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C P Lechene

Publications and source records attributed to C P Lechene.

At least 19 recordsLinked to original sources
PubMed1993

The protective action of betaine on the deleterious effects of NaCl on preimplantation mouse embryos in vitro.

The development of outbred mouse (CF1) zygotes in vitro has been studied using medium SOM in which the concentrations of NaCl (85, 105, 125 mM), glutamine (0, 1, 2 mM), and betaine (0, 1, 2 mM) were varied. The effects of the compounds were studied using a 3(3) factorial experimental arrangement. The inhibitory effect of relatively high concentrations of NaCl and the protective effect of glutamine were confirmed. Betaine, an organic osmolyte, can also protect against the deleterious effects of relatively high concentrations of NaCl. The intracellular contents of potassium and sodium have also been measured in single zygotes using X-ray electron probe spectrometry. When medium SOM contains 85 mM or 125 mM NaCl, the intracellular content of Na rises and the content of K decreases. These changes are partially reduced in the presence of 125 mM NaCl if betaine is also in the medium. Betaine has no effect on the intracellular content of K and Na if the concentration of NaCl is 85 mM. These results suggest that organic osmolytes may be required in embryo culture media to prevent excessive changes in the intracellular ionic concentration.

Animals↗
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↗
PubMed1983

The effect of spironolactone on the elemental composition of the intraluminal fluids of the seminiferous tubules, rete testis and epididymis of the rat.

Embryologically, the epididymis and renal collecting tubules develop from the mesonephric duct. Aldosterone enhances the reabsorption of sodium from the collecting tubule, and there is evidence that sodium is removed from the epididymal lumen against a concentration gradient. Rats were treated with spironolactone, an aldosterone antagonist, and the concentrations of seven elements, including sodium, were measured in intraluminal fluids from the testis and epididymis. The sodium concentration in caput epididymidal fluid rose (102.5 +/- 4.4 to 128.0 +/- 6.8 mmol./l., p less than 0.01) after 14 days of spironolactone treatment. This finding is consistent with the hypothesis that aldosterone acts on the caput epididymidis, like the collecting tubule, to augment the removal of sodium from luminal fluid.

Animals↗
PubMed1983

Role for intrarenal mechanisms in the impaired salt excretion of experimental nephrotic syndrome.

A unilateral model of puromycin aminonucleoside (PAN)-induced albuminuria was produced in Munich-Wistar rats to examine the mechanisms responsible for renal salt retention. 2 wk after selective perfusion of left kidneys with PAN (n = 8 rats) or isotonic saline (control, n = 7 rats), increases in albumin excretion and decreases in sodium excretion were demonstrated in PAN-perfused but not in nonperfused kidneys of PAN-treated rats although systemic plasma protein concentration remained at control level. Total kidney glomerular filtration rate (GFR) and superficial single nephron (SN) GFR were also reduced selectively in PAN-perfused kidneys, on average by approximately 30%, due primarily to a marked decline in the glomerular capillary ultrafiltration coefficient (Kf), which was also confined to PAN-perfused kidneys. Values for absolute proximal reabsorption (APR) were also selectively depressed in PAN-perfused kidneys, in keeping with a similarly selective decline in peritubular capillary oncotic pressure measured in these kidneys, the latter also a consequence of the fall in Kf. In a separate group of seven PAN-treated rats, however, no differences were detected between PAN-perfused and nonperfused kidneys in the absolute amount of sodium reaching the early (0.77 +/- 0.09 neq/min vs. 0.74 +/- 0.08, P greater than 0.40) and late portions of superficial distal tubules (0.31 +/- 0.02) neq/min vs. 0.32 +/- 0.05, P greater than 0.50), despite the lesser filtered load of sodium in PAN-perfused kidneys. Suppressed sodium reabsorption in both proximal convoluted tubules and short loops of Henle of PAN-perfused kidneys contributed to this equalization of sodium delivery rates to the late distal tubule, as did comparable reabsorption along distal convolutions. In two additional groups of PAN-treated rats, infusion of saralasin (0.3 mg/kg per h, i.v.) led to substantial increases in total kidney GFR and SNGFR in PAN-perfused but not in nonperfused kidneys. Despite these increases in total and SNGFR, urinary sodium excretion by PAN-perfused kidneys remained at a level far below that for nonperfused kidneys, again indicating that the antinatriuresis characterizing the PAN-perfused kidney is due to alterations in sodium handling by the tubules rather than changes in GFR. These results therefore indicate (a) that reductions in Kf and depressed sodium reabsorption by proximal tubules and Henle's loop segments in this model are brought about by intrarenal rather than circulating or systemic factors, and (b) assuming that superficial nephrons are representative of the entire nephron population, renal salt retention in this model is due primarily to intrarenal factor(s) acting beyond the distal convolution.

