Pericytes. Morphofunction, interactions and pathology in a quiescent and activated mesenchymal cell niche.

We review the morphofunctional characteristics of pericytes and report our observations. After a brief historical background, we consider the following aspects of pericytes: A) Origin in embryonic vasculogenesis (mesenchymal stem cells, neurocrest and other possible sources) and in embryonic and postnatal life angiogenesis (pre-existing pericytes, fibroblast/ myofibroblasts and circulating progenitor cells). B) Location in pericytic microvasculature and in the other blood vessels (including transitional cell forms and absence in lymphatic vessels), incidence (differences depending on species, topographical location, and type and stage of vessels) and distribution (specific polarities) in blood vessels. C) Morphology (cell body, and longitudinal and circumferential cytoplasmic processes), structure (nucleus, cytoplasmic organelles and distribution of microtubules, intermediate filaments and microfilaments) and surface (caveolae system). D) Basement membrane disposition, formation, components and functions. E) Contacts with endothelial cells (ECs) (peg and socket arrangements, adherent junctions and gap junctions) and with basal membrane (adhesion plaques). F) Molecular expression (pericyte marker identification). G) Functions, such as vessel stabilization, regulation of vascular tone and maintenance of local and tissue homeostasis (contractile capacity and vessel permeability regulation), matrix protein synthesis, macrophage-like properties, immunological defense, intervention in coagulation, participation in mechanisms that regulate the quiescent and angiogenic stages of blood vessels (including the behaviour of pericytes during sprouting angiogenesis and intussuceptive vascular growth, as well as pericyte interactions with endothelium and other cells, and with extracellular matrix) and plasticity, as progenitor cells with great mesenchymal potential, originating other pericytes, fibroblast/myofibroblasts, preadipocytes, chondroblasts, osteoblasts, odontoblasts, vascular smooth muscle and myointimal cells. This mesenchymal capacity is seen in a broad section on the perivascular mesenchymal cell niche hypothesis and in the concept of pericyte and EC "marriage and divorce". H) Peculiar pericyte types, such as hepatic stellate cells (Ito cells), bone marrow reticular cells and mesangial cells. I) Involvement in pathological processes, such as repair through granulation tissue, pericyte-derived tumors, tumor angiogenesis and tumoral cell metastasis, diabetic microangiopathy, fibrosis, atherosclerosis and calcific vasculopathy, lymphedema distichiasis, chronic venous insufficiency, pulmonary hypertension, Alzheimer disease and multiple sclerosis. J) Clinical and therapeutic implications (de-stabilization of vessels or formation of a stable vasculature).

[Study on the role of the tubule in renal vasoconstriction induced by cyclosporine]. / Papel del túbulo en la vasoconstricción renal inducida por ciclosporina.

INTRODUCTION: Cyclosporine (CyA) has proved to induce cell apoptosis on cultured proximal tubule cells. However, there is no much data about the in vivo functional consequences of this injury or the long time observed CyA-induced renal vasoconstriction. MATERIAL AND METHODS: In a swine model of subacute CyA nephrotoxicity (10 mg/ Kg. dx 15 days), we performed a right nephrectomy, followed by left renal artery, vein and ureter catheterisati8n. After inducing water diuresis, three clearance periods of 15 minutes were performed before and after a furosemide 1 mg/kg infusion. Plasma and urine electrolytes, blood gas, acid excretion, plasma renin activity and aldosterone concentration, GFR, RPF, RBF, intra-renal vascular resistances, glomerular filtration pressure, distal Cl- delivery, water clearance and TTKG were measured or estimated on 7 control and 7 treated animals. Right kidney was processed for NaKATPase activity and immunostaining. RESULTS: Treated animals presented detaching proximal cells, luminal blebbing and loss of tight junctions. Cortical but not medullar sodium pump was internalised and partially inactive. Treated animals showed much lower fractional excretions of Na+, with significantly higher distal fractional reabsorption of Cl. Distal shift in fluid load resulted in a significant rise in renal O2 consumption, and modifications in the global renal estequiometry of Na+ transport/O2 uptake. Several consequences followed this situation: preglomerular resistances increased 3 times with only minor changes in postglomerular resistances and renal blood and plasma flow were significantly reduced. Furosemide partially reversed these effects. A slight increase in fractional filtration prevented GFR differences to become statistically significant. CONCLUSION: subacute CyA treatment even al doses not modifying GFR, may cause proximal tubule Na+ transport impairment, resulting in increased rates of distal delivery and absorption of fluid load. Renal uptake of O2 may be increased and tubule glomerular feedback should be expected to be activated. Absence of changes of GFR with furosemide is an early sign of CyA renal damage.

