Tonicity Of Solution Research Articles

Osmotic response during kidney perfusion with cryoprotectant in isotonic or hypotonic vehicle solution.

Organ cryopreservation would revolutionize transplantation by overcoming the shelf-life limitations of conventional organ storage. To prepare an organ for cryopreservation, it is first perfused with cryoprotectants (CPAs). These chemicals can enable vitrification during cooling, preventing ice damage. However, CPAs can also cause toxicity and osmotic damage. It is a major challenge to find the optimal balance between protecting the cells from ice and avoiding CPA-induced damage. In this study, we examined the organ perfusion process to shed light on phenomena relevant to cryopreservation protocol design, including changes in organ size and vascular resistance. In particular, we compared perfusion of kidneys (porcine and human) with CPA in either hypotonic or isotonic vehicle solution. Our results demonstrate that CPA perfusion causes kidney mass changes consistent with the shrink-swell response observed in cells. This response was observed when the kidneys were relatively fresh, but disappeared after prolonged warm and/or cold ischemia. Perfusion with CPA in a hypotonic vehicle solution led to a significant increase in vascular resistance, suggesting reduced capillary diameter due to cell swelling. This could be reversed by switching to perfusion with CPA in isotonic vehicle solution. Hypotonic vehicle solution did not cause notable osmotic damage, as evidenced by low levels of lactate dehydrogenase (LDH) in the effluent, and it did not have a statistically significant effect on the delivery of CPA into the kidney, as assessed by computed tomography (CT). Overall, our results show that CPA vehicle solution tonicity affects organ size and vascular resistance, which may have important implications for cryopreservation protocol design.

Abiotic Factors Promote Cell Penetrating Peptide Permeability in Enterobacteriaceae Models.

Conventionally, the delivery of biomolecules into bacteria for the generation of characterized or functional mutants has relied greatly on horizontal gene transfer techniques. However, the low compatibility of these techniques with novel or hard-to-transform bacteria currently serves as a challenge to the bioengineering field. Here, we explored the use of cell penetrating peptides (CPPs) as an alternative biomolecule delivery approach by investigating the effects of the abiotic factors during CPP permeation. Using the (KFF)3K-FAM conjugate and Escherichia coli as models, we evaluated four abiotic factors where two of these factors, temperature and solution tonicity, promoted (KFF)3K-FAM permeation efficiency. Our data show that optimal (KFF)3K-FAM permeation efficiency was achieved for E. coli at approximately 98.1% under conditions of 37°C (growth optimal temperature) and 50% PBS concentration. Based on these conditions, we subsequently tested the applicability of CPP permeation in various bacterial strains by treating 10 bacterial strains from the Enterobacteriaceae family among which seven strains have no CPP permeation records with (KFF)3K-FAM. Interestingly, when compared with non-optimized conditions, all 10 strains showed a marked increase in CPP permeation ranging between 20 and 90% efficiency. Although using strains within Enterobacteriaceae that are phylogenetically close, our results hinted on the possibility that with proper optimization of the abiotic factors, CPPs could be compatible with a broad range of bacterial strains. Our efforts suggest that CPP could serve as an effective alternative approach for mutant generation and for biomolecule delivery into novel or hard-to-transform bacteria.

Effects of cryoprotectant carrier solution tonicity on transport during organ perfusion

Effects of cryoprotectant carrier solution tonicity on transport during organ perfusion

Dielectrophoretic characterization of cells in a stationary nanoliter droplet array with generated chemical gradients.

A novel design of reusable microfluidic platform that generates a stationary nanoliter droplet array (SNDA) for cell incubation and analysis, equipped with a complementary array of individually addressable electrodes for each microwell is studied. Various solute concentration gradients were generated between the wells where dielectrophoresis (DEP) was used to characterize the effect of the gradients on the cell's response. The feasibility of generating concentration gradients and observation of DEP responses was demonstrated using a gradient of salts in combination with microparticles and viable cells. L1210 Lymphoma cells were used as the model cells in these experiments. Lymphoma cells' cross-over frequency (COF) decreased with increasing stress conditions. Specifically, a linear decrease in the cell COF was measured as a function of solution tonicity and blebbistatin dose. Lymphoma cells were incubated under a gradient of the chemotherapeutic agent doxorubicin (DOX), which led to saturation in the cell-COF response at 30nM DOX, demonstrating the potential of the platform in screening of label-free drugs.

