Influence Of Tonicity Research Articles

Human CYP3A Expression under Hypertonic Stimuli In Vivo

We previously showed that human CYP3A expression in vitro is under the transcriptional control mediated by NFAT5, which is a tonicity responsive transcriptional factor. Interestingly, hypertonicity increases CYP3A expression, while hypotonicity decreases it, suggesting constitutive regulation roles of the NFAT5‐CYP3A pathway. In contrast, mouse Cyp3a was unresponsive to ambient hypertonicity in vitro. In the present study, our objective was to show if human CYP3A expression can be induced by hypertonic stimuli in vivo. The humanized CYP3A4–3A7 transgenic mice were subjected to 4 cycles of 24h water deprivation every other day, or to a 7‐day high‐salt diet (8% salt content, HSD), that mimics the dehydration and hypertonic luminal content in the intestine, respectively. In the kidney of mice undergoing the cyclic dehydration, NFAT5‐regulated BGT1 mRNA was significantly increased compared to control (5.9±1.1 fold: M±SEM; p=0.003). Fold‐changes of the CYP3A4 and 3A7 were 2.5±0.5 (p=0.02) and 0.7±0.2 (p=0.13), respectively. In mice after HSD, fold‐induction of the duodenal BGT1, CYP3A4 and 3A7 expressions were 25.2±8.2 (p=0.02), 4.7±0.9 (p=0.01) and 2.0±0.3 (p=0.08), respectively. Our findings suggest that human CYP3A transcription is under the influence of ambient tonicity. Direct involvement of NFAT5 in vivo awaits further studies.CIHR supported

Effect of Formulation Parameters on Corneal Permeability of Ofloxacin

Influence of pH, buffer, preservative, tonicity and viscosity modifiers on in vitrotranscorneal permeation of ofloxacin was studied using excised goat cornea. Permeation studies were carried out by putting 1 ml of test formulation on the cornea (0.78cm²) mounted between the donor and receptor compartments of an all glass modified Franz diffusion cell and measuring ofloxacin concentration in the receptor (containing bicarbonate ringer under stirring at 35°C) by spectrophotometry at 288 nm, at various time intervals up to 120 min. Buffering the formulation with phosphate buffer or use of combination of methyl and propyl paraben or benzalkonium chloride or combination of benzalkonium chloride and disodium edetate as preservative provided significant increase in the apparent corneal permeability coefficient of ofloxacin. While the use of phenyl mercuric acetate as preservative or tonicity adjustment with mannitol showed a significant decrease in apparent corneal permeability of ofloxacin. Raising the pH of test formulation from 6.4 to 7.2 or addition of hydroxyl-propyl-β-cyclodextrin or use of viscosity modifier had no significant effect on apparent corneal permeability of drug.

Low tonicity mediates a downregulation of cyclooxygenase-1 expression by furosemide in the rat renal papilla.

It is well known that loop diuretics enhance the renal excretion of prostanoids; therefore, this study aimed to characterize the influence of loop diuretics on the intrarenal expression of cyclooxygenases, which are the key enzymes for prostanoid formation. Male Sprague-Dawley rats were infused with furosemide (12 mg/kg per d) for 6 d, and the expression of cyclooxygenase-1 and -2 (Cox-1 and Cox-2) was analyzed in the different kidney zones. Furosemide increased Cox-2 mRNA expression approximately twofold in the cortex, but it left Cox-1 mRNA expression unaltered there. In the outer medulla, furosemide changed neither Cox-1 nor Cox-2 mRNA expression. In the inner medulla, however, furosemide decreased Cox-1 and Cox-2 mRNA levels to approximately 30% and 60% of their control levels, respectively. The downregulation of mRNA was paralleled by a decrease of Cox protein in the collecting ducts and interstitial cells. Moreover, tissue prostaglandin E(2) (PGE(2)) concentrations in the papilla were markedly decreased by furosemide to about 30% of the control level. Furosemide lowered urine osmolality from 1550 mosmol/kg to 480 mosmol/kg; therefore, further consideration was given to the influence of tonicity as a possible mediator of the effects of furosemide on the Cox expression. Water loading was therefore used to reduce the medullary tonicity by a second maneuver. Water loading led to a similar reduction in papillary Cox mRNA expression and PGE(2) content like furosemide. To investigate the influence of the osmolarity on the expression of Cox and the production of PGE(2) under defined in vitro conditions, inner medullary collecting duct cells were incubated with culture medium containing graded amounts of NaCl ranging from 200 mmol/L to 600 mmol/L, and Cox-1 and Cox-2 mRNA abundance were determined after 24 h an 48 h. Cox-1 and Cox-2 mRNA abundance changed in parallel with the osmolarity. The data suggest that loop diuretics decrease the expression of cyclooxygenases and consequently tissue PGE(2) concentrations in the kidney inner medulla. This effect could be related to the breakdown of the papillary osmotic gradient induced by loop diuretics.

