Pablo Martín-Vasallo

Na+, K+-ATPase isozyme diversity; comparative biochemistry and physiological implications of novel functional interactions.

Na+, K+-ATPase is ubiquitously expressed in the plasma membrane ofall animal cells where it serves as the principal regulator of intracellularion homeostasis. Na+, K+-ATPase is responsible for generating andmaintaining transmembrane ionic gradients that are of vital importance forcellular function and subservient activities such as volume regulation, pHmaintenance, and generation of action potentials and secondary activetransport. The diversity of Na+, K+-ATPase subunit isoforms andtheir complex spatial and temporal patterns of cellular expression suggestthat Na+, K+-ATPase isozymes perform specialized physiologicalfunctions. Recent studies have shown that the α subunit isoformspossess considerably different kinetic properties and modes of regulationand the β subunit isoforms modulate the activity, expression and plasmamembrane targeting of Na+, K+-ATPase isozymes. This review focuseson recent developments in Na+, K+-ATPase research, and in particular reportsof expression of isoforms in various tissues and experiments aimed atelucidating the intrinsic structural features of isoforms important forNa+, K+-ATPase function.

INTEGRINS AND STRETCH ACTIVATED ION CHANNELS; PUTATIVE COMPONENTS OF FUNCTIONAL CELL SURFACE MECHANORECEPTORS IN ARTICULAR CHONDROCYTES

Perception of mechanical signals and the biological responses to such stimuli are fundamental properties of load bearing articular cartilage in diarthrodial joints. Chondrocytes utilize mechanical signals to synthesize an extracellular matrix capable of withstanding high loads and shear stresses. Recent studies have shown that chondrocytes undergo changes in shape and volume in a coordinated manner with load induced deformation of the matrix. These matrix changes, together with alterations in hydrostatic pressure, ionic and osmotic composition, interstitial fluid and streaming potentials are, in turn, perceived by chondrocytes. Chondrocyte responses to these stimuli are specific and well coordinated to bring about changes in gene expression, protein synthesis, matrix composition and ultimately biomechanical competence. In this hypothesis paper we propose a chondrocyte mechanoreceptor model incorporating key extracellular matrix macromolecules, integrins, mechanosensitive ion channels, the cytoskeleton and subcellular signal transduction pathways that maintain the chondrocyte phenotype, prevent chondrocyte apoptosis and regulate chondrocyte‐specific gene expression.

ISOFORMS OF NA,K-ATPASE ALPHA AND BETA SUBUNITS IN THE RAT CEREBELLUM AND IN GRANULE CELL CULTURES

There are multiple isoforms of the Na,K-ATPase in the nervous system, three isoforms of the α subunit, and at least two of the β subunit. The α subunit is the catalytic subunit. The β subunit has several roles. It is required for enzyme assembly, it has been implicated in neuron-glia adhesion, and the experimental exchange of β subunit isoforms modifies enzyme kinetics, implying that it affects functional properties. Here we describe the specificities of antibodies against the Na,K-ATPase β subunit isoforms β1 and β2. These antibodies, along with antibodies against the α subunit isoforms, were used to stain sections of the rat cerebellum and cultures of cerebellar granule cells to ascertain expression and subcellular distribution in identifiable cells. Comparison of α and β isoform distribution with double-label staining demonstrated that there was no preferential association of particular α subunits with particular β subunits, nor was there an association with excitatory or inhibitory neurotransmission modes. Isoform composition differences were seen when Purkinje, basket, and granule cells were compared. Whether β1 and β2 are specific for neurons and glia, respectively, has been controversial, but expression of both β subunit types was seen here in granule cells. In rat cerebellar astrocytes, in sections and in culture, α2 expression was prominent, yet the expression of either β subunit was low in comparison. The complexity of Na,K-ATPase isoform distribution underscores the subtlety of its regulation and physiological role in excitable cells.

