Increase In Medium Osmolality Research Articles

Media evaporation in a dry culture incubator; effect of dish, drop size and oil on media osmolality

Modern benchtop incubators typically lack humidity, a significant difference compared to traditional big box incubators. Combined with the recent popularity of single step culture systems, media evaporation resulting in an increase in osmolality during the culture period is a legitimate concern. The objective of this study was to evaluate variables that may impact evaporation over the time course of normal human embryo culture in a dry benchtop incubator. Prospective Study. : Cleavage medium (Sage) + 10% SPS was used for all experiments. Dishes for each treatment were prepared and placed into the same chamber of a K Systems G185 incubator. After 6 days, media from microdrops in each treatment was recovered and osmolality measured on a Wescor 5600 Vapor Pressure osmometer. All experiments were repeated 3 times, with a minimum of 13 drops measured per treatment. Media prior to incubation was used as a control. In experiment 1, 30 uL drops were prepared under 3.5 mL of oil (OvOil) in a Falcon 35 mm dish and under 4.5 mL of oil in a mini-GPS dish. In experiment 2, drop volume was examined in the mini-GPS dish (20uL, 40uL and 60uL). In experiment 3, 30uL drops in a mini-GPS dish were covered with 3, 4, or 5 mL oil. In experiment 4, 30 uL drops in a mini-GPS dish were covered with 4.5ml OvOil, light oil, or heavy oil. Results are presented as mean ± SEM in mOsm. Significance was determined at p<0.01. Uninterrupted culture for 6 days in a benchtop incubator resulted in a significant increase in medium osmolality regardless of dish type (Falcon control: 275.8 ± 1.5; Falcon 6 day: 300.7 ± 2.0; GPS control: 275.9 ± 1.7; GPS 6 day: 293.1 ± 1.5). Although evaporation occurred in both types of dishes, GPS dishes resulted in a significant improvement in osmolality on D6. Evaporation resulting in increased osmolality occurred in all drop sizes (control: 271.0 ± 1.4; 20 uL: 306.6 ± 2.6; 40 uL: 292.4 ± 2.6; 60 uL: 302.4 ± 3.3), although 40 uL drops resulted in lower osmolality than 20 or 60 uL drops. Elevated osmolality also resulted regardless of oil volume (control: 272.9 ± 1.5; 3 mL: 300.6 ± 2.8; 4 mL: 298.1 ± 1.9; 5 mL: 290.6 ± 2.1). However, 5 mL of oil overlay resulted in a lower osmolality than 3 mL of oil. Finally, the type of oil had no impact on media evaporation (control: 271.3 ± 1.5; OvOil: 291.6 ± 1.5; heavy: 291.8 ± 2.2; light: 294.9 ± 2.4). Significant evaporation occurs during continuous culture for 6 days in a dry benchtop incubator. None of the dishes, drop sizes, or types of oil tested prevented evaporation, although a combination of 40 uL drops in mini-GPS dishes under 5 ml oil may result in the lowest osmolality. However, even this combination in dry incubator conditions results in osmolality (∼291) that is elevated compared to the intended osmolality of this culture medium (∼272), which may be detrimental to embryo development.

Differential regulation of TRPV4 mRNA levels by acclimation salinity and extracellular osmolality in euryhaline tilapia

Prolactin (PRL) cells of the euryhaline Mozambique tilapia, Oreochromis mossambicus, are osmoreceptors. Hyposmotically-induced PRL release is mediated by the inward movement of extracellular Ca2+ through a stretch-activated Ca2+ channel, which has been recently identified as the transient receptor potential vanilloid 4 (TRPV4). In the present study, changes in plasma PRL, as well as PRL and TRPV4 mRNA expression from the rostral pars distalis (RPD), were measured in fish transferred from seawater (SW) to fresh water (FW) and in fish transferred from FW to SW. The in vitro effects of osmolality on PRL release and on PRL and TRPV4 mRNA expression in dispersed PRL cells were compared between fish adapted to SW and FW. Both the release and expression of PRL fell when fish were transferred to SW and rose when fish were transferred to FW. By contrast, TRPV4 expression increased by 48h after fish were transferred from FW to SW and declined as early as 6h after transfer from SW to FW. A similar pattern was observed in vitro where TRPV4 expression responded positively to an increase in medium osmolality while PRL expression declined. Incubation with the Ca2+ ionophore, A23187, and the phosphodiesterase inhibitor, IBMX, stimulated PRL release. While both IBMX and A23187 inhibited TRPV4 expression, only A23187 reduced PRL expression. Together, these findings indicate that the expression of TRPV4 mRNA is osmosensitive, increasing as extracellular osmolality rises. Furthermore, these data suggest that TRPV4 expression may be regulated through the same second messenger pathways involved in hyposmotically-induced PRL release.

