Na+, K+-ATPase is ubiquitously expressed in the plasma membrane of all animal cells where it serves as the principal regulator of intracellular ion homeostasis. Na+, K+-ATPase is responsible for generating and maintaining transmembrane ionic gradients that are of vital importance for cellular function and subservient activities such as volume regulation, pH maintenance, and generation of action potentials and secondary active transport. The diversity of Na+, K+-ATPase subunit isoforms and their complex spatial and temporal patterns of cellular expression suggest that Na+, K+-ATPase isozymes perform specialized physiological functions. Recent studies have shown that the alpha subunit isoforms possess considerably different kinetic properties and modes of regulation and the beta subunit isoforms modulate the activity, expression and plasma membrane targeting of Na+, K+-ATPase isozymes. This review focuses on recent developments in Na+, K+-ATPase research, and in particular reports of expression of isoforms in various tissues and experiments aimed at elucidating the intrinsic structural features of isoforms important for Na+, K+-ATPase function.

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.

There are multiple isoforms of the Na,K-ATPase in the nervous system, three isoforms of the ␣ subunit, and at least two of the ␤ subunit. The ␣ subunit is the catalytic subunit. The ␤ subunit has several roles. It is required for enzyme assembly, it has been implicated in neuron-glia adhesion, and the experimental exchange of ␤ subunit isoforms modifies enzyme kinetics, implying that it affects functional properties. Here we describe the specificities of antibodies against the Na,K-ATPase ␤ subunit isoforms ␤1 and ␤2. These antibodies, along with antibodies against the ␣ 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 ␣ and ␤ isoform distribution with doublelabel staining demonstrated that there was no preferential association of particular ␣ subunits with particular ␤ 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 ␤1 and ␤2 are specific for neurons and glia, respectively, has been controversial, but expression of both ␤ subunit types was seen here in granule cells. In rat cerebellar astrocytes, in sections and in culture, ␣2 expression was prominent, yet the expression of either ␤ 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.

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.

Chondrocytes are the main cells in the extracellular matrix (ECM) of articular cartilage and possess a highly differentiated phenotype that is the hallmark of the unique physiological functions of this specialised load-bearing connective tissue. The plasma membrane of articular chondrocytes contains a rich and diverse complement of membrane proteins, known as the membranome, which defines the cell surface phenotype of the cells. The membranome is a key target of pharmacological agents and is important for chondrocyte function. It includes channels, transporters, enzymes, receptors, and anchors for intracellular, cytoskeletal and ECM proteins and other macromolecular complexes. The chondrocyte channelome is a sub-compartment of the membranome and includes a complete set of ion channels and porins expressed in these cells. Many of these are multi-functional proteins with "moonlighting" roles, serving as channels, receptors and signalling components of larger molecular assemblies. The aim of this review is to summarise our current knowledge of the fundamental aspects of the chondrocyte channelome, discuss its relevance to cartilage biology and highlight its possible role in the pathogenesis of osteoarthritis (OA). Excessive and inappropriate mechanical loads, an inflammatory micro-environment, alternative splicing of channel components or accumulation of basic calcium phosphate crystals can result in an altered chondrocyte channelome impairing its function. Alterations in Ca signalling may lead to defective synthesis of ECM macromolecules and aggravated catabolic responses in chondrocytes, which is an important and relatively unexplored aspect of the complex and poorly understood mechanism of OA development.

The Na+-K+-ATPase consists of alpha and beta subunits proposed to function as an alpha-beta heterodimer. Skeletal muscle is characterized by expression of alpha1, alpha2, beta1, and beta2 subunit isoforms. The relative molar proportions of each subunit or each protein isoform are not known, yet their subcellular distribution and expression in muscles of different fiber types are markedly different. In this study, the molar ratio of each pump subunit isoform was measured in purified membranes from skeletal muscle and compared with those in kidney and brain microsomes. Recombinant proteins were used as standards to quantitate each isoform by immunoblotting in combination with measurements of [3H]ouabain binding. The results indicate that in kidney microsomes, which express predominantly alpha1 and beta1 isoforms, the alpha:beta subunit molar ratio is approximately 1:1. In brain microsomes, the sum of all alpha (alpha1, alpha2, and alpha3) and all beta (beta1 and beta2) subunits also yielded a molar ratio of approximately 1:1. In contrast, in red (oxidative) skeletal muscles, the all alpha:beta subunit ratio was 0.2 in plasma membranes and 0.4 in intracellular membranes. The ratio of alpha2 subunits to alpha1 subunits ranged from 1.6 in surface membranes to up to 7 in internal membranes, while the beta1 subunits exceeded the beta2 subunits by approximately 4-fold in all membrane fractions. Thus, intracellular membranes of red skeletal muscles contain primarily alpha2 and beta1 subunits. When these intracellular membranes were further subfractionated by velocity gradient centrifugation, the alpha2:beta1 subunit ratio was 0.5 in the faster migrating (larger) membranes and 1.0 in the slower migrating (smaller) ones. This was due to a progressive decrease in abundance of the beta1 subunits without a change in the concentration of alpha2 subunits per unit protein. The Na+-K+-ATPase hydrolytic activity was higher in the larger vesicles than in the smaller ones along the sucrose gradient. These results suggest that the ratio of beta to alpha subunits may serve to regulate the catalytic activity of the Na+-K+-ATPase in skeletal muscle.

Cardiac glycosides exert a positive inotropic effect by inhibiting sodium pump (Na,K-ATPase) activity, decreasing the driving force for Na ϩ -Ca ϩϩ exchange, and increasing cellular content and release of Ca ϩϩ during depolarization. Since the inotropic response will be a function of the level of expression of sodium pumps, which are ␣␤ heterodimers, and of Na ϩ -Ca ϩϩ exchangers, this study aimed to determine the regional pattern of expression of these transporters in the heart. Immunoblot assays of homogenate from atria, ventricles, and septa of 14 nonfailing human hearts established expression of Na,K-ATPase ␣ 1, ␣ 2, ␣ 3, ␤ 1, and Na ϩ -Ca ϩϩ exchangers in all regions. Na,K-ATPase ␤ 2 expression is negligible, indicating that the human cardiac glycoside receptors are ␣ 1 ␤ 1,, ␣ 2 ␤ 1, and ␣ 3 ␤ 1. ␣ 3, ␤ 1, sodium pump activity, and Na ϩ -Ca ϩϩ exchanger levels were 30-50% lower in atria compared to ventricles and/or septum; differences between ventricles and septum were insignificant. Functionally, the EC 50 of the sodium channel activator BDF 9148 to increase force of contraction was lower in atria than ventricle muscle strips (0.36 vs. 1.54 M). These results define the distribution of the cardiac glycoside receptor isoforms in the human heart and they demonstrate that atria have fewer sodium pumps, fewer Na ϩ -Ca ϩϩ exchangers, and enhanced sensitivity to inotropic stimulation compared to ventricles. ( J. Clin. Invest. 1996. 98:1650-1658.)

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