Guard cell volume and pressure measured concurrently by confocal microscopy and the cell pressure probe

Guard cell turgor pressures in epidermal peels of broad bean (Vicia faba) were measured and controlled with a pressure probe. At the same time, images of the guard cell were acquired using confocal microscopy. To obtain a clear image of guard cell volume, a fluorescent dye that labels the plasma membrane was added to the solution bathing the epidermal peel. At each pressure, 17 to 20 optical sections (each 2 microm thick) were acquired. Out-of-focus light in these images was removed using blind deconvolution, and volume was estimated using direct linear integration. As pressure was increased from as low as 0.3 MPa to as high as 5.0 MPa, guard cell volume increased in a saturating fashion. The elastic modulus was calculated from these data and was found to range from approximately 2 to 40 MPa. The data allow inference of guard cell osmotic content from stomatal aperture and facilitate accurate mechanistic modeling of epidermal water relations and stomatal functioning.     

Cell volume regulation in mouse TA3 ascites tumor cells by exogenous ATP as measured by the Coulter Counter

Exogenous ATP can induce a marked cell enlargement in TA3 tumor cells which can be reversed or prevented by Ca and Mg. This regulatory effect on cell volume is specific for ATP. The mechanism probably involves changes in cell ionic content.     

Fed-batch culture automated by uses of continuously measured cell concentration and culture volume

An automated control method of fed-batch culture in which the nutrient feed rate was determined from continuously measured cell concentration and culture broth volume was developed. Theoretical background was elucidated, from which it was found that the method is unique in that it controls specific substrate consumption rate of the microorganism. The method was experimentally applied to the fed-batch cultures of recombinant Escherichia coli HB101. It was observed that the specific substrate feed rate affects not only the specific growth rate but also the growth yield. If some conditions are satisfied, this type of automated fedbatch culture can be applied widely to any microbial systems and seems especially useful when the culture medium is composed of natural complex nutrient(s) because their concentrations are very difficult to detect and control.     

Cell swelling impairs dye coupling in adult rat ventricular myocytes. Cell volume as a regulator of cell communication

The influence of cell swelling on cell communication was investigated in cardiomyocytes isolated from the ventricle of adult rats. Measurements of dye coupling were performed in cell pairs using intracellular dialysis of Lucifer Yellow CH. The pipette was attached to one cell of the pair and after a gig ohm seal was achieved, the membrane was ruptured by a brief suction allowing the dye to diffuse from the pipette into the cell. Fluorescence of the dye in the injected as well as in non-dialyzed cell of the pair was continuously monitored. The results indicate that in cell pairs exposed to hypotonic solution the cell volume was increased by about 60% within 35 min and the dye coupling was significantly reduced by cell swelling. Calculation of gap junction permeability (P _j) assuming an the intracellular volume accessible to intracellular diffusion of the dye as 12% of total cell volume, showed an average P _j value of 0.16 ± 0.04 × 10^?4 cm/s (n = 35) in the control and 0.89 ± 1.1 × 10^?5 cm (n = 40) for cells exposed to hypotonic solution (P < 0.05). Similar results were found assuming intracellular volumes accessible to the dye of 20 and 30% of total cell volume, respectively. Cell swelling did not change the rate of intracellular diffusion of the dye. The results which indicate that cell volume is an important regulator of gap junction permeability, have important implications to myocardial ischemia and heart failure as well as to heart pharmacology because changes in cell volume caused by drugs and transmitters can impair cell communication with consequent generation of slow conduction and cardiac arrhythmias.     

Na-pump activity in rat kidney cortex cells and its relationship with the cell volume

The present work was undertaken to evaluate whether changes in cell water content of rat kidney cortex cells can modulate the transport activity of the ouabain-insensitive Na pump as they modulate the ouabain-insensitive Na+-ATPase. It was found that there is a close relationship between the cell volume and activity of the Na pump, whereas Na,K-pump activity is not affected by variations in cell volume. When the cell water content is low, Na-pump activity (Na+ transport and Na+-ATPase activity) is minimal. Increases in cell water content produce a concomitant increase in Na-pump activity.     