The influence of tonicity and viscosity on the intranasal absorption of salmon calcitonin in rabbits

As with any drug delivery system, a clear understanding of the physicochemical and formulation factors is necessary for the rational design of a dosage form. Formulation factors need to be carefully assessed to identify those which may influence pharmacological or physiological response and thus assure optimum therapeutic activity. On the basis of physicochemical and biopharmaceutical studies a nasal peptide formulation when administered with an appropriate delivery device may be designed to provide optimal nasal activity. In the present study an attempt was made to investigate the effect of tonicity and viscosity on the intranasal absorption of salmon calcitonin (sCT). Formulations were designed as nasal sprays with viscosity at 1 and 76 cps, using 0% w/w and 1% w/w methylcellulose as the viscosity enhancing agent; and with a tonicity of 100, 300 or 600 mOsms, using sodium chloride as the tonicity adjusting agent. The low viscosity formulations were delivered using a metered nasal spray pump and the high viscosity formulations were administered using a prototype device, the nasal micron spray pump (NMSP), to facilitate a uniform distribution of the spray into the nasal cavity. Serum levels of sCT were determined in healthy male New Zealand rabbits after intranasal administration of 2000 I.U. of sCT in 200 μl. The pharmacodynamic effect of salmon calcitonin of lowering blood calcium levels was measured using a visible spectrophotometric technique. Deviation from isotonicity increased the bioavailability by 4–5 times. Variation in the viscosity did not influence the bioavailability of salmon calcitonin. Response surface methodology and the canonical analysis parameters were applied to predict the optimum formulations.

Influence of tonicity and chloramphenicol on hyperthermic cytotoxicity and cell permeability under various heating rates

The cell lethality and permeability induced in Escherichia coli E/r, Escherichia coli Bs-1 and Zygosaccharornyces bailii cells by high temperature (52°C) after heating at different rates (mean s 0.015, 0.25 and 150°C per s) and in media of different tonicity and content (isotonic YEP broth versus 0.01 M phosphate buffer, pH7.0 containing different concentrations of NaCl) and with versus without chloramphenicol (10pg/ml) have been investigated. Hyperthermic treatment in YEP broth or isotonic 0.01 M phosphate buffer resulted in markedly reduced cytotoxicity with decreasing heat rate. The heating rate effect was larger when the cells were treated in YEP broth. Chloramphenicol, which is known to inhibit expression of heat shock proteins in bacteria, did not affect the viability of cells or the development of thermotolerance in cells heated at different heating rates in isotonic phosphate buffer but prevented the development of an additional degree of thermotolerance in cells heated slowly in YEP broth. In contrast, the differential effect of heating rate on cytotoxicity and cell permeability was not demonstrated when cells were heated in hypertonic solution (1 M NaCl in 0.01 M phosphate buffer, pH 7.0). It is proposed that heat destabilization of the osmotic cell homeostasis, which is more profound after rapid heating, plays a major part in heat induced cellular lethality.

Influence of pulsing and postpulsing media tonicity on electrotransformation of intact yeast cells

The maximal transformation yield of intact yeast cells in hypotonic medium was obtained by application of nine pulses with a duration of 990 μs at 2.5 kV cm−1. Pulsation at the same electrical parameters in isotonic solution did not lead to any transformation and the electropermeability decreased by 50%. The transfer of cells, 1 min after pulsation in hypotonic medium, into media with different tonicity led to an increase of the number of transformed cells, depending on the sorbitol concentration of up to 250 mM. Further augmentation of the tonicity of postpulse medium in the range 330–1000 mM provoked strong decrease of transformation. This effect was present even when cells were resuspended in isotonic medium 30 min after pulsation.