69. All that’s needed is inside the cell: New method to measure permeability of red blood cell membrane using intrinsic fluorescence

To design effective cryopreservation procedures for cells and tissues, it is critical to know permeability of cell membrane to water and solutes. To determine the latter, one needs to measure the rate of cell volume changes when placed in anisotonic environment. Red blood cells (RBCs) respond very quickly to changes in extracellular solutes concentration, which makes it difficult to measure the rate of RBC volume change using traditional methods. Stopped-flow method captures rapid changes of RBC volume and is a common method to measure RBC osmotic permeability. There are two types of stopped-flow methods: the first measures RBC volume based on changes in the intensity of light scattered by RBCs (Sidel and Solomon, 1957), the second measures it based on the self-quenching of fluorescent dye entrapped inside RBCs (Chen et al., 1988). However, both techniques have flaws. A number of factors other than cell volume influence the intensity of scattered light (Chen et al., 1988). Absorbance of a significant portion of emitted fluorescent light by the broad spectrum of hemoglobin complicates the fluorescent quenching technique. Literature shows that RBC hemoglobin has autofluorescent properties (Hirstch, 2003). This work explored the possibility of using intrinsic fluorescence of intracellular hemoglobin to measure RBC permeability to water and solutes. Network Center for Applied Development in Vancouver provided adult RBCs for this study. We used SX20 stopped-flow reaction analyser (Applied Photophysics) to measure changes in RBC autofluorescence when mixed with sodium chloride or glycerol solutions of different concentration (excitation 280 nm, emission 314 nm). Equilibrium volumes of RBCs in sodium chloride solutions were measured on Coulter Counter Model ZBI (Coulter Electronics, Inc.). Lysed RBCs served as a negative control to verify that changes in autofluorescence intensity are caused by the changes in volume of intact RBCs. We also measured absolute values of autofluorescence as a function of hemoglobin concentration in the solution using SpectraMax GEMINI EM dual-scanning microplate spectrofluorometer (Molecular devices). Our results showed that the intensity of RBC autofluorescence and RBC volume measured on Coulter Counter were directly related. Specifically, mixing RBCs with hypotonic solution caused increase in cell volume and gradual increase in autofluorescence; cell shrinking in hypertonic solution resulted in autofluorescence decrease. On the contrary, tonicity of solution had no effect on autofluorescence of lysed RBCs. When RBCs were injected in glycerol solution, autofluorescence first dropped, then gradually increased, and finally reached a plateau. This response is similar to the well-known pattern of cell volume changes upon mixing with permeable cryoprotectant. We also found that at high (intracellular) hemoglobin concentrations fluorescence intensity and hemoglobin concentration are inversely related. Combined, our data shows that RBC autofluorescence is directly translated to RBC volume, and self-quenching of intracellular hemoglobin is responsible for the cell volume driven changes in RBC autofluorescence. Based on these findings, we are working on developing a new method to determine osmotic permeability of RBCs, which will significantly help with future design and optimization of cryopreservation protocols.

