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C P Lechene

Publications and source records attributed to C P Lechene.

At least 19 recordsLinked to original sources
PubMed1990

pH regulation in spread cells and round cells.

The aim of this work was to characterize the changes in pH regulation that lead to increased intracellular pH (pHi) in well-spread cells on tissue culture plastic relative to cells on a nonadhesive surface. Bicarbonate was not required for maintenance of a control steady state pHi or of the difference in pHi between round and spread cells. In the absence of bicarbonate, lowering the sodium content of the medium led to decreased pHi and elimination of the difference between round and spread cells. In the presence or absence of bicarbonate, adding ethylisopropyl amiloride lowered pHi and eliminated the difference between round and spread cells. Measurements of recovery from acute acidification in the absence of bicarbonate confirmed that Na+/H+ exchange was enhanced in spread cells. However, recovery from both acidification and alkalinization in the presence of bicarbonate showed that bicarbonate-dependent recovery in both directions, most likely due to sodium-dependent and -independent HCO3-/Cl- exchangers, was also stimulated in spread cells. We conclude that Na+/H+ exchange has a primary role in determining steady state pHi in 3T3 cells in serum and is responsible for the lower pHi in round cells. Bicarbonate-dependent pH regulatory mechanisms are also inhibited in round cells.

Amiloride↗
PubMed1990

Cytoplasmic pH and anchorage-independent growth induced by v-Ki-ras, v-src or polyoma middle T.

We had previously shown that spreading of normal cells on tissue culture plastic coated with extracellular matrix (ECM) proteins led to an increase in cytoplasmic pH (pHi). Since alkalinization of the cytoplasm is associated with activation of a number of signaling pathways that control growth, and is itself required for cell growth, we proposed that this phenomenon could explain, at least in part, why growth of normal cells is anchorage-dependent. Preliminary results showed that pHi in cells transformed by the ras or src oncogenes had an alkaline pHi even when completely round. To further explore the relationship between pHi and anchorage-independent growth, a series of cells transformed by mutants of the polyoma middle T oncogene, and a series of ras-transformed cells and revertants were examined. Growth in methyl cellulose was assayed, and pHi in both maximally spread and completely round cells was measured for each cell line. We found that all of the normal cells required spreading to maintain an alkaline pHi, whereas transformed cell lines had an alkaline pHi independent of spreading. There was a strong correlation between pHi in round cells and anchorage-independent growth. Thus, some plasma membrane oncogenes can substitute for cell spreading on EMC to raise pHi as well as to promote growth. These results may be relevant to understanding why transformation leads to changes not only in cellular requirements for growth factors, but also for anchorage.

Antigens, Polyomavirus Transforming↗
PubMed1989

Development of a noninvasive ultramicrofluorometric method for measuring net uptake of glutamine by single preimplantation mouse embryos.

A noninvasive ultramicrofluorometric method for measuring net uptake of glutamine by single preimplantation mouse embryos is described. A linear relationship was found between fluorescence intensity of NADH produced and glutamine concentration (R2 = 0.985). Single embryos were placed in 20 nl drops of medium containing 0.5 mM glutamine, and the medium was sampled after 2 hr incubation at 37 degrees C. Changes in net glutamine uptake were determined in one-cell, two-cell, and eight-cell embryos and blastocysts incubated in medium containing no energy substrates (glucose, pyruvate, and lactate). The median glutamine uptake increased significantly from 0.480 and 0.270 pmoles/embryo/2 hr at the one-cell and two-cell stages, respectively, to 1.610 pmoles/embryo/2 hr at the blastocyst stage. Mean glutamine uptake was compared in the presence or absence of energy substrates at several developmental stages. A highly significant reduction of glutamine uptake in the presence of substrates was observed at the one-cell and two-cell stages of development. At the eight-cell stage, glutamine uptake was only marginally reduced in the presence of substrates, and no effect was found at the blastocyst stage. These data may partially explain the beneficial effect of glutamine on the culture of early mouse embryos through the two-cell block of development.

Animals↗
PubMed1989

Use of ultramicrofluorometric methods for the study of single preimplantation embryos.

Ultramicrofluorescence and video fluorescence microscopy are highly sensitive techniques for studying embryo metabolism and developmental potential. These techniques are attractive in that they allow one to study single embryos and thus to detect more subtle changes in embryo metabolism or intracellular pH which, using less sensitive techniques or batches of embryos, would likely go undetected. This is especially important in those species where few embryos can be recovered from a single mother. An additional advantage of ultramicrofluorescence techniques lies in their lack of invasiveness to the embryo under study. When measuring nutrient uptake, several measurements can be made on a single embryo that can then be placed back into culture for evaluation of its developmental potential. Alternatively, test embryos can be transferred back into a recipient for determination of embryo viability. This flexibility further illustrates the beneficial aspects of using ultramicrofluorescence techniques for studying embryo metabolism and development.

Animals↗
PubMed1979

Phosphate transport along the inner medullary collecting duct of the rat.