Influence of pulsing and postpulsing media tonicity on electrotransformation of intact yeast cells.

The maximal transformation yield of intact yeast cells in hypotonic medium was obtained by application of nine pulses with a duration of 990 microseconds at 2.5 kV cm-1. Pulsation at the same electrical parameters in isotonic solution did not lead to any transformation and the electropermeability decreased by 50%. The transfer of cells, 1 min after pulsation in hypotonic medium, into media with different tonicity led to an increase of the number of transformed cells, depending on the sorbitol concentration of up to 250 mM. Further augmentation of the tonicity of postpulse medium in the range 330-1000 mM provoked strong decrease of transformation. This effect was present even when cells were resuspended in isotonic medium 30 min after pulsation.

Formulation influence on conjunctival penetration of four beta blockers in the pigmented rabbit: a comparison with corneal penetration.

The objective of this study was to compare the influence of pH, tonicity, benzalkonium chloride, and EDTA on the conjunctival and corneal penetration of four beta blockers--atenolol, timolol, levobunolol, and betaxolol. Drug penetration was evaluated using the isolated pigmented rabbit conjunctiva and cornea in the modified Ussing chamber. The conjunctiva was more permeable than the cornea to all four beta blockers. Formulation changes caused larger changes in corneal than in conjunctival drug penetration, especially for the hydrophilic beta blockers, atenolol and timolol. Raising the solution pH to 8.4 caused the largest increase in corneal penetration for all drugs except atenolol. This increase was greater than that obtained by removing the corneal epithelium. The same formulation also increased conjunctival drug penetration, although to a lesser extent. In the case of timolol, the formulation changes evaluated brought about similar changes in its ocular and systemic absorption with good in vitro-in vivo correlations. The above findings indicate that in making formulation changes to maximize corneal drug penetration, it is necessary to evaluate possible changes in conjunctival drug penetration, hence systemic absorption. Moreover, because the conjunctiva plays an active role in the noncorneal route of ocular drug absorption, the relative contribution of the noncorneal to the corneal routes to ocular drug absorption may also be altered by formulation changes.

Factorial designs in the evaluation of preservative efficacy

An approach to evaluate preservative efficacy using a 23 factorial design is presented. The influence of pH and tonicity of the solution and the presence of EDTA on the antimicrobial activity of phenol was studied. C. albicans and E. coli were employed as test organisms. Statistically significant effects ( P < 0.005) of pH and the presence of EDTA on the efficacy of phenol were obtained with both organisms. The tonicity of the solution had a significant effect (if P < 0.005) only with E. coli. Significant interactions ( P < 0.005) were found between pH and tonicity of the solution with both organisms, and between tonicity of the solution and the presence of EDTA only with C. albicans. Furthermore, interactions among all 3 factors were detected with C. albicans (P < 0.005). The application of a factorial design study can be useful in the evaluation of preservative efficacy at the preformulation stage.

Control of thermoregulatory sweating during exercise in the heat.

The purposes of this study were the following: 1) to determine whether erythrocyte infusion alters the control of thermoregulatory sweating and 2) to demonstrate how increases and decreases of both plasma tonicity and blood volume influence the thermoregulatory control parameters of threshold temperature and sweating sensitivity. Six non-heat-acclimated and five heat-acclimated males attempted heat stress tests (HSTs) both before and shortly after (48-96 h) autologous erythrocyte infusion. The non-heat-acclimated subjects were euhydrated for both HSTs, whereas the heat-acclimated subjects were studied in a euhydrated and a hypohydrated (-5% body wt) condition both pre- and postinfusion (500 ml of solution containing approximately 60% hematocrit of autologous erythrocytes). The HSTs consisted of treadmill exercise (335 W.m-2) in a hot (35 degrees C, 45% relative humidity) environment, and esophageal temperature and local sweating rate were continuously measured during 25 min of exercise. These experiments resulted in a matrix of conditions where both plasma tonicity and blood volume were increased or decreased relative to control conditions (euhydration, preinfusion). The findings concerning thermoregulatory sweating during exercise in the heat were summarized as follows: 1) acute polycythemia decreases the threshold temperature and increases the sweating sensitivity, 2) both threshold temperature and sweating sensitivity are increased or decreased from control levels dependent on the combined influence of plasma tonicity and blood volume, and 3) equations are presented that describe how plasma tonicity and blood volume alter threshold temperature and sweating sensitivity values.

