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Introduction: Pompe disease (PD), glycogen storage disease Type II (GSD II), is an autosomal recessive inherited lysosomal storage disease caused by pathogenic variants in the GAA gene that encodes lysosomal acid ?-glucosidadase (GAA) enzyme. The incidence of the disease varies from country to country. PD is mainly presents as two groups of phenotypes as infantile-onset Pompe disease (IOPD) and late-onset Pompe disease. Objective: The aim of this study is to discuss the molecular and clinical characteristics of infantile-onset Pompe disease (IOPD) and late-onset pompe disease (LOPD) followed-up in our center. Method: A total of 10 patients diagnosed with IOPD and 4 patients diagnosed with LOPD in Izmir Dr. Behcet Uz Pediatric Health and Diseases and Surgery Training and Research Hospital Pediatric Metabolism Unit between 06.01.2015 and 06.01. 2019 were included in the study. The patients' demographic characteristics, clinical findings at the time of diagnosis and during the folllow-up period, biochemical findings, muscle biopsy data, results of enzymatic analyses and molecular-genetic characteristics were recorded retrospectively. Results: A total of 10 patients were included in the study. 7 patients were diagnosed with IOPD and 3 patients with LOPD. The median follow-up period of all patients was 26 months (range: 6-42 months). The c.896 C> T (8/32, 25%) is detected as the most common variant. 1237G>T (p.Asp413Tyr), c.2019 C>A (p.Asn673Lys), c.418A>T (p.Asn140Tyr) variants were detected for the first time. Conclusion: Pompe disease is one of the most important congenital metabolic diseases in which early diagnosis and treatment are of great importance. Despite the significant improvement in disease prognosis with the introduction of enzyme replacement therapy, there are still patients with poor prognosis despite early diagnosis. Phenotype-genotype studies are crucial in this respect. © Copyright Health Sciences University Bakırköy Sadi Konuk Training and Research Hospital. This journal published by Logos Medical Publishing. Licenced by Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

The regulation of body fluid balance is a key concern in health and disease and comprises three concepts. The first concept pertains to the relationship between total body water (TBW) and total effective solute and is expressed in terms of the tonicity of the body fluids. Disturbances in tonicity are the main factor responsible for changes in cell volume, which can critically affect brain cell function and survival. Solutes distributed almost exclusively in the extracellular compartment (mainly sodium salts) and in the intracellular compartment (mainly potassium salts) contribute to tonicity, while solutes distributed in TBW have no effect on tonicity. The second body fluid balance concept relates to the regulation and measurement of abnormalities of sodium salt balance and extracellular volume. Estimation of extracellular volume is more complex and error prone than measurement of TBW. A key function of extracellular volume, which is defined as the effective arterial blood volume (EABV), is to ensure adequate perfusion of cells and organs. Other factors, including cardiac output, total and regional capacity of both arteries and veins, Starling forces in the capillaries, and gravity also affect the EABV. Collectively, these factors interact closely with extracellular volume and some of them undergo substantial changes in certain acute and chronic severe illnesses. Their changes result not only in extracellular volume expansion, but in the need for a larger extracellular volume compared with that of healthy individuals. Assessing extracellular volume in severe illness is challenging because the estimates of this volume by commonly used methods are prone to large errors in many illnesses. In addition, the optimal extracellular volume may vary from illness to illness, is only partially based on volume measurements by traditional methods, and has not been determined for each illness. Further research is needed to determine optimal extracellular volume levels in several illnesses. For these reasons, extracellular volume in severe illness merits a separate third concept of body fluid balance.

We report a morphological manipulation of cell division which was achieved by changing the environment from isotonic to highly hypotonic. Cells at telophase were observed to undergo a morphological reversal to anaphase, with the contractile ring being reopened and the cell shape reversing from dumb-bell back to spherical. Once restored to isosmotic environment, the reversed cells would either continue to divide or instead to form binuclear cells that further proliferated in runaway fashions. The immunofluorescent staining of tubulins and myosin II indicated that the hypotonic stress affected the accumulation of tubulins and myosin II at the contractile ring. Distinct from previous studies using specific chemical reagents, the present study provides a simple method to manipulate cell division. The morphological reversal is the adaption of dividing cells to the environmental change. The observation opens a new window to understand cell division mechanisms and runaways.

