A transgenic animal model of osmotic cataract. Part 1: over-expression of bovine Na+/myo-inositol cotransporter in lens fibers.
(9/1433)
PURPOSE: Intracellular osmotic stress is believed to be linked to the advancement of diabetic cataract. Although the accumulation of organic osmolytes (myo-inositol, sorbitol, taurine) is thought to protect the lens by maintaining osmotic homeostasis, the physiologic implication of osmotic imbalance (i.e., hyperosmotic stress caused by intracellular over-accumulation of organic osmolytes) on diabetic cataract formation is not clearly understood. Studies from this laboratory have identified several osmotic compensatory mechanisms thought to afford the lens epithelium, but not the lens fibers, protection from water stress during intervals of osmotic crisis. This model is founded on the supposition that the fibers of the lens are comparatively more susceptible to damage by osmotic insult than is the lens epithelium. To test this premise, several transgenic mouse lines were developed that over-express the bovine sodium/myo-inositol cotransporter (bSMIT) gene in lens fiber cells. METHODS: Of the several transgenic mouse lines generated, two, MLR14 and MLR21, were analyzed in detail. Transgenic mRNA expression was analyzed in adult and embryonic transgenic mice by a coupled reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization on embryonic tissue sections, respectively. Intralenticular myo-inositol content from individual mouse lenses was quantified by anion exchange chromatography and pulsed electrochemical detection. Ocular histology of embryonic day 15.5 (E15.5) embryos from both transgenic (TG) families was analyzed and compared to their respective nontransgenic (NTG) littermates. RESULTS: Both RT-PCR and in situ hybridization determined that transgene expression was higher in line MLR21 than in line MLR14. Consistent with this, intralenticular myo-inositol from MLR21 TG mice was markedly higher compared with NTG littermates or MLR14 TG mice. Histologic analysis of E15.5 MLR21 TG embryos disclosed a marked swelling in the differentiating fibers of the bow region and subcapsular fibers of the central zone, whereas the lens epithelium appeared morphologically normal. The lenticular changes, initiated early during lens development in TG MLR21 embryos, result in severe bilateral nuclear cataracts readily observable in neonates under normal rearing and dietary conditions. In contrast, TG MLR14 pups reared under standard conditions produced no lens opacity. CONCLUSIONS: Lens fiber swelling and related cataractous outgrowth positively correlated to the degree of lens bSMIT gene expression and intralenticular myo-inositol content. The affected (i.e., swollen) lens fibers appeared to be unable to cope with the water stress generated by the transgene-induced over-accumulation of myo-inositol and, as a result of this inability to osmoregulate, suffered osmotic damage due to water influx. (+info)
Functional characterization of the interaction of Ste50p with Ste11p MAPKKK in Saccharomyces cerevisiae.
(10/1433)
The Saccharomyces cerevisiae Ste11p protein kinase is a homologue of mammalian MAPK/extracellular signal-regulated protein kinase kinase kinases (MAPKKKs or MEKKs) as well as the Schizosaccharomyces pombe Byr2p kinase. Ste11p functions in several signaling pathways, including those for mating pheromone response and osmotic stress response. The Ste11p kinase has an N-terminal domain that interacts with other signaling molecules to regulate Ste11p function and direct its activity in these pathways. One of the Ste11p regulators is Ste50p, and Ste11p and Ste50p associate through their respective N-terminal domains. This interaction relieves a negative activity of the Ste11p N terminus, and removal of this negative function is required for Ste11p function in the high-osmolarity glycerol (HOG) pathway. The Ste50p/Ste11p interaction is also important (but not essential) for Ste11p function in the mating pathway; in this pathway binding of the Ste11p N terminus with both Ste50p and Ste5p is required, with the Ste5p association playing the major role in Ste11p function. In vitro, Ste50p disrupts an association between the catalytic C terminus and the regulatory N terminus of Ste11p. In addition, Ste50p appears to modulate Ste11p autophosphorylation and is itself a substrate of the Ste11p kinase. Therefore, both in vivo and in vitro data support a role for Ste50p in the regulation of Ste11p activity. (+info)
Impaired osmoregulatory responses in rats with area postrema lesions.