Papel del túbulo en la vasoconstricción renal inducida por ciclosporina / Study of the role of the tubule in renal vasoconstriction induced by cyclosporine

Introducción: Ciclosporina es el primero de una familia de potentes inmunosupresores con capacidad anticalcineurínica que, sin embargo, presentan como limitación terapéutica una nefrotoxicidad que puede aparecer desde periodos tempranos. Los primeros datos funcionales sugerían la existencia de vasoconstricción renal, si bien estudios posteriores han mostrado un efecto tóxico directo de ciclosporina sobre el túbulo proximal. Materiales y Métodos: En este artículo se presenta un modelo porcino de nefrotoxicidad por ciclosporina a medio plazo, analizándose los cambios hemodinámicos intrarrenales y las funciones tubulares, así como la relación entre ambos. Resultados: Ciclosporina produce específicamente una internalización e inactivación de la Na+, K+-ATPasa basolateral del túbulo proximal, determinando el desplazamiento axial de la carga filtrada hacia regiones distales (asa de Henle), con transportes activos más intensos y no modificados por ciclosporina. El resultado es un aumento en la reabsorción fraccional distal de cloro que condiciona un estímulo del “feed-back” túbulo glomerular, vasoconstricción aferente pero no eferente, aumento de las resistencias renales y caída de la presión intraglomerular. Furosemida restaura parcialmente la situación basal. La consecuencia inmediata es un aumento en el consumo de O2 por unidad de sodio trasportado. A nivel del túbulo colector no se observan cambios en la secreción de agua libre, pero la secreción de K+ parece limitada, y no se corrige al aumentar la oferta distal de sodio con furosemida, ni con aldosterona, cuyos valores fueron más altos en los animales tratados con ciclosporina. Conclusión: En resumen, el efecto vasoconstrictor de ciclosporina sobre el riñón se debe en parte a una retroalimentación túbulo glomerular activada por el desplazamiento de solutos filtrados de sus sitios de reabsorción proximal a otros sitos más distales. Las dietas con alto contenido en sodio pueden aumentar el efecto deletéreo de ciclosporina sobre el filtrado glomerular

Introduction: Cyclosporine (CyA) has proved to induce cell apoptosis on cultured proximal tubule cells. However, there is no much data about the in vivo functional consequences of this injury or the long time observed CyA-induced renal vasoconstriction. Material and Methods: In a swine model of subacute CyA nephrotoxicity (10 mg/ Kg. d x 15 days), we performed a right nephrectomy, followed by left renal artery, vein and ureter catheterisation. After inducing water diuresis, three clearance periods of 15 minutes were performed before and after a furosemide 1 mg/kg infusion. Plasma and urine electrolytes, blood gas, acid excretion, plasma renin activity and aldosterone concentration, GFR, RPF, RBF, intra-renal vascular resistances, glomerular filtration pressure, distal Cl- delivery, water clearance and TTKG were measured or estimated on 7 control and 7 treated animals. Right kidney was processed for NaKATPase activity and immunostaining. Results: Treated animals presented detaching proximal cells, luminal blebbing and loss of tight junctions. Cortical but not medullar sodium pump was internalised and partially inactive. Treated animals showed much lower fractional excretions of Na+, with significantly higher distal fractional reabsorption of Cl-. Distal shift in fluid load resulted in a significant rise in renal O2 consumption, and modifications in the global renal estequiometry of Na+ transport/O2 uptake. Several consequences followed this situation: preglomerular resistances increased 3 times with only minor changes in postglomerular resistances and renal blood and plasma flow were significantly reduced. Furosemide partially reversed these effects. A slight increase in fractional filtration prevented GFR differences to become statistically significant. Conclusión: subacute CyA treatment even al doses not modifying GFR, may cause proximal tubule Na+ transport impairment, resulting in increased rates of distal delivery and absorption of fluid load. Renal uptake of O2 may be increased and tubule glomerular feedback should be expected to be activated. Absence of changes of GFR with furosemide is an early sign of CyA renal damage