Heterogeneity of endothelial cells of adult rat liver as resolved by sedimentation velocity and flow cytometry

Sinusoidal cells isolated from adult rat liver were fractionated by velocity sedimentation at 1 X g ( primarily on the basis of size) and the various cell fractions were further analysed by flow cytometry on the basis of forward and perpendicular light scattering and autofluorescence. Cell volume was also measured electronically using a Coulter counter. At least four enriched cell populations were resolved after velocity sedimentation. They corresponded to cells having a modal diameter of 6.5, 7.5, 9, and 11 microns, respectively. Transmission electron microscopy (TEM) analysis of the various cell populations revealed that the 7.5- and 9-microns cell fractions represented two distinct classes of endothelial cells while the 11-microns cells corresponded to Kupffer cells. The 6.5-microns cells were identified as lymphocytes. Fat-storing cells, identified by their autofluorescence and lipid content, were included in the Kupffer population. Further information about the nature of the two physically distinct endothelial cell populations was obtained by TEM. It demonstrated that the smaller endothelial cells possessed quantitatively and relatively less retracted sieve plates than the larger ones. This ultrastructural feature can be possibly correlated to a differential localization of the two classes of endothelial cells within the liver acinus.     

Blood volume measurement in baboons: simultaneous estimation of red cell volume and plasma volume

A method is described for frequent sequential blood volume estimation in baboons using 32P for red cell volume measurements and 125I-albumin for simultaneous plasma volume measurements. Values for red cell, plasma, and total blood volumes are reported. Close correlations of the volumes to bodyweight were demonstrated. Circulatory half-lives of the isotopes, determined from disappearance curves, confirmed their suitability for serial measurements in these baboons.     

Post-ejaculatory changes in the metabolic status of rat spermatozoa as measured by GC-MS

Analysis of low molecular weight or metabolic compounds can provide deeper insights into the biochemical pathways regulating normal cell function. Here we report, for the first time, a method to extract a large number of metabolites from rat spermatozoa, which were analysed using gas chromatography mass spectrometry. Based on the retention time index and at least 3–5 fragment qualifier ions, we positively identified 71 compounds, 2 of which we could not find any match to the database. Several different classes of metabolic compounds including amino acids, sugars, fatty acids, sterols and lipids were found. In order to gain insight into sperm function, we extracted metabolites from sperm cells that were in the initial stages of the post-testicular sperm maturation process known as capacitation, and compared the relative intensity of each compound to non-capacitated spermatozoa through the use of an internal standard. We could clearly demonstrate significant down regulation of cholesterol, a hallmark of capacitating cells, being less abundant in the more mature cells. In addition, several monosaccharides including glucose, fructose, sorbitol, galactose and the polyol myo-Inositol decreased in their abundance as sperm begin to capacitate. Interestingly, galactose was able to support sperm motility and an increase in the level of tyrosine phosphorylation, however this came at the expense of longevity of these cells when compared to glucose.     

Glucose and octanoate utilization by isolated adult rat heart cells

Rat heart muscle cells continue to beat in the isolated state apparently independent of any innervation. The oxidation of ¹⁴C-glucose to ¹⁴CO₂ was linear for at least 60 minutes of incubation. The rate of glucose oxidation rose rapidly up to a medium glucose concentration of 2.5 mM and then plateaued. Lactate production reached a maximum at 5 mM glucose. Glucose uptake was linearly related to the concentration up to 40 mM. The addition of octanoate reduced, but did not eliminate, glucose oxidation. Octanoate utilization increased with increasing concentration and reached a maximum at 2 mM. The oxidation of octanoate was linearly related to the time of incubation for at least 90 minutes. The presence of glucose, at a concentration of 1.25 mM or higher, increase the oxidation of octanoate by the heart cells. The metabolic parameters measured with the isolated heart cells gave values comparable to those obtained with the perfused rat heart. Decreasing or increasing the concentration of sodium, potassium or magnesium did not effect the oxidation of either glucose or octanoate with the exception that when sodium was increased above 200 mM, a significant increase in glucose oxidation was observed. In contrast, the addition of calcium to a calcium free medium increased glucose oxidation, reaching a maximum at 0.2 mM calcium. The oxidation of octanoate reached a maximum at 0.2 mM and then decreased significantly with increasing calcium concentration. The metabolic activity appears to be independent of the concentration of sodium, potassium or magnesium. In contrast, the isolated heart cell is very sensitive to a change in calcium concentration.