The role of carbon monoxide and nitric oxide in hyperosmolality-induced atrial natriuretic peptide release by hypothalamus in vitro

We evaluated the participation of the nitrergic and carbon monoxide (CO) systems in the atrial natriuretic peptide (ANP) release induced by osmotic stimulation of the rat anterior and medial basal hypothalamus (BH) fragments in vitro. The increase in the medium osmolality (NaCl, 340 mOsm/kg H 2O) induced an elevated ANP release, which was associated with a decrease in nitric oxide synthase (NOS) activity ( p<0.001), nitric oxide (NO) production and nitrate ( p<0.001) release into the medium. The NO donors sodium nitroprusside (SNP, 300 μM), S-nitroso- N-acetylpenicillamine (SNAP, 300 μM) and 3-morpholinylsydnoneimine chloride (SIN-1, 300 μM) promoted a significant decrease in ANP release in response to hyperosmolality ( p<0.001). ANP release observed in the present study did not result from injury to the BH caused by the increase in medium osmolality nor a toxic effect of the NO donors as demonstrated by the ANP release after incubation with KCl (56 mM). Furthermore, hyperosmolality or NO donors did not increase the LDH content in the medium. The hyperosmotic-induced ANP release and reduction of NOS activity were prevented by the heme oxygenase inhibitor, zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG). In conclusion, these results suggest that NO, the production of which is dependent on CO, modulates the osmolality-induced ANP release by BH fragments.

A chimeric Anabaena/ Escherichia coli KdpD protein (Anacoli KdpD) functionally interacts with E. coli KdpE and activates kdp expression in E. coli.

The kdpFABC operon, coding for a high-affinity K(+)-translocating P-type ATPase, is expressed in Escherichia coli as a backup system during K(+) starvation or an increase in medium osmolality. Expression of the operon is regulated by the membrane-bound sensor kinase KdpD and the cytosolic response regulator KdpE. From a nitrogen-fixing cyanobacterium, Anabaena sp. strain L-31, a kdpDgene was cloned (GenBank accession no. AF213466) which codes for a KdpD protein (365 amino acids) that lacks both the transmembrane segments and C-terminal transmitter domain and thus is shorter than E. coli KdpD. A chimeric kdpD gene was constructed and expressed in E. coli coding for a protein (Anacoli KdpD), in which the first 365 amino acids of E. coli KdpD were replaced by those from Anabaena KdpD. In everted membrane vesicles, this chimeric Anacoli KdpD protein exhibited activities, such as autophosphorylation, transphosphorylation and ATP-dependent dephosphorylation of E. coli KdpE, which closely resemble those of the E. coli wild-type KdpD. Cells of E. coli synthesizing Anacoli KdpD expressed kdpFABC in response to K(+) limitation and osmotic upshock. The data demonstrate that Anabaena KdpD can interact with the E. coliKdpD C-terminal domain resulting in a protein that is functional in vitro as well as in vivo.