Defective cellular trafficking of aquaporin-5 (AQP5) to the apical plasma membrane (APM) in salivary glands is associated with the loss of salivary fluid secretion. To examine mechanisms of α₁-adrenoceptor (AR)-induced trafficking of AQP5, immunoconfocal microscopy and Western blot analysis were used to analyze AQP5 localization in parotid tissues stimulated with phenylephrine under different osmolality. Phenylephrine-induced trafficking of AQP5 to the APM and lateral plasma membrane (LPM) was mediated via the α1A-AR subtype, but not the α1B- and α1D-AR subtypes. Phenylephrine-induced trafficking of AQP5 was inhibited by ODQ and KT5823, inhibitors of nitric oxide (NO)-stimulated guanylcyclase (GC) and protein kinase (PK) G, respectively, indicating the involvement of the NO/ soluble (c) GC/PKG signaling pathway. Under isotonic conditions, phenylephrine-induced trafficking was inhibited by La(3+), implying the participation of store-operated Ca(2+) channel. Under hypotonic conditions, phenylephrine-induced trafficking of AQP5 to the APM was higher than that under isotonic conditions. Under non-stimulated conditions, hypotonicity-induced trafficking of AQP5 to the APM was inhibited by ruthenium red and La(3+), suggesting the involvement of extracellular Ca(2+) entry. Thus, α1A-AR activation induced the trafficking of AQP5 to the APM and LPM via the Ca(2+)/ cyclic guanosine monophosphate (cGMP)/PKG signaling pathway, which is associated with store-operated Ca(2+) entry.

Studies in Caenorhabditis elegans have revealed osmoregulatory systems engaged when worms experience hypertonic conditions, but less is known about measures employed when faced with hypotonic stress. Inactivation of fmo-4, which encodes flavin-containing monooxygenase-4, results in dramatic hypoosmotic hypersensitivity; worms are unable to prevent overwhelming water influx and swell rapidly, finally rupturing due to high internal hydrostatic pressure. fmo-4 is expressed prominently in hypodermis, duct and pore cells but is excluded from the excretory cell. Thus, FMO-4 plays a crucial osmoregulatory role by promoting clearance of excess water that enters during hypotonicity, perhaps by synthesizing an osmolyte that acts to establish an osmotic gradient from excretory cell to duct and pore cells. C. elegans FMO-4 contains a C-terminal extension conserved in all nematode FMO-4s. The coincidently numbered human FMO4 also contains an extended C-terminus with features similar to those of FMO-4. Although these shared sequence characteristics suggest potential orthology, human FMO4 was unable to rescue the fmo-4 osmoregulatory defect. Intriguingly, however, mammalian FMO4 is expressed predominantly in the kidney – an appropriate site if it too is, or once was, involved in osmoregulation., Summary: C. elegans lacking fmo-4 fail to clear body water following hypotonic exposure resulting in rapid swelling and rupture, indicating that FMO-4 is required in the hypoosmotic stress response.

It is well established that hypotonicity generates a marked and unexpected increase in active Na+ efflux in frog muscle fibers as well as in other cells like cardiac myocytes, astrocytes, brain synaptosomes and renal cells. The effect of hypotonicity on the electrical activity of skeletal muscle related to Na+ and K+ voltage-gated channels, however, has not been specifically addressed. The results of the present investigation show that the changes in resting and action potentials produced by hypotonicity can be fully explained by the reduction of intracellular [Na+] and [K+] due to the increase in cellular water content., Centro de Investigaciones Cardiovasculares

Background: The water channel protein aquaporin 4 (AQP4) controls water permeability of the blood-brain barrier. Results: Hypotonicity induces rapid relocalization of AQP4 in a calcium-, calmodulin-, and kinase-dependent manner. Conclusion: AQP4 can be relocalized between the cell membrane and intracellular compartments. Significance: Pharmacological modulation of AQP4 membrane localization could provide a new approach to treating brain edema., The aquaporin family of integral membrane proteins is composed of channels that mediate cellular water flow. Aquaporin 4 (AQP4) is highly expressed in the glial cells of the central nervous system and facilitates the osmotically driven pathological brain swelling associated with stroke and traumatic brain injury. Here we show that AQP4 cell surface expression can be rapidly and reversibly regulated in response to changes of tonicity in primary cortical rat astrocytes and in transfected HEK293 cells. The translocation mechanism involves PKA activation, influx of extracellular calcium, and activation of calmodulin. We identify five putative PKA phosphorylation sites and use site-directed mutagenesis to show that only phosphorylation at one of these sites, serine 276, is necessary for the translocation response. We discuss our findings in the context of the identification of new therapeutic approaches to treating brain edema.