Búsqueda de nuevos autoantígenos en el síndrome de Sjögren / The search for new autoantigens in Sjögren’s syndrome

Objetivo: Determinar la existencia de nuevos autoantígenos en el síndrome de Sjögren (SS) así como estudiar la prevalencia de éstos en pacientes y población sana. Material y métodos: Se procedió a realizar un muestreo con el suero de una enferma afectada de SS mediante la utilización de una genoteca de cerebro humano (técnica SEREX). Se determinó que este suero expresaba autoantígenos ya conocidos y proteínas no descritas previamente, y también se confirmó la presencia de una proteína desconocida hasta ahora. De entre ellas, se seleccionó a esta última (hIscA) y a la proteína Tau (hallada en el muestreo) para ser transformadas en sendos plásmidos de expresión para conseguir su síntesis recombinante. Resultados: Mediante técnica de inmunotransferencia se testó la existencia de anticuerpos anti-Tau y anti-hIscA en 19 pacientes y 20 sujetos sanos. No se encontró diferencia estadísticamente significativa entre pacientes y controles en la expresión de anticuerpos anti-Tau y se halló que los pacientes expresaban, de forma significativa, valores inferiores de anticuerpos anti-hIscA. Conclusión: Se ha identificado a las proteínas Tau y hIscA como nuevos autoantígenos en el SS. Se ha hallado que los pacientes con SS expresan valores inferiores de anticuerpos anti-hIscA en comparación con controles y, aunque no se ha encontrado ninguna diferencia entre sanos y enfermos en relación con la presencia de anticuerpos anti-Tau, ésta es la primera vez en que anticuerpos contra esta proteína se han detectado en el SS(AU)

Objective: To identify new autoantigens related to Sjögren’s syndrome and to determine their prevalence in patients and healthy individuals. Material and methods: Serological sampling was performed in a patient with Sjögren’s syndrome through the use of a human brain expression genotec (SEREX technique) to determine expression of known autoantigens and previously undescribed proteins. The presence of a previously unknown protein was found. Several proteins were obtained and two were selected to be studied (a human protein called Tau and an unknown protein described by our group and named hlscA). Both Tau and hIscA cDNA were transformed into an expression plasmid to obtain their recombinant proteins. Results: Using a Western-blot technique we investigated the presence of anti-Tau and anti-hlscA autoantibodies in the sera of 19 patients with Sjögren’s syndrome and in the sera of 20 controls. No statistically significant differences were found in the expression of anti-Tau antibodies between patients with Sjögren’s syndrome and controls but values of anti-hlscA autoantibodies were significantly lower in patients with Sjögren’s syndrome. Conclusion: We identified Tau and hIscA proteins as new autoantigens in Sjögren’s syndrome. Anti-hlscA antibody values were significantly lower in patients with Sjögren’s syndrome than in healthy controls. Although no statistically significant differences in values of anti-Tau antibodies were found between Sjögren’s syndrome patients and controls, this is the first time antibodies against this protein have been detected in Sjögren’s síndrome(AU)

[The search for new autoantigens in Sjögren's syndrome]. / Búsqueda de nuevos autoantígenos en el síndrome de Sjögren.

OBJECTIVE: To identify new autoantigens related to Sjögren's syndrome and to determine their prevalence in patients and healthy individuals. MATERIAL AND METHODS: Serological sampling was performed in a patient with Sjögren's syndrome through the use of a human brain expression genotec (SEREX technique) to determine expression of known autoantigens and previously undescribed proteins. The presence of a previously unknown protein was found. Several proteins were obtained and two were selected to be studied (a human protein called Tau and an unknown protein described by our group and named hlscA). Both Tau and hIscA cDNA were transformed into an expression plasmid to obtain their recombinant proteins. RESULTS: Using a Western-blot technique we investigated the presence of anti-Tau and anti-hlscA autoantibodies in the sera of 19 patients with Sjögren's syndrome and in the sera of 20 controls. No statistically significant differences were found in the expression of anti-Tau antibodies between patients with Sjögren's syndrome and controls but values of anti-hlscA autoantibodies were significantly lower in patients with Sjögren's syndrome. CONCLUSION: We identified Tau and hIscA proteins as new autoantigens in Sjögren's syndrome. Anti-hlscA antibody values were significantly lower in patients with Sjögren's syndrome than in healthy controls. Although no statistically significant differences in values of anti-Tau antibodies were found between Sjögren's syndrome patients and controls, this is the first time antibodies against this protein have been detected in Sjögren's syndrome.