Animals↗
PubMed1983

The effects of cyproterone acetate on the elements in the intraluminal fluids of the testis and epididymis of the rat.

Spermatozoal maturation and the integrity of the epididymis are dependent on the action of androgen. Micropuncture techniques and electron probe microanalysis were used to investigate the effect of cyproterone acetate on the concentrations of seven elements in intraluminal fluids of the testis, rete testis, and epididymis. After 4 weeks of treatment with cyproterone acetate, the sodium concentration rose in the corpus epididymidis (74.4 +/- 5.4 to 102 +/- 6 mmol/L, p less than 0.01). The concentration of potassium rose in seminiferous tubular fluid (33.9 +/- 2.7 to 49.4 +/- 4.4 mmol/L, p less than 0.01) and caput epididymidal fluid (15.9 +/- 1.4 to 25.7 +/- 0.9 mmol/L, p less than 0.01) after 8 weeks of cyproterone acetate treatment. The chloride concentration rose in the caput (23.7 +/- 0.8 to 65.4 +/- 12.5 mmol/L, p less than 0.01) and distal cauda (39.4 +/- 4.6 to 74.7 +/- 10.8 mmol/L, p less than 0.01) epididymidis after 8 weeks of treatment. Cyproterone acetate had no effect on the concentrations of phosphorus, calcium, and magnesium. The sulfur concentration rose in seminiferous tubular fluid (4.3 +/- 0.3 to 7.3 +/- 0.3 mmol/L, p less than 0.01) and distal cauda fluid (5.3 +/- 0.5 to 14.0 +/- 1.6 mmol/L, p less than 0.01) after 8 weeks of treatment with cyproterone acetate. Four weeks of treatment also increased the sulfur concentration in the distal cauda (6.8 +/- 1.6 to 13.4 +/- 1.5 mmol/L, p less than 0.01). Cyproterone acetate treatment did not affect the concentration of any element in rete testis fluid or serum.

Animals↗
PubMed1980

Calcium and magnesium transport along the inner medullary collecting duct of the rat.

We have measured calcium and magnesium transport along the inner medullary collecting duct (IMCD) in rats by the microcatheterization technique. The fraction of filtered divalent cation ((F/P)Ca,Mg/In) as a function of IMCD length was analyzed by linear regression. In eight control rats calcium reabsorption was demonstrated, inasmuch as there was a significant correlation and slope, P less than 0.001. (F/P)Ca/In decreased from 2.2 +/- 0.3% at the beginning of the IMCD to 0,84 +/- 0.1% in the urine. (F/P)Mg/In was not changed along the IMCD, After acute thyroparathyroidectomy (TPTX) (F/P)Ca/In increased to 4.6 +/- 0.4% at the beginning of the IMCD and 1.55 +/- 0.1% in the urine. The slope was not significantly different from the control group. (F/P)Mg/In was unchanged and was not different from the control group. Accordingly, net Ca reabsorption occurs along the IMCD and the fractional Ca reabsorption is not altered by TPTX. Net Mg transport along the IMCD was absent in both control and TPTX rats; The increased calciuresis after TPTX is not dependent on IMCD function nor is Mg excretion under the conditions studied.

Animals↗
PubMed1980

Elemental composition of fluid in the human Fallopian tube.

Relatively undisturbed samples of human oviduct fluid were obtained by aspiration into a Silastic catheter inserted through the fimbriated end of the oviduct during surgical laparotomy. The concentrations of Na, Cl, K, Ca, Mg, S and P were determined in picolitre aliquots of this fluid using X-ray spectrometry by electron probe excitation. Human tubal fluid has a unique elemental composition characterized by high concentrations of K and Cl, but low concentrations of Ca, relative to the range of normal human serum values. Na and Mg concentrations in oviduct fluid are similar to serum levels.

Adult↗
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↗