The TRPV4 cation channel is expressed in a broad range of tissues where participates in the generation of a Ca2+ signal. TRPV4 participation in osmo- and mechanotransduction contributes to important functions such as cellular and systemic volume homeostasis, among others. TRPV4 also responds to temperature and 4α-phorbol 12,13-didecanoate (4αPDD) thus showing multiple modes of activation. Besides, dozens of TRPV4 mutations have been linked to osteoarticular and neuromuscular diseases. However, little is known about the domains relevant for TRPV4 activation by different stimuli. This Thesis research aims to elucidate the participation of the N-terminal cytosolic tail in the gating of TRPV4 by physiological stimuli and the channel implication in cell motility. I provide evidences that activation of TRPV4 by hypotonic shocks and temperature requires PIP2 binding within the sequence 121KRWRK125 of the N-terminus tail and rearrangement of the cytosolic tails. My results also point to the participation of TRPV4 in cell migration by modulating the dynamics of trailing adhesions, a function that may require the interplay of TRPV4 with other cation channels present at the focal adhesion sites. Resumen El canal TRPV4 es un canal, El canal TRPV4 es un canal catiónico capaz de generar señales intracelulares de Ca2+ en diversos tejidos. La participación del canal TRPV4 en procesos de mecano-osmotransducción le implica en funciones tan importantes como la regulación del volumen celular y sistémico. El TRPV4 también se activa en respuesta a calor y al agonista sintético 4αPDD, lo que implica la presencia de varios modos de activación. Además, existen numerosas mutaciones en el TRPV4 que se han encontrado en pacientes que sufren de patología osteoarticular y neuromuscular. Sin embargo, aún se desconocen aspectos de su función relacionados con los mecanismos de activación. Mi trabajo de Tesis doctoral aborda el estudio de la región N-terminal del TRPV4, su participación en la activación del canal por estímulos fisiológicos y la relevancia del canal en proceso de migración celular. Esta Tesis doctoral proporciona evidencias de que el TRPV4 necesita unir PIP2 a través de la secuencia 121KRWRK125 de la cola N-terminal y que las colas se reorganicen para que el canal se abra en respuesta a estímulos osmóticos y de calor. Mis estudios también sugieren que el canal TRPV4 participa en la modulación de la adherencia de la cola durante el proceso de la migración celular, posiblemente interaccionando con otros canales presentes en las adhesiones focales.

Background Sensory nerves innervating the airways play an important role in regulating various cardiopulmonary functions, maintaining homeostasis under healthy conditions and contributing to pathophysiology in disease states. Hypo-osmotic solutions elicit sensory reflexes, including cough, and are a potent stimulus for airway narrowing in asthmatic patients, but the mechanisms involved are not known. Transient receptor potential cation channel, subfamily V, member 4 (TRPV4) is widely expressed in the respiratory tract, but its role as a peripheral nociceptor has not been explored. Objective We hypothesized that TRPV4 is expressed on airway afferents and is a key osmosensor initiating reflex events in the lung. Methods We used guinea pig primary cells, tissue bioassay, in vivo electrophysiology, and a guinea pig conscious cough model to investigate a role for TRPV4 in mediating sensory nerve activation in vagal afferents and the possible downstream signaling mechanisms. Human vagus nerve was used to confirm key observations in animal tissues. Results Here we show TRPV4-induced activation of guinea pig airway–specific primary nodose ganglion cells. TRPV4 ligands and hypo-osmotic solutions caused depolarization of murine, guinea pig, and human vagus and firing of Aδ-fibers (not C-fibers), which was inhibited by TRPV4 and P2X3 receptor antagonists. Both antagonists blocked TRPV4-induced cough. Conclusion This study identifies the TRPV4-ATP-P2X3 interaction as a key osmosensing pathway involved in airway sensory nerve reflexes. The absence of TRPV4-ATP–mediated effects on C-fibers indicates a distinct neurobiology for this ion channel and implicates TRPV4 as a novel therapeutic target for neuronal hyperresponsiveness in the airways and symptoms, such as cough.