Expression of the β-subunit isoforms of the Na, K-ATpase in rat embryo tissues, inner ear and choroid plexus

Summary— We report evidence of the apical localization of the two Na, K‐ATPase β‐subunit isoforms in cells of the inner ear and of the choroid plexus of the rat. To this end, we generated isoform‐specific antisera against the human Na, K‐ATPase β1 and β2 subunits. These polyclonal rabbit antisera were raised against truncated β‐isoform proteins that were made in E coli with pET expression vectors. Deglycosylation of the native antigen with N‐endoglycosidase F shows four bands in the β1 isoform and five bands in the β2 iso‐form immunoblots. In E15 rat embryos, the β1 isoform was detected in brain, heart and kidney and the β2 isoform only in brain. While β‐subunit mRNA expression (Watts AG, Sanchéz‐Watts G, Emanuel JR, Levenson R 1991 Proc Natl Acad Sci USA 88, 7425–7429), and immunoblotting and enzymatic activity have been determined (Zlokovic BV, Mackic JB, Wang L, McComb JG, McDonough A 1993 J Biol Chem 268, 8019–8025), very little is known about the specific localization of each β‐isoform in the epithelia of choroid plexus and inner ear. Immunocytochemical preparations of 15‐day‐old whole rat embryos and adult rat brain showed an enhanced staining for the β1 and β2 isoforms in the apical membrane of the ampullary crests of the inner ears semicircular ducts and in the cuboidal cells of the choroid plexus

ATPase pumps in osteoclasts and osteoblasts.

Osteoblasts, osteocytes and osteoclasts are specialised cells of bone that play crucial roles in the formation, maintenance and resorption of bone matrix. Bone formation and resorption critically depend on optimal intracellular calcium and phosphate homeostasis and on the expression and activity of plasma membrane transport systems in all three cell types. Osteotropic agents, mechanical stimulation and intracellular pH are important parameters that determine the fate of bone matrix and influence the activity, expression, regulation and cell surface abundance of plasma membrane transport systems. In this paper the role of ATPase pumps is reviewed in the context of their expression in bone cells, their contribution to ion homeostasis and their relation to other transport systems regulating bone turnover.

Expression of the β1 and β2(AMOG) subunits of the Na,K-ATPase in neural tissues: Cellular and developmental distribution patterns

Abstract We have used isoform-specific antisera against the Na,K-ATPase β1 (SpETb1) and β2(AMOG) (SpETb2) subunit isoforms in order to establish their specific cellular and subcellular localization in several developmental stages of the rat central nervous system. Immunocytochemical preparations revealed β1 isoform protein in most neural cells, being predominantly located in the soma of neurons and astrocytes, with no appreciable developmental variations. In the newborn rat, β2(AMOG) immunoreactivity was present in cellular processes of astroglia and in the somas of neurons and decreasing in intensity with maturation until adulthood, where no β2 isoform was detected in neurons. The diffenential location of these isoforms, both developmentally and at the cellular level suggest a complex regulation of their genes expression and mechanisms of subcellular distribution, as well as functional differences.

Novel interactions of CLN3 protein link Batten disease to dysregulation of fodrin-Na+, K+ ATPase complex.

Juvenile neuronal ceroid lipofuscinosis (JNCL, Batten disease) is the most common progressive neurodegenerative disorder of childhood. CLN3, the transmembrane protein underlying JNCL, is proposed to participate in multiple cellular events including membrane trafficking and cytoskeletal functions. We demonstrate here that CLN3 interacts with the plasma membrane-associated cytoskeletal and endocytic fodrin and the associated Na(+), K(+) ATPase. The ion pumping activity of Na(+), K(+) ATPase was unchanged in Cln3(-/-) mouse primary neurons. However, the immunostaining pattern of fodrin appeared abnormal in JNCL fibroblasts and Cln3(-/-) mouse brains suggesting disturbances in the fodrin cytoskeleton. Furthermore, the basal subcellular distribution as well as ouabain-induced endocytosis of neuron-specific Na(+), K(+) ATPase were remarkably affected in Cln3(-/-) mouse primary neurons. These data suggest that CLN3 is involved in the regulation of plasma membrane fodrin cytoskeleton and consequently, the plasma membrane association of Na(+), K(+) ATPase. Most of the processes regulated by multifunctional fodrin and Na(+), K(+) ATPase are also affected in JNCL and Cln3-deficiency implicating that dysregulation of fodrin cytoskeleton and non-pumping functions of Na(+), K(+) ATPase may play a role in the neuronal degeneration in JNCL.