(11/1433)
Area postrema lesions (APX) in adult male rats produced a robust spontaneous intake of 0.5 M NaCl, as reported previously. The largest NaCl intakes (up to 108 ml/day) were observed when there was little incidental damage in the medial subnucleus of the nucleus of the solitary tract adjacent to the caudal and middle portions of the area postrema. Rats with discrete APX also drank substantial amounts of 0.5 M NaCl when access to saline was restricted to 7 h/day (up to 30 ml in 1 h, 48 ml in 7 h). Such large NaCl intakes stimulated considerable water ingestion and renal sodium excretion, but together these responses usually were insufficient for osmoregulation during the 7-h test period. After systemic administration of hypertonic NaCl solution, rats with APX excreted less Na(+) in urine and secreted less vasopressin and oxytocin than control rats did. The prominent salt appetite, insufficient thirst and natriuresis in response to an ingested NaCl load, and blunted natriuresis and neurohypophysial hormone secretion in response to an injected NaCl load, all indicate that osmoregulatory responses are impaired in rats after APX. (+info)
Effects of missense mutations on rat aquaporin-2 in LLC-PK1 porcine kidney cells.
(12/1433)
BACKGROUND: Mutations in the aquaporin-2 (AQP2) gene have been found in families with nephrogenic diabetes insipidus (NDI), but the pathophysiological mechanisms of how mutant AQP2 causes the disease are still not clear. METHODS: Wild-type (WT) AQP2 and four mutants-T126M, A147T, R187C, and S216P-were transiently expressed in LLC-PK1 cells. The osmotic water permeability of LLC-PK1 cells expressing AQP2 mutants was determined by stopped-flow light-scattering microphotometry. Cell surface expression, subcellular localization, and effects of vasopressin stimulation were examined by surface biotin labeling and confocal immunohistochemistry. RESULTS: The osmotic water permeability (Pf) of cells expressing WT increased significantly after vasopressin treatment, whereas the Pf of cells expressing T126M A147T, R187C, and S216P was not significantly different from that of the control even after vasopressin stimulation. Confocal immunohistochemistry demonstrated distribution of WT and A147T in early/recycling endosomal compartments and vasopressin-responsive translocation and surface expression. In contrast, stainings of T126M, R187C, and S216P were similar to that of Grp78, indicating that these mutants were misassembled and retarded in the endoplasmic reticulum. CONCLUSION: Our results indicated that the intracellular distribution and vasopressin-regulated trafficking of A147T is intact, in contrast to the other three mutants, of which both were impaired. Thus, it is conceivable that the disruption of the AQP2 channel function accounts for the pathogenesis of A147T NDI, whereas trafficking defects account for that of the other types, suggesting that the pathophysiology of AQP2-related NDI is heterogeneous. (+info)
Structural equivalents of latency for lysosome hydrolases.
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1. Structure-linked latency, a trait for most lysosome hydrolase activities, is customarily ascribed to the permeability-barrier function performed by the particle-limiting membrane, which shields enzyme sites from externally added substrates. 2. The influence of various substrate concentrations on the reaction rate has been measured for both free (non-latent) and total (completely unmasked by Triton X-100) hydrolase activities in rat liver cell-free preparations. The substrates were: beta-glycerophosphate, phenolphthalein mono-beta-glucuronide. p-nitrophenyl N-acetyl-beta-D-glucosaminide and p-nitrophenyl beta-D-galactopyranoside. The ratio (free activity/total activity) X 100 is called fractional free activity at any given substrate concentration. 3. The fractional free activity of beta-glucuronidase and beta-N-acetylglucosaminidase were clearly independent of substrate concentration, over the range examined, in both homogenates and lysosome-rich fractions. The fractional free activity of acid phosphatase appeared to be either unaffected (homogenate) or even depressed (lysosome-rich fraction) by increasing the beta-glycerophosphate concentration. The fractional free activity of beta-galactosidase consistently showed a non-linear increase with increasing substrate concentration in both homogenates and lysosome-rich fractions. 4. Procedures such as treatment with digitonin, hypo-osmotic shock and acid autolysis, although effective in causing varying degrees of resolution of the latency of lysosome hydrolase activities, were unable to modify appreciably the pattern of dependence or independence of their fractional free activities on substrate concentration, as compared with that exhibited by control preparations. Ouabain did not affect the free beta-N-acetylglucosaminidase activity of liver homogenates at all. 5. Preincubation of control preparations with beta-glycerophosphate or p-nitrophenyl beta-galactoside did not result in any significant stimulation of the free hydrolytic activity toward these substrates. 6. The results consistently support the view that the membrane of "intact" lysosomes is virtually impermeable to all the substrates tested, except for p-nitrophenyl beta-galactoside, for which the evidence is contradictory. Moreover the progressive unmasking of the hydrolase activities produced by these procedures in vitro reflects the increasing proportion of enzyme sites that are fully accessible to their substrates rather than a graded increase in the permeability of the lysosomal membrane. (+info)
Osmosis, osmometry, and osmoregulation.