Defective cellular trafficking of aquaporin-5 (AQP5) to the apical plasma membrane (APM) in salivary glands is associated with the loss of salivary fluid secretion. To examine mechanisms of α1-adrenoceptor (AR)-induced trafficking of AQP5, immunoconfocal microscopy and Western blot analysis were used to analyze AQP5 localization in parotid tissues stimulated with phenylephrine under different osmolality. Phenylephrine-induced trafficking of AQP5 to the APM and lateral plasma membrane (LPM) was mediated via the α1A-AR subtype, but not the α1B- and α1D-AR subtypes. Phenylephrine-induced trafficking of AQP5 was inhibited by ODQ and KT5823, inhibitors of nitric oxide (NO)-stimulated guanylcyclase (GC) and protein kinase (PK) G, respectively, indicating the involvement of the NO/ soluble (c) GC/PKG signaling pathway. Under isotonic conditions, phenylephrine-induced trafficking was inhibited by La3+, implying the participation of store-operated Ca2+ channel. Under hypotonic conditions, phenylephrine-induced trafficking of AQP5 to the APM was higher than that under isotonic conditions. Under non-stimulated conditions, hypotonicity-induced trafficking of AQP5 to the APM was inhibited by ruthenium red and La3+, suggesting the involvement of extracellular Ca2+ entry. Thus, α1A-AR activation induced the trafficking of AQP5 to the APM and LPM via the Ca2+/ cyclic guanosine monophosphate (cGMP)/PKG signaling pathway, which is associated with store-operated Ca2+ entry.

TRPV4 protein forms a Ca2+-permeable channel that is sensitive to osmotic and mechanical stimuli and responds to warm temperatures, and expresses widely in various kinds of tissues. As for cardiac myocytes, TRPV4 has been detected only at the mRNA level and there were few reports about subcel-lular localization of the protein. The purpose of the present study was to investigate the expression profile of TRPV4 protein in cultured neonatal rat ventricular myocytes. Using Western blots, immunofluorescence, confocal microscopy and immuno-electron microscopy, we have shown that TRPV4 protein was predominantly located in the nucleus of cultured neonatal myocytes. Furthermore, cardiac myocytes responded to hypotonic stimulation by translocating TRPV4 protein out of the nucleus. The significance and mechanism concerning the unusual distribution and translocation of TRPV4 protein in cardiac myocytes remain to be clarified.

Background: Aquaporin channels ensure appropriate membrane permeability to water in all cells. Results: Following a hypotonic stimulus, subcellular localization of aquaporin 1 occurs via a mechanism dependent on transient receptor potential channels, extracellular calcium influx, calmodulin, and the phosphorylation of two threonines (157 and 239) of aquaporin 1. Conclusion: Rapid translocation of aquaporin 1 regulates membrane water permeability. Significance: This mechanism may serve as a prototype for the rapid regulation of aquaporin function., The control of cellular water flow is mediated by the aquaporin (AQP) family of membrane proteins. The structural features of the family and the mechanism of selective water passage through the AQP pore are established, but there remains a gap in our knowledge of how water transport is regulated. Two broad possibilities exist. One is controlling the passage of water through the AQP pore, but this only has been observed as a phenomenon in some plant and microbial AQPs. An alternative is controlling the number of AQPs in the cell membrane. Here, we describe a novel pathway in mammalian cells whereby a hypotonic stimulus directly induces intracellular calcium elevations through transient receptor potential channels, which trigger AQP1 translocation. This translocation, which has a direct role in cell volume regulation, occurs within 30 s and is dependent on calmodulin activation and phosphorylation of AQP1 at two threonine residues by protein kinase C. This direct mechanism provides a rationale for the changes in water transport that are required in response to constantly changing local cellular water availability. Moreover, because calcium is a pluripotent and ubiquitous second messenger in biological systems, the discovery of its role in the regulation of AQP translocation has ramifications for diverse physiological and pathophysiological processes, as well as providing an explanation for the rapid regulation of water flow that is necessary for cell homeostasis.

Voltage-gated sodium channels (VGSCs) play an important role in the control of membrane excitability. We previously reported that the excitability of nociceptor was increased by hypotonic stimulation. The present study tested the effect of hypotonicity on tetrodotoxin-sensitive sodium current (TTX-S current) in cultured trigeminal ganglion (TG) neurons. Our data show that after hypotonic treatment, TTX-S current was increased. In the presence of hypotonicity, voltage-dependent activation curve shifted to the hyperpolarizing direction, while the voltage-dependent inactivation curve was not affected. Transient Receptor Potential Vanilloid 4 receptor (TRPV4) activator increased TTX-S current and hypotonicity-induced increase was markedly attenuated by TRPV4 receptor blockers. We also demonstrate that inhibition of PKC attenuated hypotonicity-induced inhibition, whereas PKA system was not involved in hypotonic-response. We conclude that hypotonic stimulation enhances TTX-S current, which contributes to hypotonicity-induced nociception. TRPV4 receptor and PKC intracellular pathway are involved in the increase of TTX-S current by hypotonicity.