Effects of l-valine on growth and polyamine metabolism in human colon carcinoma cells

HT-29 cells, originating from a human colon carcinoma, can proliferate in standard culture conditions with an absolute requirement for polyamines. The major precursor provided in the culture medium for polyamine biosynthesis is l-arginine. l-Arginine conversion to l-ornithine by arginase is followed by stepwise conversion of this latter amino acid to putrescine, spermidine and spermine. The aim of the present work was to document the consequences of a total inhibition of l-arginine flux through arginase, resulting in a decreased l-ornithine availability, on HT-29 cell proliferation and polyamine metabolism. l-Valine, a known arginase inhibitor, when used at a high concentration, i.e., 100 mM, inhibits l-arginine flux through arginase almost totally. The addition in the culture medium of 100 mM l-valine or 50 mM NaCl used to mimic the l-valine induced increase in medium osmolality both reduced equally cellular growth. Cell viability, protein synthesis or oxidative metabolism measured in isolated cells were unaffected by the l-valine treatment, suggesting that decreased proliferation was not associated with an acute toxic effect of this aminoacid, but was rather due to the increase in the medium osmolality. l-Valine treated cells displayed an altered polyamine metabolism when compared with control cells grown in the absence of the amino acid. After 4 days of treatment with 100 mM l-valine, l-ornithine flux through ornithine decarboxylase was significantly higher as well as putrescine and spermidine cellular uptakes in treated cells. However, the changes in polyamine metabolism led to similar polyamine cell contents in untreated and l-valine treated cells. In conclusion, we propose that the observed alterations of polyamine metabolism may reflect an adaptative response of HT-29 cells to the presence of l-valine which contribute together with the low amount of l-ornithine present in the culture medium to polyamine homeostasis.

Glycosylation of CHO-derived recombinant tPA produced under elevated pCO2.

Carbon dioxide is a metabolic byproduct of mammalian cell metabolism that can accumulate in poorly ventilated cultures. A buildup of CO2 at constant pH will be accompanied by an increase in medium osmolality. We have examined the glycosylation of tissue plasminogen activator (tPA) produced under serum-free conditions by recombinant Chinese hamster ovary (CHO) cells (MT2-1-8 cell line) in response to elevated pCO2 at constant or elevated osmolality. The proportion of sialic acids comprising N-glycolylneuraminic acid decreased from 2.3-4.0% under 36 mmHg pCO2 to 1.5-2.2% under 250 mmHg pCO2. No changes were observed in the total sialic acid content, the content of other monosaccharides, the relative amounts of type I and type II tPAs, the distribution of surface charges, or the proportion of high-mannose oligosaccharides-even though these conditions have previously been shown to inhibit the specific growth rate of MT2-1-8 cells by 30-40% and the specific tPA production rate by as much as 40%. These results suggest robust glycosylation of tPA by CHO cells.

Effect of substrate concentration on ethanol production by Zymomonas mobilis on cellulose hydrolysate

A cellulose hydrolysate from Aspen wood, containing mainly glucose, was fermented into ethanol by a thermotolerant strain MSN77 of Zymomonas mobilis. The effect of the hydrolysate concentration on fermentation parameters was investigated. Growth parameters (specific growth rate and biomass yield) were inhibited at high hydrolysate concentrations. Catabolic parameters (specific glucose uptake rate, specific ethanol productivity and ethanol yield) were not affected. These effects could be explained by the increase in medium osmolality. The results are similar to those described for molasses based media. Strain MSN77 could efficiently ferment glucose from Aspen wood up to a concentration of 60 g/l. At higher concentration, growth was inhibited.

Batch fermentation kinetics of ethanol production by Zymomonas mobilis on cellulose hydrolysate

Growth and ethanol production by three strains (MSN77, thermotolerant, SBE15, osmotolerant and wild type ZM4) of the bacterium Zymomonas mobilis were tested in a rich medium containing the hexose fraction from a cellulose hydrolysate (Aspen wood). The variations of yield and kinetic parameters with fermentation time revealed an inhibition of growth by the ethanol produced. This inhibition may result from the increase in medium osmolality due to ethanol formation from glucose.