Effects of solution tonicity on crossbridge properties and myosin lever arm disposition in intact frog muscle fibres

The aims of this study were to investigate the effects of solution tonicity on muscle properties, and to verify their consistence with the lever arm theory of force generation. Experiments were made in single muscle fibres and in fibre bundles from the frog, using both fast stretches and time-resolved X-ray diffraction, in isotonic Ringer solution (1T), hypertonic (1.4T) and hypotonic (0.8T) solutions. Fast stretches (0.4-0.6 ms duration and 16-25 nm per half-sarcomere (nm hs(-1)) amplitude) were applied at various tensions during the force development in isometric tetani. Force increased during the stretch up to a peak (critical tension, Pc) at which it started to fall, in spite of continued stretching. In all solutions, Pc was proportional to the initial isometric tension developed. For a given isometric tension, Pc increased with solution tonicity and occurred at a precise sarcomere elongation (critical length, Lc) which also increased with tonicity. M3 meridional layer line intensity (I M3) was measured during the application of sinusoidal length oscillations (1 kHz frequency, and about 2% fibre length amplitude) at tetanus plateau. I M3 changed during the length oscillations in a sinusoidal manner in phase opposition to length changes, but a double peak distortion occurred at the peak of the release phase. The presence of the distortion, which decreased with tonicity, allowed calculation of the mean position of the myosin head (S1) during the oscillation cycle. In agreement with the lever arm theory, both X-ray diffraction and mechanical data show that solution tonicity affects S1 mean position and consequently crossbridge individual extension and force, with no effect on crossbridge number. The force needed to break the single crossbridge was insensitive to solution tonicity suggesting a non-ionic nature of the actomyosin bond.

Hypertonicity of the challenge solution may increase the diagnostic accuracy of histamine challenge

There is significant overlap in the responsiveness to direct airway challenges, such as the histamine challenge, between asthmatic and non-asthmatic subjects, which decreases their accuracy in the diagnosis of asthma. To minimise this overlap, a new test, hypertonic histamine challenge, was developed. Fifteen healthy subjects, 16 subjects with steroid-naive asthma, and 16 asthmatic subjects undergoing inhaled corticosteroid treatment underwent inhalation challenges with hypertonic saline, isotonic histamine, and hypertonic histamine, using an ultrasonic nebuliser and 2-min tidal breathing method. The increase in histamine solution tonicity decreased the histamine PC20 values only in the steroid-naive asthmatic subjects (1.1 (0.5-2.7) vs. 0.5 (0.2-1.2) mg/ml, P = 0.047). Using 1mg/ml as the cut-off value, the sensitivity, specificity, and accuracy of the hypertonic histamine challenge to detect steroid-naive asthma was 81%, 100%, and 90%. The respective values for the isotonic histamine challenge were 56%, 100%, and 77%. Furthermore, there was a statistically significant difference in the hypertonic histamine PC20 between steroid-naive and steroid-treated asthmatic subjects, which could not be detected in the isotonic histamine PC20. The hypertonic histamine PC20 was highly repeatable, with a single determination 95% range of +/-1.35 doubling concentrations. The hypertonic histamine challenge was safe but provoked more cough and throat irritation than the other two challenges. In conclusion, compared with a conventional, isotonic histamine challenge, hypertonic histamine challenge may be more accurate in the diagnosis of asthma and also, more capable to detect the effects of inhaled corticosteroid treatment.

Pilocarpine permeability across ocular tissues and cell cultures: influence of formulation parameters.

In vitro permeation studies of drugs across biological barriers are promising tools for estimating the quality and quantity of drug transport in vivo. The objective of this work was to compare the permeability of the hydrophilic model drug pilocarpine-HCl (P-HCl) through different ocular tissues and cell cultures: isolated pig cornea (PCr) and sclera (PSc), rabbit conjunctiva (RCo), and rabbit conjunctival (RCoEC) or corneal epithelial cell culture (RCrEC). Furthermore, the study included investigations about the influence of the excipients benzalkonium chloride (BAC) and ethylene diamine tetra acetic acid disodium salt (EDTA) on the permeability of the small drug. In general, BAC caused a facilitated drug transport, while EDTA hardly influenced the P-HCl concentration on the acceptor side, except for RCoEC. Additionally, the impact of variation in buffer solution pH and tonicity on drug transport in both cell cultures was tested. The higher the tonicity of the buffer solution (80, 300, and 600 mOsm/kg) the lower the permeability coefficient (P(eff)). At different pH values (6.4, 7.4, and 8.4) the P(eff) showed a directly proportional demeanor. In summary, a good correlation between the isolated tissues and cell cultures with regard to P-HCl transport could be observed.