Cilastatin protects against cisplatin-induced nephrotoxicity without compromising its anticancer efficiency in rats.

Cisplatin is an anticancer agent marred by nephrotoxicity; however, limiting this adverse effect may allow the use of higher doses to improve its efficacy. Cilastatin, a small molecule inhibitor of renal dehydropeptidase I, prevents proximal tubular cells from undergoing cisplatin-induced apoptosis in vitro. Here, we explored the in vivo relevance of these findings and the specificity of protection for kidney cells in cisplatin-treated rats. Cisplatin increased serum blood urea nitrogen and creatinine levels, and the fractional excretion of sodium. Cisplatin decreased the glomerular filtration rate, promoted histological renal injury and the expression of many pro-apoptotic proteins in the renal cortex, increased the Bax/Bcl2 ratio, and oxidative stress in kidney tissue and urine. All these features were decreased by cilastatin, which preserved renal function but did not modify the pharmacokinetics of cisplatin area under the curve. The cisplatin-induced death of cervical, colon, breast, and bladder-derived cancer cell lines was not prevented by cilastatin. Thus, cilastatin has the potential to prevent cisplatin nephrotoxicity without compromising its anticancer efficacy.

Epithelial Na, K-ATPase expression is down-regulated in canine prostate cancer; a possible consequence of metabolic transformation in the process of prostate malignancy

BackgroundAn important physiological function of the normal prostate gland is the synthesis and secretion of a citrate rich prostatic fluid. In prostate cancer, citrate production levels are reduced as a result of altered cellular metabolism and bioenergetics. Na, K-ATPase is essential for citrate production since the inward Na+ gradients it generates are utilized for the Na+ dependent uptake of aspartate, a major substrate for citrate synthesis. The objective of this study was to compare the expression of previously identified Na, K-ATPase isoforms in normal canine prostate, benign prostatic hyperplasia (BPH) and prostatic adenocarcinoma (PCa) using immunohistochemistry in order to determine whether reduced citrate levels in PCa are also accompanied by changes in Na, K-ATPase expression.ResultsExpression of Na, K-ATPase α1 and β1 isoforms was observed in the lateral and basolateral plasma membrane domains of prostatic epithelial cells in normal and BPH prostates. Canine kidney was used as positive control for expression of Na, K-ATPase α1 and γ isoforms. The α1 isoform was detected in abundance in prostatic epithelial cells but there was no evidence of α2, α3 or γ subunit expression. In advanced PCa, Na, K-ATPase α1 isoform expression was significantly lower compared to normal and BPH glands. The abundant basolateral immunostaining observed in normal and BPH tissue was significantly attenuated in PCa.ConclusionThe loss of epithelial structure and function and the transformation of normal epithelial cells to malignant cells in the canine prostate have important implications for cellular metabolism and are accompanied by a down regulation of Na, K-ATPase.

Oligodendrocytes in brain and optic nerve express the β3 subunit isoform of Na,K-ATPase

The Na,K‐ATPase, which catalyzes the active transport of Na+ and K+, has two principal subunits (α and β) that have several genetically distinct isoforms. Most of these isoforms are expressed in the nervous system, but certain ones are preferentially expressed in glia and others in neurons. Of the β isoforms, β1 predominates in neurons and β2 in astrocytes, although there are some exceptions. Here we demonstrate that β3 is expressed in rat and mouse white matter oligodendrocytes. Immunofluorescence microscopy identified β3 in oligodendrocytes of rat brain white matter in typical linear arrays of cell bodies between fascicles of axons. The intensity of stain peaked at 20 postnatal days. β3 was identified in cortical oligodendrocytes grown in culture, where it was expressed in processes and colocalized with antibody to galactocerebroside. In the mouse and rat optic nerve, β3 stain was seen in oligodendrocytes, where it colocalized with carbonic anhydrase II. For comparison, optic nerve was stained for the β1 and β2 subunits, showing distinct patterns of labelling of axons (β1) and astrocytes (β2). The C6 glioma cell line was also found to express the β3 isoform preferentially. Since β3 was not found at detectable levels in astrocytes, this suggests that C6 is closer to oligodendrocytes than astrocytes in the glial cell lineage. GLIA 31:206–218, 2000.