Regeneration influences expression of the Na+, K+-atpase subunit isoforms in the rat peripheral nervous system.

Neural injury triggers changes in the expression of a large number of gene families. Particularly interesting are those encoding proteins involved in the generation, propagation or restoration of electric potentials. The expression of the Na+, K+-ATPase subunit isoforms (alpha, beta and gamma) was studied in dorsal root ganglion (DRG) and sciatic nerve of the rat in normal conditions, after axotomy and during regeneration. In normal DRG, alpha1 and alpha2 are expressed in the plasma membrane of all cell types, while there is no detectable signal for alpha3 in most DRG cells. After axotomy, alpha1 and alpha2 expression decreases evenly in all cells, while there is a remarkable onset in alpha3 expression, with a peak about day 3, which gradually disappears throughout regeneration (day 7). beta1 Is restricted to the nuclear envelope and plasma membrane of neurons and satellite cells. Immediately after injury, beta1 shows a homogeneous distribution in the soma of neurons. No beta2 expression was found. Beta3 Specific immunofluorescence appears in all neurons, although it is brightest in the smallest, diminishing progressively after injury until day 3 and, thereafter, increasing in intensity, until it reaches normal levels. FXYD7 is expressed weakly in a few DRG neurons (less than 2%) and Schwann cells. It increases intensely in satellite cells immediately after axotomy, and in all cell types at day 3. Transient switching of members of the Na+, K+-ATPase isoform family elicited by axotomy suggests variations in the sodium pump isozymes with different affinities for Na+, K+ and ATP from those in intact nerve. This adaptation may be important for regeneration.

Human articular chondrocytes, synoviocytes and synovial microvessels express aquaporin water channels; upregulation of AQP1 in rheumatoid arthritis.

Recent studies have shown that aquaporin water channels are expressed in human Meckel's cartilage. The aim of the present investigation was to determine if human articular chondrocytes and synoviocytes express aquaporin 1 (AQP1) water channels and to establish if there are any alterations in AQP1 expression in osteoarticular disorders such as osteoarthritis (OA) and rheumatoid arthritis (RA). Immunohistochemistry was employed semi-quantitatively to compare the expression of AQP1 in human chondrocytes derived from normal, OA and RA joints. PCR, cloning and sequencing confirmed the presence of AQP1 transcripts in chondrocytes. Normal human tissue microarrays including samples of kidney, choroid plexus and pancreas were used as positive controls for AQP1 expression. In most tissues AQP1 was expressed along endothelial barriers. In the kidney AQP1 was present in the glomerular capillary endothelium, proximal tubule and descending thin limbs. AQP1 was also localized to pancreatic ducts and acini and the apical membrane domain of the choroid plexus. Immunohistochemistry showed that AQP1 is expressed in synovial micro-vessels, synoviocytes and predominantly in chondrocytes located in the deep zone of articular cartilage. Image analysis of normal, OA and RA cartilage suggested that AQP1 may be upregulated in RA. This is the first report of AQP1 mRNA and protein expression in articular chondrocytes and synoviocytes. These findings suggest a potential role for AQP1 and possibly other members of the AQP gene family in the movement of extracellular matrix and metabolic water across the membranes of chondrocytes and synoviocytes for the purposes of chondrocyte volume regulation and synovial homeostasis.

Expression and cellular localization of Na,K-ATPase isoforms in the rat ventral prostate.