Influence of increase in osmotic pressure with sucrose on relaxation and cyclonucleotides levels in isolated rat aorta

The influence of increases in osmolarity by addition of sucrose were investigated on relaxation and changes in adenosine 3′:5′-cyclic monophosphate (cyclic AMP) and guanosine 3′:5′-cyclic monophosphate (cyclic GMP) levels in isolated rat aortic rings. Isoprenaline-mediated relaxations were attenuated in hypertonic (341±0.4 mOsmol) (mean±S.E.M.) solution. The concentration–response curve to isoprenaline was displaced to the right. The EC 50 (0.16±0.05 vs. 1.14±0.5 μM) significantly ( n=6; P<0.05) increased without any changes to the maximum response. Hypertonic solution also attenuated methacholine-mediated relaxations resulting in a significant increase in the EC 50 (0.28±0.04 vs. 0.52±0.04 μM) and reduced the maximal response (73±5% vs. 51±8%). In contrast, an increase in tonicity did not have any influence on sodium nitroprusside, forskolin or pinacidil concentration–response curves. Hypertonic solution also did not affect either basal cyclic AMP or cyclic GMP production. In addition, an increase in osmolarity did not affect isoprenaline-stimulated increases in the levels of cyclic AMP. However, an increase in the tonicity of Krebs solution significantly inhibited methacholine-stimulated (58%–34%) accumulation of cyclic GMP. The present data indicated that an increase in the tonicity of Krebs solution impaired endothelium-dependent relaxation and the associated increase in cyclic GMP production without affecting basal levels of this nucleotide. The inhibitory effects of high osmolarity on β-adrenoceptor-mediated relaxation did not appear to be due to a reduction in cyclic AMP generation, or the result of inhibition of pinacidil-sensitive K ATP + channels. Moreover, an increase in the tonicity of Krebs solution did not influence relaxation induced by direct activation of adenylate cyclase or guanylate cyclase by forskolin and sodium nitroprusside, respectively.

Mechanism of hemolysis of human erythrocytes exposed to monosodium urate monohydrate crystals. Preliminary characterization of membrane pores

Microcrystals of monosodium urate monohydrate (MSUM) have the ability to cause rapid hemolysis of erythrocytes. The nature of the initial MSUM crystal-erythrocyte membrane binding interaction was investigated over a range of different ionic strength media. There was negligible binding of MSUM to erythrocyte ghost membranes in low ionic strength media such as isotonic mannitol but binding was dramatically increased in isotonic NaCl/mannitol solutions or isotonic mannitol containing 1 mM Ca 2+. Hemolysis induced by MSUM crystals was preceded by the leakage of K + from the cells suggesting a colloid-osmotic mechanism of hemolysis. The inclusion of large (oligosaccharide) molecules in the extracellular media or the modulation of the extracellular solution tonicity inhibited both the rate and extent of hemolysis supporting the concept of MSUM-induced pores followed by colloid osmotic hemolysis.

The Effect Of Solution Tonicity On The Eye

A randomised, double-blind study was performed to determine the effect of exposing the anterior eye to solutions of varying tonicities. Discomfort, conjunctival hyperaemia and corneal epithelial integrity were recorded on an analog scale for eight subjects after five-second exposures to nine different sodium chloride solutions, ranging in concentration from 0.0 to 7.0 per cent. Both hypotonic and hypertonic solutions produced discomfort, and the range of 0.9 to 2.0 per cent was the most comfortable. Interpolation of a curve fit to the data indicated that 1.3 per cent NaCl solution produced the least discomfort. Hyperaemia was induced at concentrations above 2.0 per cent NaCl but did not occur with hypotonic solutions. No cases of corneal staining with fluorescein were recorded, suggesting that, at least with respect to variation in solution tonicity, both discomfort and conjunctival hyperaemia will be present before damage to the corneal tissue. (Clin Exp Optom 1993; 76: 1: 17–21)