Sucrose medium osmolality as a regulator of anabolic and catabolic parameters in Zymomonas culture

AbstractThis study focuses on the growth of Zymomonas mobilis strain 113 S and its ethanol and levan production under the conditions of increasing sucrose medium osmolality caused by NaCl, KCl, sorbitol or maltose. The increase in medium osmolality (700–1,500 mosml/kg) was accompanied by the inhibition of growth (growth rate, biomass yield) and ethanol production (specific productivity and yield) In contrast, levan synthesis was less affected or even stimulated and, as a consequence, levan specific productivity was increased significantly. A decrease in the anabolic growth parameters correlated with a parallel inhibition of glucose‐6‐P dehydrogenase and alcohol dehydrogenase (isoenzyme ADH II) activities. A significant inverse linear relationship (r = − 0.932, 1 − P = 0.01) was observed between the values of the specific productivities of ethanol and levan. This relationship was confirmed independently by a controlled reduction of growth and ethanol productivity (3.75–4.75 mM sodiumbisulphite as an acceptor of acetaldehyde formed in the pyruvate decarboxylase reaction). As further support of this relationship, a significant inverse correlation was observed between levan specific productivity and ATP concentration in Zymomonas mobilis cells, most probably demonstrating that a reduced level of energetic metabolism is favourable for levan production.

Volume-dependent regulation of sodium and potassium fluxes in cultured vascular smooth muscle cells: dependence on medium osmolality and regulation by signalling systems.

To identify ion transport systems involved in the maintenance of vascular smooth muscle cell volume the effects of incubation medium osmolality and ion transport inhibitors on the volume and 86Rb and 22Na transport in cultured smooth muscle cells from rat aorta (VSMC) have been studied. A decrease of medium osmolality from 605 to 180 mosm increased intracellular water volume from 0.6 to 1.3 microliters per 10(6) cells. Under isosmotic conditions, cell volume was decreased by ouabain (by 10%, P less than 0.005) but was not influenced by bumetanide, furosemide, EIPA and quinidine. These latter compounds were also ineffective in cell volume regulation under hypotonic buffer conditions. Under hyperosmotic conditions, cell volume was decreased by bumetanide (by approximately 7%, P less than 0.05) and by ethylisopropyl amiloride (by approximately 13%, P less than 0.005). Ouabain-sensitive 86Rb influx was decreased by 30-40% under hypoosmotic conditions. An increase in medium osmolality from 275 to 410 mosm resulted in an approximately eightfold increase in bumetanide-inhibited 86Rb influx and 86Rb efflux. The (ouabain and bumetanide)-insensitive component of 86Rb influx was not dependent on the osmolality of the incubation medium. However (ouabain and bumetanide)-insensitive 86Rb efflux was increased by approximately 1.5-2 fold in VSMC incubated in hypotonic medium. Ethylisopropyl amiloride-inhibited 22Na influx was increased by approximately sixfold following osmotic-shrinkage of VSMC. The data show that both Na+/H+ exchange and Na+/K+/2Cl- cotransport may play a major role in the regulatory volume increase in VSMC. Basal and shrinkage-induced activities of Na+/K+/2Cl- cotransport in VSMC were similarly sensitive to inhibition by either staurosporin, forskolin, R24571 or 2-nitro-4-carboxyphenyl N,N-diphenylcarbomate (NCDC). In contrast basal and shrinkage-induced Na+/K+/2Cl- cotransport were differentially inhibited by NaF (by 30 and 65%, respectively), suggesting an involvement of guanine nucleotide binding proteins in the volume-sensitive activity of this carrier. Neither staurosporin, forskolin, R24571 nor NCDC influenced shrinkage-induced Na+/H+ exchange activity. NaF increased Na+/H+ exchanger activity under both isosmotic and hyperosmotic conditions. These data demonstrate that different intracellular signalling mechanisms are involved in the volume-dependent activation of the Na+/K+/2Cl- cotransporter and the Na+/H+ exchanger.