OBJECTIVE: To determine the expression and plasma membrane domain location of isoforms of Na,K-ATPase in the rat ventral prostate. MATERIALS AND METHODS: Ventral prostate glands from adult male rats were dissected, cryosectioned (7 micro m) and attached to poly-l-lysine coated glass slides. The sections were then fixed in methanol and subjected to indirect immunofluorescence and immunoperoxidase procedures using a panel of well-characterized monoclonal and polyclonal antibodies raised against known Na,K-ATPase subunit isoforms. Immunofluorescence micrographs were digitally captured and analysed by image analysis software. RESULTS: There was expression of Na,K-ATPase alpha1, beta1, beta2 and beta3 subunit isoforms in the lateral and basolateral plasma membrane domains of prostatic epithelial cells. The alpha1 isoform was abundant but there was no evidence of alpha2, alpha3 or gamma isoform expression in epithelial cells. The alpha3 isoform was not detected, but there was a relatively low level of alpha2 isoform expression in the smooth muscle and stroma. CONCLUSION: Rat prostate Na,K-ATPase consists of alpha1/beta1, alpha1/beta2 and alpha1/beta3 isoenzymes. These isoform proteins were located in the lateral and basolateral plasma membrane domains of ventral prostatic epithelial cells. The distribution and subcellular localization of Na,K-ATPase is different in rodent and human prostate. Basolateral Na,K-ATPase probably contributes to the establishment of transepithelial ionic gradients that are a prerequisite for the uptake of metabolites by secondary active transport mechanisms and active citrate secretion.

Glucose transport and metabolism in chondrocytes: a key to understanding chondrogenesis, skeletal development and cartilage degradation in osteoarthritis.

Despite the recognition that degenerative cartilage disorders like osteoarthritis (OA) and osteochondritis dissecans (OCD) may have nutritional abnormalities at the root of their pathogenesis, balanced dietary supplementation programs have played a secondary role in their management. This review emphasizes the importance and role of nutritional factors such as glucose and glucose-derived sugars (i.e. glucosamine sulfate and vitamin C) in the development, maintenance, repair, and remodeling of cartilage. Chondrocytes, the cells of cartilage, consume glucose as a primary substrate for ATP production in glycolysis and utilize glucosamine sulfate and other sulfated sugars as structural components for extracellular matrix synthesis and are dependent on hexose uptake and delivery to metabolic and biosynthetic pools. Data from several laboratories suggests that chondrocytes express multiple isoforms of the GLUT/SLC2A family of glucose/polyol transporters. These facilitative glucose transporter proteins are expressed in a tissue and cell-specific manner, exhibit distinct kinetic properties, and are developmentally regulated. They may also be regulated by endocrine factors like insulin and insulin-like growth factor I (IGF-I) and cytokines such as interleukin 1 beta (IL-1 beta) and tumour necrosis factor alpha (TNF-alpha). Recent studies suggest that degeneration of cartilage may be triggered by metabolic disorders of glucose balance and that OA occurs coincident with metabolic disease, endocrine dysfunction and diabetes mellitus. Based on these metabolic, endocrine and developmental considerations we present a novel hypothesis regarding the role of glucose transport and metabolism in cartilage physiology and pathophysiology and speculate that supplementation with sugar-derived vitamins and nutraceuticals may benefit patients with degenerative joint disorders.

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 in human prostate; specificity of expression and apical membrane polarization.

The cellular distribution of Na+, K+-ATPase subunit isoforms was mapped in the secretory epithelium of the human prostate gland by immunostaining with antibodies to the alpha and beta subunit isoforms of the enzyme. Immunolabeling of the alpha1, beta1 and beta2 isoforms was observed in the apical and lateral plasma membrane domains of prostatic epithelial cells in contrast to human kidney where the alpha1 and beta1 isoforms of Na+, K+-ATPase were localized in the basolateral membrane of both proximal and distal convoluted tubules. Using immunohistochemistry and PCR we found no evidence of Na+, K+-ATPase alpha2 and alpha3 isoform expression suggesting that prostatic Na+, K+-ATPase consists of alpha1/beta1 and alpha1/beta2 isozymes. Our immunohistochemical findings are consistent with previously proposed models placing prostatic Na+, K+-ATPase in the apical plasma membrane domain. Abundant expression of Na+, K+-ATPase in epithelial cells lining tubulo-alveoli in the human prostate gland confirms previous conclusions drawn from biochemical, pharmacological and physiological data and provides further evidence for the critical role of this enzyme in prostatic cell physiology and ion homeostasis. Na+, K+-ATPase most likely maintains an inwardly directed Na+ gradient essential for nutrient uptake and active citrate secretion by prostatic epithelial cells. Na+, K+-ATPase may also regulate lumenal Na+ and K+, major counter-ions for citrate.