La neteja del moc i dels patògens dels pulmons, i el transport de gàmets i embrions en els òrgans reproductius de les femelles són funcions clau en els epitelis ciliats, tals com aquells que es troben presents en les vies respiratòries i l'oviducte. La taxa de transport mucociliar és funció de la freqüència de batut ciliar (CBF) i aquesta freqüència és augmentada per increments en la concentració de Ca2+ intracelul·lar. El canal catiònic "transient potential vanilloid 4" (TRPV4) intervé en l'entrada de Ca2+ en resposta a estímuls mecànics i osmòtics. L'expressió del TRPV4 en l'epiteli ciliat de les vies respiratòries i de l'oviducte és confirmada mitjançant la localització per immunofluorescència del canal iònic a la membrana apical de l'epiteli ciliat i polaritzat, allà on la senyalització de Ca2+ és requerida per la regulació de la CBF. Cèl·lules ciliades de la tràquea de ratolins TRPV4-/- no expressen el canal TRPV4, no responen a l'activador específic del TRPV4, el 4α-phorbol 12,13-didecanoate (4α-PDD) i presenten respostes de Ca2+ reduïdes a temperatures mitjanes (~25ºC- 8ºC), un altre estímul dels canals TRPV4. L'activació dels canals TRPV4 per solucions altament viscoses i per hypotonicitat depèn de l'activació de la via de la fosfolipasa A2(PLA2)i la subseqüent producció de àcid epoxieicosatrienoic (EET). En condicions de baixa activació de la PLA2, estímuls mecànics i hipotònics alliberen ATP per a l'activació de la via de la fosfolipasa C (PLC)-inositol trifosfat (IP3) per contribuir a l'activació dels canals TRPV4. Descrivim que el metabòlit IP3 sense ser un agonista per ell mateix, sensibilitza el TRPV4 per a l'activació de EET, essent aquest un mecanisme general. L'acoblament funcional entre els canals TRPV4 de la membrana plasmàtica i els receptors de IP3 (IP3R) és necessari tant per iniciar com mantenir la senyalització oscil·latòria del Ca2+ desencadenada per estímuls viscosos i hipotònics. Un dels principals activadors de la CBF, la adenosina-5'-trifosfat (ATP), desencadena una resposta cel·lular mediada per Ca2+ en la que es desencadena tant l'alliberament de Ca2+ des dels dipòsits intracel·lulars com l'entrada de Ca2+. És destacable la contribució de el TRPV4 en l'augment de la CBF mediada per ATP. És més, el nostre treball implica als canals TRPV4 exclusivament en l'entrada de Ca2+ activada per receptor (ROCE). Tot plegat, aquesta tesi doctoral mostra el paper dels canals TRPV4 en l'acoblament d'estímuls fisiològics tipus mecànic, osmòtic i químic a la regulació de la CBF en l'epiteli ciliat destinat al transport mucociliar., Clearance of mucus and pathogenic agents from lungs and the transport of gametes and embryos in the female reproductive organs are key functions of ciliated epithelia such as those present in the airways and the oviduct. The rate of mucociliary transport is a function of ciliary beat frequency (CBF) and this, in turn, is increased by increases in intracellular calcium. Transient potential vanilloid 4 (TRPV4)cation channel mediates Ca2+ influx in response to mechanical and osmotic stimuli. TRPV4 expression in ciliated epithelia from airways and oviduct is confirmed by immunofluorescence localization of the channel at the apical membrane of the polarized ciliated epithelia, where the Ca2+ signalling is required for CBF regulation. Ciliated tracheal cells from TRPV4-/-mice show no TRPV4 expression, neither increases in intracellular Ca2+ and CBF in response to the TRPV4-specific activator 4α- phorbol 12,13- idecanoate (4α-PDD), and reduced responses to mild temperatures (~25ºC - 38ºC), another TRPV4-activating stimulus. TRPV4 gating by high viscous loads and hypotonicity depends on phospholipase A2 (PLA2) pathway activation and subsequent production of epoxyeicosatrienoic acid (EET). Under conditions of low PLA2 activation, mechanical and hypotonic stimuli use extracellular ATP release-mediated activation of phospholipase C (PLC)-inositol triphosphate(IP3)signalling to support TRPV4 gating. We describe that IP3, without being an agonist itself, sensitizes TRPV4 to EET activation. Besides, the functional coupling between plasma membrane TRPV4 channels and IP3 receptors (IP3R) is required to initiate and maintain the cellular oscillatory Ca2+ signal triggered by high viscous loads and hypotonic stimuli. One of the main CBF activators, adenosine-5'-triphosphate (ATP), triggers both Ca2+ release from intracellular Ca2+ stores and Ca2+ entry. Interestingly, TRPV4 contributes to ATP-induced increase in CBF. Furthermore, our work implicates TRPV4 channel exclusively in receptor-operated Ca2+ entry. Collectively, this PhD thesis shows the role of TRPV4 channels coupling physiologically relevant mechanical, hypotonic and chemical stimuli to CBF regulation in motile ciliary epithelia.