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The maintenance of adequate body fluid volume and the correct distribution of this fluid between the body compartments is a critical part of homeostasis. The process of osmosis plays an important role in movement of fluid within the body and the use of osmometry is an important part of the management of many patients. In addition to the application of osmometry to the measurement of body fluids, most commonly plasma and urine, osmotic action plays a part in some therapeutic actions of drugs and its strength needs to be quantified in fluids administered to patients. Unfortunately confusion often exists in the various terms that are used in the field of osmometry. This review aims to explain the different terms used, the laboratory methodology involved in osmometry, and the clinical application and interpretation of the results obtained. (+info)
Inhibitory effect of high [Mg2+] on the vasopressin-stimulated hydroosmotic permeability of the isolated perfused cortical collecting duct.
(15/1433)
High magnesium concentration inhibits the effect of arginine vasopressin (AVP) on smooth muscle contraction and platelet aggregation and also influences hepatocyte AVP receptor binding. The aim of this study was to determine the role of magnesium concentration [Mg2+] in AVP-stimulated water transport in the kidney collecting duct. The effect of low and high peritubular [Mg2+] on the AVP-stimulated osmotic water permeability coefficient (Pf) was evaluated in the isolated perfused rabbit cortical collecting duct (CCD). Control tubules bathed and perfused with standard Ringer bicarbonate solution containing 1 mM Mg2+ presented a Pf of 223.9 +/- 27.2 microm/s. When Mg2+ was not added to the bathing solution, an increase in the AVP-stimulated Pf to 363.1 +/- 57.2 microm/s (P<0. 05) was observed. An elevation of Mg2+ to 5 mM resulted in a decrease in Pf to 202.9 +/- 12.6 microm/s (P<0.05). This decrease in the AVP-stimulated Pf at 5 mM Mg2+ persisted when the CCDs were returned to 1 mM Mg2+, Pf = 130.2 +/- 20.3 microm/s, and was not normalized by the addition of 8-[4-chlorophenylthio]-adenosine 3', 5'-cyclic monophosphate, a cAMP analogue, to the preparation. These data indicate that magnesium may play a modulatory role in the action of AVP on CCD osmotic water permeability, as observed in other tissues. (+info)
Permeability characteristics of human oocytes in the presence of the cryoprotectant dimethylsulphoxide.
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Equilibration of oocytes with cryoprotectants is a prerequisite of low temperature storage. However, cryoprotectant exposure may induce damage via osmotic stress. Knowledge of cell membrane permeability characteristics and their temperature dependence would facilitate the design of cryopreservation protocols in which osmotic stress is minimized and the incidence of intracellular freezing is reduced. To obtain such data, the volume change of donated human oocytes following exposure to cryoprotectant was measured at a variety of temperatures. After removal of cumulus cells, each oocyte was placed in a 5 microl droplet of phosphate-buffered medium. The oocyte was held in position by suction generated using a fine pipette and perfused with 1 ml 1.5 mol/l dimethylsulphoxide (DMSO) at 30, 24 or 10 degrees C. The volume of the oocyte before, during and after perfusion was recorded by videomicroscopy. Oocyte volume was calculated from radius measurements and the Kedem-Katchalsky (K-K) passive coupled transport coefficients, namely L(p) (hydraulic permeability), P(DMSO) (permeability to DMSO) and sigma (reflection coefficient) were derived. The resulting coefficients were L(p) = 1. 65 +/- 0.15, 0.70 +/- 0.06 and 0.28 +/- 0.04 microm/min.atm; P(DMSO) = 0.79 +/- 0.10, 0.25 +/- 0.04 and 0.06 +/- 0.01 microm/s and sigma = 0.97 +/- 0.01, 0.94 +/- 0.03 and 0.96 +/- 0.01 at 30, 24 and 10 degrees C respectively. The activation energy for L(p) was 14.70 and for P(DMSO) was 20.82 kcal/mol. The permeability parameters of human oocytes are higher than those of murine oocytes, suggesting that they require a shorter period of exposure to DMSO with concomitantly reduced toxic effects. (+info)