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

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.

Oligodendrocytes in brain and optic nerve express the beta3 subunit isoform of Na,K-ATPase.

The Na,K-ATPase, which catalyzes the active transport of Na(+) and K(+), has two principal subunits (alpha and beta) 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 beta isoforms, beta1 predominates in neurons and beta2 in astrocytes, although there are some exceptions. Here we demonstrate that beta3 is expressed in rat and mouse white matter oligodendrocytes. Immunofluorescence microscopy identified beta3 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. beta3 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, beta3 stain was seen in oligodendrocytes, where it colocalized with carbonic anhydrase II. For comparison, optic nerve was stained for the beta1 and beta2 subunits, showing distinct patterns of labelling of axons (beta1) and astrocytes (beta2). The C6 glioma cell line was also found to express the beta3 isoform preferentially. Since beta3 was not found at detectable levels in astrocytes, this suggests that C6 is closer to oligodendrocytes than astrocytes in the glial cell lineage.

Sodium transport systems in human chondrocytes. I. Morphological and functional expression of the Na+,K(+)-ATPase alpha and beta subunit isoforms in healthy and arthritic chondrocytes.

The chondrocyte is the cell responsible for the maintenance of the articular cartilage matrix. The negative charges of proteoglycans of the matrix draw cations, principally Na+, into the matrix to balance the negative charge distribution. The Na+,K(+)-ATPase is the plasma membrane enzyme that maintains the intracellular Na+ and K+ concentrations. The enzyme is composed of an alpha and a beta subunit, so far, 4 alpha and 3 beta isoforms have been identified in mammals. Chondrocytes are sensitive to their ionic and osmotic environment and are capable of adaptive responses to ionic environmental perturbations particularly changes to extracellular [Na+]. In this article we show that human fetal and adult chondrocytes express three alpha (alpha 1, alpha 2 and the neural form of alpha 3) and the three beta isoforms (beta 1, beta 2 and beta 3) of the Na+,K(+)-ATPase. The presence of multiple Na+,K(+)-ATPase isoforms in the plasma membrane of chondrocytes suggests a variety of kinetic properties that reflects a cartilage specific and very fine specialization in order to maintain the Na+/K+ gradients. Changes in the ionic and osmotic environment of chondrocytes occur in osteoarthritis and rheumatoid arthritis as result of tissue hydration and proteoglycan loss leading to a fall in tissue Na+ and K+ content. Although the expression levels and cellular distribution of the proteins tested do not vary, we detect changes in p-nitrophenylphosphatase activity "in situ" between control and pathological samples. This change in the sodium pump enzymatic activity suggests that the chondrocyte responds to these cationic environmental changes with a variation of the active isozyme types present in the plasma membrane.

Sodium transport systems in human chondrocytes. II. Expression of ENaC, Na+/K+/2Cl- cotransporter and Na+/H+ exchangers in healthy and arthritic chondrocytes.

In this article, the second of two, we continue our studies of sodium-dependent transport systems in human cartilage from healthy individuals and with osteoarthritis (OA) and rheumatoid arthritis (RA). We demonstrate the presence of the epithelial sodium channel (ENaC), previously undescribed in chondrocytes. This system is composed of three subunits, alpha, beta and gamma. We have shown that the human chondrocytes express at least the alpha and the beta subunit of ENaC. The expression of these subunits is altered in arthritic chondrocytes. In RA samples the quantity of alpha and beta is significantly higher than in control samples. On the other hand, ENaC alpha and beta subunits are absent in the chondrocytes of OA cartilage. Human chondrocytes also possess three isoforms of the Na+/H+ exchanger (NHE), NHE1, NHE2 and NHE3. The NHE system is composed of a single protein and is believed to participate in intracellular pH regulation. Furthermore, our studies indicate that at least one isoform of the electroneutral Na+/K+/2Cl- cotransporter (NKCC) is present in human chondrocytes. There are no obvious variations in the relative expression of NHE isoforms or NKCC between healthy and arthritic cartilage. Our data suggests that chondrocytes from arthritic cartilage may adapt to changes in their environmental sodium concentration through variations in ENaC protein levels. ENaC is also likely to serve as a major sodium entry mechanism, a process that, along with cytoskeletal proteins, may be part of mechanotransduction in cartilage.