Hypotonicity produces a marked activation of the Na⁺ pump in frog sartorius muscle. The increase in net Na⁺ efflux under hypotonic conditions occurs despite the reductions in [Na⁺]i that are due to fibre swelling and Na⁺ loss. The pump density (ouabain binding) increases not only upon reduction of the medium osmotic pressure (π) from its normal value (π = 1) to one-half (π = 0.5), but also in muscles that are returned to π = 1 after equilibration in π = 2 medium. The equilibration in π = 2 medium does not affect pump density. Ouabain-binding increments cannot be ascribed to a rise in the Na⁺–K⁺ exchange rate of a fixed number of pumps: they also occurred in the continued presence of a saturating concentration of ouabain (50 μM). Under those conditions, the π = 1 → π = 0.5 transfer produced a 43 % increase in pump sites, while the π = 2 → π = 1 transfer induced a rise of 46 %. Actinomycin D did not alter the stimulation of Na⁺ extrusion elicited by hypotonicity, suggesting that de novo synthesis of pumps was not involved in the increase of the apparent number of pump sites. Disruption of microtubules by colchicine (100 μM) and intermediate filaments by acrylamide (4 mM) did not alter the hypotonic effect. Likewise, genistein (100 μM), a specific inhibitor of tyrosine kinase, did not affect significantly the hypotonic response. Microfilament-disrupting agents like cytochalasin B (5 μM) and latrunculin B (10 μM) reduced the increase in Na⁺ efflux induced by π = 1 → π = 0.5 transfer by about 35 % and 72 %, respectively. Latrunculin B reduced the increases in pump density generated by π = 1 → π = 0.5 and π = 2 → π = 1 transfers by about 79 % and 91 %, respectively. The results suggest that the membrane stretch due to hypotonic fibre volume increase would promote a microfilament-mediated insertion of submembranous spare Na⁺ pumps in the sarcolemma and, consequently, the rise in active Na⁺ transport., Facultad de Ciencias Médicas, Centro de Investigaciones Cardiovasculares

The purpose of this work was to determine if hypotonicity, in addition to the stimulation of active Na+ transport (Venosa, R.A., 1978, Biochim. Biophys. Acta 510:378-383), promoted changes in (i) active K+ influx, (ii) passive Na+ and K+ fluxes, and (iii) the number of 3H-ouabain binding sites. The results indicate that a reduction of external osmotic pressure (pi) to one-half of its normal value (pi = 0.5) produced the following effects: (i) an increase in active K+ influx on the order of 160%, (ii) a 20% reduction in Na+ influx and K+ permeability (PK), and (iii) a 40% increase in the apparent density of ouabain binding sites. These data suggest that the hypotonic stimulation of the Na+ pump is not caused by an increased leak of either Na+ (inward) or K+ (outward). It is unlikely that the stimulation of active Na+ extrusion and the rise in the apparent number of pump sites produced by hypotonicity were due to a reduction of the intracellular ionic strength. It appears that, at least in part, the stimulation of active Na+ transport takes place whenever muscles are transferred from one medium to another of lower tonicity even if neither one was hypotonic (for instance pi = 2 to pi = 1 transfer). Comparison of the present results with those previously reported indicate that in addition to the number of pump sites, the cycling rate of the pump is increased by hypotonicity. Active Na+ and K+ fluxes were not significantly altered by hypertonicity (pi = 2)., Facultad de Ciencias Médicas