The question of phosphate transport along the collecting duct remains controversial inasmuch as no data from direct in vivo evaluation of this nephron segment have been reported. We measured net phosphate transport along the inner medullary collecting duct (IMCD) using the collecting duct microcatheterization technique in five groups of rats. In control rats no net phosphate transport was found and 9.4% of the fraction of filtered phosphate (FFP) entered the IMCD and was excreted. After acute thyroparathyroidectomy (TPTX) there was a striking reduction in the FFP entering the IMCD, 1.8%, and significant reabsorption occurred, 0.5% being excreted. With acute TPTX and parathormone infusion, delivery increased to 33% without significant change along the IMCD. With acute TPTX and phosphate infusion, delivery was increased to control levels but no change was found in net phosphate transport. In rats studied 5-7 days after uninephrectomy alone, phosphate delivery was greater than in control, 25%, and no net phosphate transport was found. These studies demonstrate that phosphate absorption occurs along the IMCD in acutely TPTX rats when the delivery of phosphate to the IMCD is markedly reduced. The increase in phosphaturia which occurs after a reduction in renal mass cannot be accounted for by changes in net phosphate transport along the IMCD.

Animals↗
PubMed1979

Elemental composition of rabbit antral fluid during preovulatory follicular swelling.

Electron probe microanalysis was used to determine the concentrations of Na, Cl, K Ca, Mg, S and P in samples of follicular fluid, ovarian vein serum and peripheral venous serum obtained from virgin rabbits at 2-h intervals up to 10 h after injection of hCG. Throughout this 10-h period the elemental composition of follicular fluid was essentially the same as that of blood serum. However, there was a significant drop in follicular fluid Ca relative to blood during the 10-h period which may reflect Ca involvement in the regulation of oocyte maturation. Significant differences were also found between follicles within rabbits for K and P concentrations.

Animals↗
PubMed1978

Ouabain-sensitive fluid accumulation and ion transport by rabbit blastocysts.

1. By incubating 6-day post-coitum (p.c.) blastocysts in medium supplemented with either 20 mM-sucrose or 10 mM-KCl it was possible to show that the trophectoderm of the blastocyst prevents the concentration of K in the blastocoele fluid rising to external concentrations. 2. The concentrations of K in the blastocoele fluid are maintained predominantly by leakage of K from the trophoblast cells into the blastocoele and by ouabain-sensitive transport of K into the trophoblast cells from the blastocoele fluid. 3. Exposure of blastocysts to ouabain on the juxtacoelic, but not abcoelic, surface of the trophectoderm may inhibit blastocoele fluid accumulation.

Animals↗
PubMed1977

Electron probe microanalysis of the chemical elemental content of human follicular fluid.

Follicular fluid samples were obtained by puncturing follicles of ovaries in situ from patients undergoing laparotomy. Sodium, potassium, chloride, magnesium, calcium, phosphorus, and sulfur concentrations measured by electron probe microanalysis were similar to those of blood, with minimal differences. This suggested that culture media in which these electrolytes are added in concentrations similar to those of serum are appropriate for culture of the human oocyte.

Calcium↗
PubMed1977

Electron probe microanalysis of chemical elemental content of single human red cells.

Sodium, potassium, iron and sulfur contents of single human red cells were measured using electron microprobe microanalysis. Three preparative procedures were compared, and the most reliable technique was found to be spraying of cells onto polished pyrolytic graphite by atomization. Primary standards were prepared by adjusting the intracellular electrolyte content of red cells, eliminating the need to correct for X-ray absorption. Samples were stable under the electron beam during analysis, and could be stored for long periods of time. Strong correlations were found between the X-ray intensities of iron and sulfur and between potassium and sodium. X-ray intensities of potassium and sodium were found to be directly proportional to internal ionic content. Large populations of single cells could be analyzed and the distribution of their elemental content studied.

Electron Probe Microanalysis↗
PubMed1977

Absence of significant secretory flux of phosphate in the proximal convoluted tubule.

The presence of phosphate secretion by the renal tubule in mammals has been controversial. Recently, in a micropuncture and microperfusion study, net secretion of phosphate was reported for the proximal tubule of rats which were infused with parathyroid hormone (PTH) and phosphate. Since the finding of net secretion of phosphate by the proximal tubule is in contradiction to other reports, the question was reinvestigated with the use of microperfusion techniques that were modified to allow identification of sample contamination. Studies were performed in intact dogs, phosphate- and PTH-loaded intact rats, and phosphate-loaded, acutely thyroparathyroidectomized rats. After exclusion of contaminated samples, no significant influx of phosphate was found in any of the three groups. Neither increased plasma phosphate concentration nor the infusion of parathyroid hormone unmasked a secretory process for phosphate. It is concluded that phosphate transport in the proximal tubule is essentially a unidirectional phenomenon, i.e., reabsorption is not opposed by significant backflux of phosphate.

Animals↗
PubMed1977

Electron probe microanalysis: its present, its future.

Electron probe microanalysis (EPM) is a method of elemental ultramicroanalysis based on the spectrometry of the characteristic X rays which are emitted when a target sample is excited by an electron beam. In the same sample many more elements (from carbon and up in the periodic table) in much smaller volumes (less than 1 micronm3 or 10(-15) liter) can be analyzed with EPM than with any other ultramicroanalytical method. Quantitation down to 10(-15) g is relatively easy. Any application of EPM in biology depends on the development of suitable methods for biological sample preparation. Liquid droplets of 10(-11)-liter volumes are routinely analyzed. Elemental content in isolated cells can be quantitated. The analysis of cellular, extracellular, and intercellular content in the same preparation is in sight: a general method for the analysis of diffusible elements in different compartments in tissue relies on the manipulation of frozen hydrated tissue near liquid nitrogen temperature. Localization in the same preparation of several organic compounds at high resolution will be possible by use of appropriate tags. All fields of microphysiology are likely to benefit from this new discipline.

Cells↗