Insulin stimulation of K+ uptake in 3T3-L1 fibroblasts involves phosphatidylinositol 3-kinase and protein kinase C-zeta.

Despite the important physiological role of insulin in the regulation of ionic homeostasis, primarily mediated by the Na+/K(+)-ATPase and Na+/K+/2Cl- cotransporter, the intracellular signalling molecules mediating this effect of insulin have not been elucidated. Treatment of 3T3-L1 fibroblasts with insulin increased total 86Rb+ (K+) uptake from 0.8 +/- 0.04 to 1.02 +/- 0.05 nmol.mg-1.protein-1.min-1 (p < 0.005). These changes in K+ flux, though small, can alter the membrane potential. Uptake occurred through both the Na+/K(+)-ATPase and Na+/K+/2Cl- cotransporter and both were stimulated by insulin. Interestingly, when bumetanide was used to inhibit the Na+/K+/2Cl- cotransporter prior to insulin action, no increase in 86Rb+ uptake via the Na+/K(+)-ATPase was observed. The structurally distinct phosphatidylinositol 3-kinase inhibitors wortmannin (50-200 nmol/l) and LY294002 (50 mumol/l) attenuated both total insulin-stimulated 86Rb+ uptake as well as uptake via the Na+/K(+)-ATPase and Na+/K+/2Cl- cotransporter. Neither the inhibitor of p70.S6 kinase activation, rapamycin (30 ng/ml) nor the mitogen activated protein kinase kinase inhibitor, PD098059 (50 mumol/l), had any effect on insulin's stimulation of K+ influx. A 10 mumol/l concentration of the protein kinase C (PKC) inhibitor bisindolylmaleimide attenuated insulin action but at 1 mumol/l it was ineffective, suggesting involvement of the atypical PKC-zeta isoform. We conclude that insulin-stimulated K+ uptake in 3T3-L1 fibroblasts appears to involve concerted regulation of both the Na+/K(+)-ATPase and Na+/K+/2Cl- cotransporter and we show for the first time that this process is signalled via a pathway involving phosphatidylinositol 3-kinase and PKC-zeta.

Ion transport in chondrocytes: membrane transporters involved in intracellular ion homeostasis and the regulation of cell volume, free [Ca2+] and pH.

Chondrocytes exist in an unusual and variable ionic and osmotic environment in the extracellular matrix of cartilage and are responsible for maintaining the delicate equilibrium between extracellular matrix synthesis and degradation. The mechanical performance of cartilage relies on the biochemical properties of the matrix. Alterations to the ionic and osmotic extracellular environment of chondrocytes have been shown to influence the volume, intracellular pH and ionic content of the cells, which in turn modify the synthesis and degradation of extracellular matrix macromolecules. Physiological ion homeostasis is fundamental to the routine functioning of cartilage and the factors that control the integrity of this highly evolved and specialized tissue. Ion transport in cartilage is relatively unexplored and the biochemical properties and molecular identity of membrane transport mechanisms employed by chondrocytes in the control of intracellular ion concentrations and pH is not fully defined and this review focuses on these processes. Chondrocytes have been shown to express voltage and stretch activated ion channels, passive exchangers and ATP dependent ion pumps. In addition, recent studies of transport systems in chondrocytes have demonstrated the presence of isozyme diversity that includes Na+/H+ exchange (NHE1, NHE3), Na+, K(+)-ATPase (several isoforms) and others each of which possess considerably different kinetic properties and modes of regulation. This multitude of isozyme diversity indicates the highly specialized handling of ions and protons in order to accomplish a fine regulation of their transmembrane fluxes. The complexities of these transport systems and their patterns of isoform expression underscore the subtlety of ion homeostasis and pH regulation in normal cartilage. Perturbations in these mechanisms may affect the physiological turnover of cartilage and thus increase the susceptibility to degenerative joint disease.