Formulation Influence on Ocular and Systemic Absorption of Topically Applied Atenolol in the Pigmented Rabbit

The objective of this study was to determine the relative efficacy of various formulations in maximizing the ratio of ocular to systemic absorption of topically applied atenolol following solution instillation in the pigmented rabbit. Formulations of various pH's and tonicities and containing various preservatives and polymers were tested. Ocular absorption was determined by monitoring atenolol concentrations in various anterior segment tissues at 30 min following solution instillation, while systemic absorption was determined by monitoring the time course of atenolol concentration over 480 min. Reversed phase HPLC was the analytical methodology. All formulations except those containing 0.025% benzalkonium chloride or 0.5% EDTA showed similar drug concentration vs. time profiles in plasma, attaining a peak concentration of 30-50 ng/ml at about 100 min. For benzalkonium chloride and EDTA, there was an undesirable increase in systemic absorption, although ocular absorption was also increased. By contrast, lowering the solution tonicity to 80 mOsm/kg increased the ratio of aqueous humor to plasma peak concentrations 2 times and the ratio of iris-ciliary body to plasma peak concentrations 3 times. Incorporation of 3.75% poly(vinyl alcohol) into the formulation afforded yet a larger increase in the iris-ciliary body to plasma drug concentration ratio (52 times.) It may therefore be concluded that, for a hydrophilic drug like atenolol, formulation changes that increase membrane permeability and/or enhance noncorneal drug access may be more promising than those that increase drug residence in the conjunctival sac with respect to maximizing the ratio of ocular to systemic drug absorption.

Improving the safety of topically applied timolol in the pigmented rabbit through manipulation of formulation composition

The objective of this study was to compare the efficiency of various formulations in maximizing the ratio of ocular to systemic absorption of topically applied timolol in the pigmented rabbit. Formulations of various pHs, tonicities, and concentrations of benzalkonium chloride, EDTA, poly(vinyl alcohol), poly(vinylpyrrolidone), hydroxypropylcellulose, and Na hyaluronate were tested. Ocular absorption was determined by monitoring the timolol concentration in various anterior segment tissues 30 min after instillation of timolol solution, while systemic absorption was determined by monitoring the time course of timolol concentration over 120 min. Timolol was assayed by reversed-phase HPLC. Lowering the solution tonicity to 80 mosmol kg −1 and incorporating polymers into the formulation were the only approaches that promised to improve the safety of topically applied timolol, since they afforded the desired increase in ocular absorption and reduction in systemic absorption. Lowering the solution pH to 6·4 and increasing the tonicity of the solution to 600 mosmol kg −1 reduced systemic absorption but caused either no change or a decrease in ocular absorption. Raising the solution pH to 8·4 and incorporating 0·025% benzalkonium chloride and 0·5% EDTA into the formation led to an undesirable increase in systemic absorption although ocular absorption was also increased. In the final analysis, the net effect of formulation changes on the ratio of ocular to systemic absorption depended on the interplay of changes in solution drainage; permeability of the cornea, conjunctiva, and nasal mucosa; and fraction of drug in the preferentially absorbed form.

Na+ channel activity in cultured renal (A6) epithelium: regulation by solution osmolarity.