Effects of osmotic pressure on prolactin and growth hormone secretion from organ-cultured eel pituitary

Effects of medium osmotic pressure on the release of prolactin (PRL) and growth hormone (GH) from the pituitary of the Japanese eel, Anguilla japonica, were examined during long-term organ culture in a defined medium. Prolactin and GH release, as measured by homologous radioimmunoassays, increased gradually for 7 days during incubation in isosmotic medium (295 mOsmolal). On day 7, 3 to 5 times more PRL and GH were released than on day 1. The amount of GH released was about 100 times greater than that of PRL. Electron microscopic observation revealed that both PRL and GH cells were in good condition after 7 days incubation. The reduction of medium osmotic pressure from 295 (isosmotic) to 235 or 260 mOsmolal significantly stimulated PRL release for 4 days. By contrast, an increase in medium osmolality from 295 to 360 mOsmolal was without effect. These treatments produced no significant alterations in GH release. The stimulatory effect of hyposmotic medium (235 mOsmolal) was no longer evident by 12 h after the pituitaries were returned to isosmotic medium. The isosmotic but low-sodium medium, prepared by adding mannitol to the hyposmotic medium, did not stimulate PRL release from the pituitary. These results indicate that plasma osmolality may be an important physiological factor controlling PRL release during freshwater adaptation of the eel.

Rat renal papillary tissue explants survive and produce epithelial monolayers in culture media made hyperosmotic with sodium chloride and urea

The capacity of papillary cells to adapt to elevated osmotic concentrations is unusual among mammalian cells. This capacity was evaluated by using primary tissue culture. Viability and growth of cells in rat renal papillary tissue explants were assessed after culture in media adjusted with urea and sodium chloride to various osmotic concentrations between 300 and 1,500 mOsm/kg water. The survival of cells, including cells resembling those of the collecting ducts and the loop of Henle, was greatest in medium adjusted to 1,000 mOsm with equiosmolar amounts of the two solutes. At 1,500 mOsm only cuboidal tubular epithelium resembling collecting duct epithelial cells survived. In contrast, cells of cortical tissue survived and grew at 300 and 640 mOsm, but not at 1,000 mOsm or above. Epithelial monolayers appeared to proliferate from collecting ducts and spread over the surface of the explants as well as onto the glass surface in the culture dish. Epithelial growth of medullary tissue was most rapid at 300 mOsm and was slower at 700 and 1,000 mOsm. Monolayers did not form at 1,500 mOsm; however, epithelial overgrowth of explants did occur. Hydropenia in the donor animal did not significantly affect the viability or growth of cultured papillary tissue. Explants cultured for 5 days at 300 mOsm followed by a stepwise increase in medium osmolality to 1,100 or 1,500 mOsm and cultured for 3 more days showed low or no survival whereas explants cultured at 700 mOsm survived such increases. Explants cultured for 5 days at 1,500 mOsm survived and grew monolayers when lowered to 300 mOsm. Poor viability and no epithelial proliferation were observed in explants cultured in medium adjusted to 900 mOsm with either urea or sodium chloride alone, suggesting that a mixture of the two solutes in the extracellular space, as found in vivo, may be essential in achieving elevated osmolalities.

Parathyroid hormone action on phosphate transport is inhibited by high osmolality

Parathyroid hormone (PTH) produces rapid inhibition of Na(+)-phosphate cotransport, characterized by a decreased maximal rate of transport, and the inhibition is independent of de novo protein synthesis. The present study determined whether the action of PTH on Na(+)-phosphate cotransport is mediated, at least in part, by rapid endocytic internalization of Na(+)-phosphate cotransporters present in the plasma membrane. Horseradish peroxidase, a fluid-phase marker, was used to demonstrate the presence of endocytosis in opossum kidney (OK) epithelial cells in monolayer culture. An increase in medium osmolality to 500 mosmol/kgH2O, by addition of sucrose, produced 80% inhibition of endocytosis within 1 h. The inhibition was reversed on returning the cells to normal medium. Incubation of OK cell monolayers with PTH (10(-8) M) for 3 h at normal osmolality (281 mosmol/kgH2O) inhibited Na(+)-phosphate cotransport (4 min uptakes) by 56-67%. In hyperosmolar medium (513 mosmol/kgH2O), when endocytosis was inhibited, PTH inhibited Na(+)-phosphate cotransport by only 25-39%, a change that was significantly different from the inhibition in normal medium. Hyperosomolality had no effect on PTH inhibition of Na(+)-H+ exchange or on PTH stimulation of intracellular adenosine 3',5'-cyclic monophosphate. We conclude that the full inhibitory action of PTH on Na(+)-phosphate cotransport may require an intact endocytic mechanism.

