelial adjustments to altered salinity in the gill and opercular epithelium of a cichlid fish (Oreochromis mossambicus)

Morphological features of the gill and opercular epithelia of tilapia (Oreochromis mossambicus) have been compared in fish acclimated to either fresh water (FW) or hypersaline water (60‰ S) by scanning electron and fluorescence microscopy. In hyperosmoregulating, i.e., FW-acclimated, tilapia only those mitochondria-rich (MR) cells present on the filament epithelium of the gill were exposed to the external medium. After acclimation of fish to hypersaline water these cells become more numerous, hypertrophy extensively, and form apical crypts not only in the gill filament but also in the opercular epithelium. Regardless of salinity, MR cells were never found to be exposed to the external medium on the secondary lamellae. In addition, two types of pavement cells were identified having distinct morphologies, which were unaffected by salinity. The gill filaments and the inner operculum were generally found to be covered by pavement cells with microridges, whereas the secondary lamellae were covered exclusively by smooth pavement cells.

Maintenance of genomic integrity in mammalian kidney cells exposed to hyperosmotic stress

Changes in environmental salinity/osmolality impose an osmotic stress upon cells because, if left uncompensated, such changes will alter the conserved intracellular ionic milieu and macromolecular density, for which cell metabolism in most extant cells has been optimized. Cell responses to osmotic stress include rapid posttranslational and slower transcriptional events for the compensatory regulation of cell volume, intracellular electrolyte concentrations, and protein stability/activity. Changes in external osmolality are perceived by osmosensors that control the activation of signal transduction pathways giving rise to the above responses. We have recently shown that the targets of such pathways include cell cycle-regulatory and DNA damage-inducible genes (reviewed in Kültz, D., 2000. Environmental stressors and gene responses, Elsevier, Amsterdam. pp 157-179). Moreover, recent evidence suggests that hyperosmotic stress causes chromosomal aberrations and DNA double-strand breaks in mammalian cells. We propose that the modulation of cell cycle checkpoints and the preservation of genomic integrity are important aspects of cellular osmoprotection and as essential for cellular osmotic stress resistance as the capacity for cell volume regulation and maintaining inorganic ion homeostasis and protein stability/activity.

Cellular osmoregulation: beyond ion transport and cell volume

All cells are characterized by the expression of osmoregulatory mechanisms, although the degree of this expression is highly variable in different cell types even within a single organism. Cellular osmoregulatory mechanisms constitute a conserved set of adaptations that offset antagonistic effects of altered extracellular osmolality/environmental salinity on cell integrity and function. Cellular osmoregulation includes the regulation of cell volume and ion transport but it does not stop there. We know that organic osmolyte concentration, protein structure, cell turnover, and other cellular parameters are osmoregulated as well. In this brief review two important aspects of cellular osmoregulation are emphasized: 1) maintenance of genomic integrity, and 2) the central role of protein phosphorylation. Novel insight into these two aspects of cellular osmoregulation is illustrated based on two cell models, mammalian kidney inner medullary cells and teleost gill epithelial cells. Both cell types are highly hypertonicity stress-resistant and, therefore, well suited for the investigation of osmoregulatory mechanisms. Damage to the genome is discussed as a newly discovered aspect of hypertonic threat to cells and recent insights on how mammalian kidney cells deal with such threat are presented. Furthermore, the importance of protein phosphorylation as a core mechanism of osmosensory signal transduction is emphasized. In this regard, the potential roles of the 14-3-3 family of phospho-protein adaptor molecules for cellular osmoregulation are highlighted primarily based on work with fish gill epithelial cells. These examples were chosen for the reader to appreciate the numerous and highly specific interactions between stressor-specific and non-specific pathways that form an extensive cellular signaling network giving rise to adaptive compensation of hypertonicity. Furthermore, the example of 14-3-3 proteins illustrates that a single protein may participate in several pathways that are non-specific with regard to the type of stress and, at the same time, in stress-specific pathways to promote cell integrity and function during hypertonicity.

Chapter 12 - Osmotic regulation of DNA activity and the cell cycle

This chapter discusses osmotic effects on DNA conformation, DNA activity, and the cell cycle with emphasis on their interdependent relationship and adaptive value during osmotic stress. Osmotic stress has multiple effects on DNA conformation and DNA activity in all biological kingdoms. An important part of the cellular response to osmotic stress is the regulation of the cell cycle, which is closely linked to DNA conformation and DNA activity. For a better understanding of the osmosensory signal transduction networks that control DNA activity and the cell cycle during osmotic stress, it is needed to identify more of the elements and mechanisms that determine the ways in which cells respond to changes in osmolality. Important questions that remain to be answered concern the interplay between signaling mechanisms necessary for cell cycle delay, cellular repair, and apoptosis in response to osmotic stress. Given the complex interactions between osmo-protective mechanisms and cell cycle regulation, integrative approaches to study cellular adaptation during osmotic stress will be instrumental for identifying the mechanisms that hold the key for the outcome of the cellular osmotic stress response. Approaches based on proteomics and gene chip technology should provide significant insight into these mechanisms in the future.

Evolution of Osmosensory MAP Kinase Signaling Pathways

SYNOPSIS. Mitogen-activated protein (MAP) kinases constitute a large family of proteins with many functions. They are represented by a multitude of paralogous isoforms in yeast, vertebrates, and other eukaryotes. A phylogenetically conserved function of MAP kinases is to carry osmotic signals from sensory to target elements of cells. Even though this function of MAP kinases is ubiquitous and characteristic of unicellular and multicellular eukaryotes alike the contingencies between individual MAP kinases, sensor elements, and target elements have been subject to vast modification during evolution. Extensive networking of MAP kinase cascades with other signaling pathways is reflected by the large number of diverse signals that can be carried by a single MAP kinase pathway and flexible activation kinetics. It is emerging that the most important function of MAP kinase networks may not be signal amplification but integration of information about the setpoint of environmental parameters (including osmolality) with other physiological processes to control cell function. Insight into how this cellular integration of information is achieved by MAP kinase networks will shed light on the principles of cell dynamics and adaptation.