Influence of tonicity on the viscoelastic properties of blood during isovolemic dilution.

The influence of isotonic or hypotonic dilution on viscoelastic properties of blood is examined. The viscous, as well as the elastic, properties of blood samples diluted with isotonic saline or pure water, respectively, and of undiluted whole blood samples are compared by means of a dynamic capillary viscosimeter (OCR-D, A. Paar K.G., Austria). The dilution (approximately 17% of the total blood volume) was performed isovolemically and retained the same rbc count. The rbc swelling observed as a consequence of changes in plasma osmolarity was tracked by the high resolution density measurement, according to the mechanical oscillator technique. Since no significant rbc swelling was found in the dilution with isotonic saline, viscoelastic resistance of blood was efficiently reduced in the observed range of shear rates (2 s-1-100 s-1). This decrease is due to reduced plasma protein concentration, which also lowers plasma viscosity by approximately 18%. Although plasma viscosity is significantly decreased in hypotonic dilutions (-12%), flow properties of the rbc suspensions are, in general, significantly impaired. This is due to the osmotic rbc swelling (hematocrit = +8%), which increases viscous resistance within the suspending fluid, as well as elastic resistance of the rbc due to a loss in rbc deformability. It can be concluded that isotonic dilution leads to a decrease in the viscosity of blood, whereas hypotonic dilution--in an order of magnitude which may occur during resorption of water--leads to increased viscous and elastic resistance of the blood.

INFLUENCE OF POLYSACCARIDE CAPSULE AND IONIC STRENGTH ON BUOYANT DENSITY OF GROUP B STREPTOCOCCI

Density fluctuation during the cell cycle was investigated with group B streptococcus (GBS), type Ia, in isotonic medium. Maximum density occurred at late lag-phase, with a minimum at the middle of logarithmic growth. During the stationary phase the density remained stable. The buoyant density of serotypes Ia, Ib, II, III and a rough variant was determined by means of gradient centrifugation. In an isotonic milieu at the early stationary phase, the density of the strains varied in the range between 1.06 and 1.11 g/ml. Low ionic strength reduced the density of all strains. Neuraminidase-treatment increased the buoyant density of types Ia, Ib and III. The same pattern was seen in isotonic and hypotonic medium, although low ionic strength markedly augmented these differences. The influence of tonicity and neuraminidase-treatment was most prominent with types Ia and III, which may be due to relatively thicker polysaccaride capsules in these types.

The influence of varied tonicity of the extracellular medium on the depressant effect of active shortening in vertebrate striated muscle.

The depressant effect of active shortening was studied during isometric twitch contraction in intact single muscle fibres of the frog at varied tonicity of the extracellular medium. The shortening effect was calculated as the difference in peak redeveloped force after a small (control) and a larger (test) release step and was expressed in per cent of the isometric tetanic force. The solutions were made hypertonic by addition of sucrose (relative tonicity 1.22T and 1.44T) and hypotonic by reduction of NaCl (relative tonicity 0.81T and 0.62T). The shortening induced depression decreased from 13.0 +/- 1.2% in normal Ringer solution to 7.8 +/- 1.3% after immersion of the fibre in 1.22T solution (mean +/- SE, n = 7). This reduction of the depressant effect by shortening was less than half the size of that obtained in 1.44T solution. An increased force depression by shortening, from 12.7 +/- 1.2% to 17.1 +/- 1.5% (mean +/- SE, n = 7), was obtained when normal Ringer was replaced by 0.81T solution. This enhancement was further augmented in 0.62T solution. Experimental evidence is presented supporting the view that the influence of tonicity on the depressant effect of shortening is not due to tonicity induced changes in fibre width. The effect of varied tonicity on the shortening induced depression appears to be essentially related to alterations in intracellular ionic strength.