Solution osmolarity is known to affect Na+ transport rates across tight epithelia but this variable has been relatively ignored in studies of cultured renal epithelia. Using electrophysiological methods to study A6 epithelial monolayers, we observed a marked effect of solution tonicity on amiloride-sensitive Na+ currents (I(sc)). I(sc) for tissues bathed in symmetrical hyposmotic (170 mOsm), isosmotic (200 mOsm), and hyperosmotic (230 or 290 mOsm) NaCl Ringer's solutions averaged 25 +/- 2, 9 +/- 2, 3 +/- 0.4, and 0.6 +/- 0.5 microA/cm2, respectively. Similar results were obtained following changes in the serosal tonicity: mucosal changes did not significantly affect I(sc). The changes in I(sc) were slow and reached steady-state within 30 min. Current fluctuation analysis measurements indicated that single-channel currents and Na+ channel blocker kinetics were similar for isosmotic and hyposmotic conditions. However, the number of conducting Na+ channels was approximately threefold higher for tissues bathed in hyposmotic solutions. No channel activity was detected during hyperosmotic conditions. The results suggest that Na+ channels in A6 epithelia are highly sensitive to relatively small changes in serosal solution tonicity. Consequently, osmotic effects may partly account for the large variability in Na+ transport rates for A6 epithelia reported in the literature.

Performance characteristics of the DeVilbiss Ultraneb 99 ultrasonic nebuliser with reference to use in sputum induction

The DeVilbiss Ultraneb 99 ultrasonic nebuliser is frequently used for sputum induction in AIDS patients undergoing investigation of suspected Pneumocystis carinii pneumonia. We set out to characterise this machine under a wide range of operating conditions so that the efficiency of the technique might be optimised. The range and frequency of particle sizes remained reasonably constant and gave a Gaussian distribution pattern (mean MMD = 5.05 μm, SD = 0.34 μm) below a critical volume of nebulised solution, this volume being related to the intensity setting of the nebuliser. Beyond this volume, the particle size distribution adopted a bimodal pattern, and nebuliser output subsequently tailed off altogether. The volume of couplant within the nebuliser chamber was critical for efficient nebuliser output. Changes in surface tension and tonicity of solution to be nebulised did not affect the performance of the nebuliser substantially. Successful sputum induction may rely on both proximal and distal airway deposition, which may be enhanced by changes in both the nebuliser and nebulised solution.

Light diffraction intensity from muscle fibres in different osmotic solutions: measurement of equilibration time.

A new method for evaluating the osmotic equilibration time of striated muscle fibres is described. Single fibres from the semitendinosus muscles of frog were illuminated normally with a He-Ne laser. The first-order diffraction intensity was measured continuously after the tonicity of the fibre's bathing solution was changed abruptly. Hypertonicity decreased while hypotonicity increased the intensity. The time course of the intensity decrease or increase followed closely a simple exponential with a single time constant. It is proposed that the time constant associated with the diffraction intensity transient is a true representation of the equilibration process. A simple diffraction model provides a relationship between diffraction intensity and the diameter of the muscle fibre. This relationship is used to explain the diffraction intensity of fibres in different osmotic solutions.

Factors affecting victorin-induced root cap cell death: temperature and plasmolysist

Victorin, the pathotoxin produced by Helminthosporium victoriae, causes false plasmolysis and death of root cap cells, and this effect is selective for oat cultivars susceptible to the fungus. The development of a procedure for the isolation of root cap cells made possible a study of victorin's effects on populations of intact cells uniformly exposed to the toxin. Isolated root cap cells from resistant and susceptible cultivars were used to examine the effects of temperature and plasmolysis on victorin-induced cell death. Cell viability was determined by the ability to stain with fluorescein diacetate. Victorin-induced cell death was highly temperature dependent with a temperature coefficient of 3·5. At 10°C 96 to 98% of the cells remained alive for over 30 days when incubated in up to 50 units of toxin per ml. Plasmolysis also inhibited cell death; this protective effect was dependent upon solution tonicity. Non-permeating media, mannitol and sorbitol, protected more than sucrose in which cells slowly deplasmolyzed. Urea, a rapidly-permeating osmoticum, did not delay victorin-induced cell death. The modification of victorin's lethal effects by temperature and plasmolysis may explain variability in reported effects of the toxin on isolated protoplasts.