Osmoregulatory fluxes of myo-inositol and betaine in renal cells.

Renal medullary cells contain high concentrations of "compatible" organic osmolytes, such as myo-inositol, betaine, sorbitol, and glycero-phosphorylcholine. These organic osmolytes accumulate as an osmoregulatory response to the high and variable interstitial NaCl concentration that is part of the urinary concentrating mechanism. Madin-Darby canine kidney (MDCK) cells in culture were previously shown to accumulate myo-inositol and betaine in response to increased NaCl. These organic osmolytes are taken up by sodium-dependent active transport into the cells from the medium. The maximum concentration is not reached until 2-4 days after an increase in medium osmolality. The purpose of this study was to characterize the response to a decrease in medium osmolality of cells that had been grown at a high osmolality. The initial response to decreased osmolality was a rapid, transient efflux of both myo-inositol and betaine from the cells. Efflux was greatest during the first 15 min and resulted in a reduction of cell myo-inositol and betaine by almost 13 and 22%, respectively, after 3 h. Active myo-inositol and betaine influx fell more slowly, reaching a lower limit after approximately 1-2 days. The reduced influx was followed by progressive decrease in cell myo-inositol and betaine to approximately 30% of the initial value after 6 days. Thus, after a decrease in medium osmolality, MDCK cell myo-inositol and betaine fell because of a rapid, transient increase in efflux and a slow, sustained decrease in active influx.

A simple method for continuous measurement of the volume of cells in suspension.

A simple electrical method for continuous monitoring of the volume of cells in suspension is described. The procedure consists of placing cells in a medium containing an impermeant quaternary ammonium ion, such as tetramethylammonium (TMA), which behaves as an extracellular volume marker, and monitoring the activity of this ion with a quaternary ammonium ion-specific minielectrode. Changes in the activity of the cell impermeant TMA ion in the extracellular medium quantitatively reflect movements of water into, or out of, cells and, therefore, provide a direct measure of alterations in cell volume. A direct linear relation between the fractional decrease in rat red blood cell volume and the fractional increase in medium osmolality was observed using this method. The osmotically active water volume of these red blood cells, calculated from the slope of this relationship, was 73.6 +/- 0.7% of total cell volume, a value similar to that defined by other techniques. This method should be easily adaptable for use with virtually any type of separated cell, including epithelial cells.

Role of calcium in osmotic and nonosmotic release of vasopressin from rat organ culture.

In the present study the role of calcium (Ca) in the stimulation of arginine vasopressin (AVP) release from the cultured rat hypothalamoneurohypophyseal complex (HNC) was examined in response to three different stimuli, 56 mM potassium chloride, an increase in medium osmolality from 290 to 310 mosmol/kg H2O, or 1 X 10(-6) M angiotensin II (ANG II). With all three stimuli AVP release from rat HNC explants was enhanced by increasing Ca concentration in the medium from 0 to 1.8 mM Ca. However, high concentrations of Ca (8 mM) inhibited the response of AVP release to either hyperosmolality or angiotensin II. Chemically dissimilar blockers of cellular Ca uptake, verapamil (5.2 X 10(-6) or 5.2 X 10(-5) M) or nifedipine (5.8 X 10(-6) or 5.8 X 10(-5) M), completely abolished AVP release from rat HNC explants in response to the three different stimuli in 1.8 mM Ca. In a normal concentration of medium Ca (1.8 mM) a Ca ionophore, A23187 (3.8 X 10(-5) M), significantly enhanced the osmotic and nonosmotic (ANG II-stimulated) release of AVP from rat HNC explants compared with controls without Ca ionophore. This effect of Ca ionophore to enhance AVP release was more evident in a lower Ca medium (0.9 mM Ca in the hyperosmolality study and 0.3 mM Ca in the ANG II study). These results therefore indicate that cellular Ca uptake is an important modulator of osmotic and nonosmotic AVP release from the intact rat hypothalamoneurohypophyseal system. The influence of extracellular Ca on the osmotic and nonosmotic release of AVP is also demonstrated.