Influence of sarcomere length, tonicity, and external sodium concentration on conduction velocity in frog muscle fibres.

1. Using an optical technique, conduction velocity in isolated frog muscle fibres has been measured at different sarcomere lengths and in solutions of altered tonicity and Na content. 2. Conduction velocity (in m/s) in normal Ringer solution is found to be independent of sarcomere length in the range of 2-5 microns. 3. Fibre cross-section appears to become circular with stretch to sarcomere lengths exceeding 4 microns. The data on fibre diameter and length are in agreement with the assumption that constant fibre volume is maintained during passive length changes. 4. In Na-deficient solutions, conduction velocity is reduced, in agreement with predictions based on action potential parameters. 5. In solutions of half or twice the normal tonicity, the conduction velocity is proportional to the square root of the measured fibre diameter. After correcting the bias involved in estimating fibre cross-section from only one measurement of fibre diameter, the data suggest an increase in specific internal resistance (Ri) by about 8% in twice hypertonic solution and a decrease by about 5% in half normal tonicity. 6. Releasing and stretching a fibre in hypertonic solution has no effect on conduction velocity as long as the initial sarcomere length is not exceeded. Stretching the fibre beyond the sarcomere length at which it was transferred to hypertonic solution reversibly increases conduction velocity.

Influence of medium tonicity on respiration by suspensions of human platelets.

We describe the use of graded decrements of medium osmolarity to progressively unmask respiratory capacities of whole human platelets in suspension. This departure led to the first demonstration that human platelet mitochondria are capable of tightly coupled respiration that responds to addition of mitochondrial substrates, ADP, and inhibitors in a way that other mammalian mitochondria are expected to behave. In 300 mosM media added alpha-glycerophosphate (G3P), succinate, or ADP effected only slight stimulation of base-line O2 consumption. At 180 mosM O2 consumption peaked and was not significantly affected by succinate or ADP. At 80 mosM base-line O2 consumption fell precipitously and was restored by G3P or succinate prior to being raised to its highest levels by ADP. Added NADH had no effect on O2 consumption at 80 mosM but sharply stimulated it when platelet suspensions were exposed to 60 mosM media by pretreatment with distilled water. At 80 mosM, selected compounds that inhibit or uncouple oxidative phosphorylation of isolated mammalian mitochondria from a variety of cells exerted similar influences on while platelets.

Studies on the subcutaneous absorption in mice. II. Influence of tonicity on the dynamics of subcutaneous absorption.

Studies on the subcutaneous absorption in mice. II. Influence of tonicity on the dynamics of subcutaneous absorption.

Influence of ions and tonicity on RNA metabolism in Tetrahymena pyriformis.

AbstractNet RNA degradation occurs in Tetrahymem pyrifmmis when this ciliate is suspended in a non‐nutrient medium. The quantity and quality of the excretion products is at least partially under the control of the ionic content and the tonicity of the cellular environment. The excretion of ultraviolet‐absorbing materials was found to be elevated by sodium ions in a medium isotonic to the culture fluid, or by a hypertonic environment. Magnesium counteracted these effects.In isotonic suspension, sodium and magnesium ions lowered orthophosphate excretion; however, sodium altered the nature of the phosphate products so that acidlabile phosphates were also excreted rather than solely orthophosphate. Similar results were obtained in a hypertonic environment with or without sodium.The degree of purine and pyrimidine loss from the cells in all conditions of suspension was reflected in the amount of RNA degraded. The ion and tonicity effects apparently reflect events which alter the stability of the RNA and the properties of the membrane system, resulting in changes in both the rate of RNA degradation and the nature of the excreted products. The rates of orthophosphate excretion appear to be affected by changes in the acid‐base balance within the cell which may be governed by the cation levels. The manipulation of the ionic content and tonicity of the medium offers a convenient method for obtaining cells reduced in RNA content.

Influence of Tonicity on Mitotic Recovery in X-irradiated Grasshopper Neuroblasts

Influence of Tonicity on Mitotic Recovery in X-irradiated Grasshopper Neuroblasts