The Measurement of Lymphocyte Volume: Importance of Reference Particle Deformability and Counting Solution Tonicity

AbstractWe have determined the influence of reference particle deformability and suspending buffer tonicity on the measurement of lymphocyte volume by an electronic particle volume analyzer. When the volume analyzer was standardized with latex spherules having a shape factor (fe) of 1.5, red cell volume was 96 cu µm and lymphocyte volume was 289 cu µm. The red cell volume corresponded closely to the true red cell volume; the true lymphocyte volume, however, was 218 cu µm when measured by the lymphocytocrit/lymphocyte count and 203 cu µm by wet lymphocyte weight and density (mean ~ 210 cu µm). The difference between the electronic volume (Ve) of 289 cu µm and true lymphocyte volume of 210 cu µm was due to the influence of lymphocyte deformability (shape factor) as it traverses the sizing aperture. Since the true volume equals the Ve/Ve, the red cells with a shape factor near 1.0 were sized appropriately by this method. In contrast, the lymphocyte shape factor was 1.38; thus, the true lymphocyte volume was 289 cu µm/1.38 or 210 cu µm. The tonicity of the suspending solution also influenced the measurement of particle volume when osmotically inactive standard particles (e.g., latex spherules) were used as a reference. Whereas the true lymphocyte volume was 210 cu µm at 286 mosmole/liter, it was 194 cu µm at 330 and 229 cu µm at 250 mosmole/liter. The standard counting solution, Isoton, is hyperosmolar (330 mosmole/liter) and causes an 8% shrinkage of osmotically active cells.

The measurement of lymphocyte volume: importance of reference particle deformability and counting solution tonicity

We have determined the influence of reference particle deformability and suspending buffer tonicity on the measurement of lymphocyte volume by an electronic particle volume analyzer. When the volume analyzer was standardized with latex spherules having a shape factor (fe) of 1.5, red cell volume was 96 cu micron and lymphocyte volume was 289 cu micron. The red cell volume corresponded closely to the true red cell volume; the true lymphocyte volume, however, was 218 cu micron when measured by the lymphocytocrit/lymphocyte count and 203 cu micron by wet lymphocyte weight and density (mean approximately 210 cu micron). The difference between the electronic volume (Ve) of 289 cu micron and true lymphocyte volume of 210 cu micron was due to the influence of lymphocyte deformability (shape factor) as it traverses the sizing aperture. Since the true volume equals the Ve/fe, the red cells with a shape factor near 1.0 were sized appropriately by this method. In contrast, the lymphocyte shape factor was 1.38; thus, the true lymphocyte volume was 289 cu micron/1.38 or 210 cu micron. The tonicity of the suspending solution also influenced the measurement of particle volume when osmotically inactive standard particles (e.g., latex spherules) were used as a reference. Whereas the true lymphocyte volume was 210 cu micron at 286 mosmole/liter, it was 194 cu micron at 330 and 229 cu micron at 250 mosmole/liter. The standard counting solution, Isoton, is hyperosmolar (330 mosmole/liter) and causes an 8% shrinkage of osmotically active cells.

Bathing Solution Tonicity and Potassium Transport by the Midgut of the Tobacco Hornworm Manduca Sexta

ABSTRACT Potassium transport by the isolated midgut of Manduca larvae, as measured by the short circuit current, is inhibited by substitution of small organic solutes (M.W. &amp;lt; 340) for the sucrose normally included in bathing solution formulated for this tissue. Other solutes of molecular weight equal to or greater than sucrose are essentially as effective as sucrose in promoting the short circuit current. Equilibration of midgut in solutions containing the small solute mannitol results in a decrease in the dry weight/wet weight ratio of the tissue, suggesting that the small solutes can penetrate into areas of the tissue which are not accessible to sucrose. Histological studies suggest that sites of swelling in the presence of mannitol include both cytoplasm and goblet cell lumen. The inhibition of the short circuit current is rapidly reversible on return to bathing solution containing sucrose or another large solute. The effect of small solutes probably does not involve compromise of the energy source for potassium transport since oxygen uptake is unchanged in the presence of a small solute.