Oocyte quality assessment in marine invertebrates: a novel approach by fluorescence spectroscopy

BACKGROUND: The assessment of oocyte quality is, nowadays, a major challenge in aquaculture, oocyte cryopreservation, and environmental science. Oocyte quality is a determining factor in fertilization and embryo development; however, there is still a lack of rapid and sensitive cellular markers for its assessment. Currently, its estimation is pre-dominantly based on morphological analysis, which is subjective and does not consistently reflect the developmental competence of the oocytes. Despite several recent studies investigating molecular markers related to oocyte quality, methods currently available for their determination pose various technical challenges and limitations. In this study, we developed a novel approach based on fluorescence spectroscopy to assess different intrinsic physiological parameters that can be employed to evaluate egg quality in marine invertebrates that are widely used as animal models such as sea urchins and mussels. RESULTS: Different physiological parameters, such as viability, mitochondrial activity, intracellular ROS levels, plasma membrane lipid peroxidation, and intracellular pH, for egg quality evaluation have been successfully assessed in sea urchins and mussels by using specific fluorescent dyes and detecting the fluorescent signals in eggs through fluorescence spectroscopy. CONCLUSIONS: Based on our findings, we propose these physiological markers as useful predictors of egg quality in marine invertebrates; they can be estimated rapidly, selectively, and sensitively by employing this novel approach, which, due to the speed of analysis, the low cost, and easy use can be considered a powerful analytical tool for the egg quality assessment.

Study on denervated rat skeletal muscle by 7.0 T 31P-MR phosphorus spectroscopy / 中华放射学杂志

Objective@#To investigate the changes of 31P-MRS in denervated skeletal muscle at 7.0 T MR system.@*Methods@#In the experiment group, a total of 18 male Sprague-Dawley rats aged 6-8 weeks old and weighing 200-250 g were obtained. The right posterior femoral nerve were transected, and the proximal stumps were ligated by using 5-0 nylon stitches to preclude spontaneous repair. A sham surgery (incision and exploration of the nerve) was performed at the same time (n=6). Before rat model established and at varying times after initial surgery (3 d, and 1, 2, 4, 6, and 8 weeks). 31P-MR spectra of rat quadriceps femoris were acquired on 7.0 T Agilent small animal MR imaging system. We quantified the phosphocreatine (PCr), adenosine triphosphate(β-ATP), and inorganic phosphate (Pi) using Creatine phosphate disodium salt(10 mmol/L) as an external standard. The ratios, include PCr/Pi, Pi/β-ATP, PCr/β-ATP, PCr/(PCr+Pi) and Pi/(PCr+Pi) were quantified in jMRUI. All data were analyzed by one way analysis of variance (ANOVA) with posttest inter-group comparisons using Bonferroni test. Comparative analysis methods between the experimental and control group were performed via independent samples t test. P<0.05 was considered as statistically significance.@*Results@#The average measured concentrations of β-ATP, PCr, and Pi in control groups were (6.654±0.178) μmol/g, (25.656±0.738) μmol/g, and (1.594±0.096) μmol/g, respectively. There were significant statistically differences in β-ATP, PCr, Pi, PCr/Pi, PCr/PCr+Pi,Pi/PCr+Pi between the study and control group(P<0.05) at any measurement time point after denervation. Pi/β-ATP,PCr/β-ATP and pHi in the experimental group significantly statistically differed from those of the control group (all P<0.05)at each time point except on day 3 after operation. The total concentrations of β-ATP and PCr were reduced by 11.5% and 19.7% respectively on day 3. Thereafter, β-ATP and PCr declined rapidly by 63.1% and 68.8% at week 4 respectively, then decreased slowely by 74.0%和82.3% till week 10. The change of PCr/Pi is similar to β-ATP and PCr, but more remarkable. Pi, Pi/PCr+Pi, Pi/ATP showed a progressively increase till week 10. The intra-cellular pH (pHi) of normal rat muscles was 7.033±0.017, While the pHi gently in experiment rat muscles decreased during the entire experiment.@*Conclusion@#31P-MRS with 7.0 T can quantify the temporal changes of energy metabolism and pHi in normal and denervated rat muscles. It shows that the dysfunction of energy metabolism are progressive with time and that they begin within a short period following the nerve section, the change of β-ATP, PCr, PCr/Pi take place primarily within 4 weeks after denervation. β-ATP, PCr, and PCr/Pi may be potential biomarkers of energy metabolism in the evaluation of denervated muscle atrophy.

Effects of Dopamine on Intracellular pH in Opossum Kidney Cells

Na+/H+ exchanger (NHE) has a critical role in regulation of intracellular pH (pHi) in the renal proximal tubular cells. It has recently been shown that dopamine inhibits NHE in the renal proximal tubules. Nevertheless, there is a dearth of information on the effects of long-term (chronic) dopamine treatment on NHE activities. This study was performed to elucidate the pHi regulatory mechanisms during the chronic dopamine treatments in renal proximal tubular OK cells. The resting pHi was greatly decreased by chronic dopamine treatments. The initial rate and the amplitude of intracellular acidification by isosmotical Na+ removal from the bath medium in chronically dopamine-treated cells were much smaller than those in control. Although it seemed to be attenuated in Na+-dependent pH regulation system, Na+-dependent pHi recovery by NHE after intracelluar acid loading in the dopamine-treated groups was not significantly different from the control. The result is interpreted to be due to the balance between the stimulation effects of lower pHi on the NHE activity and counterbalance by dopamine. Our data strongly suggested that chronic dopamine treatment increased intrinsic intracellular buffer capacity, since higher buffer capacity was induced by lower resting pHi and this effect could attenuate pHi changes under extracellular Na+-free conditions in chronically dopamine-treated cells. Our study also demonstrated that intracellular acidification induced by chronic dopamine treatments was not mediated by changes in NHE activity.

Intracellular pH is a Critical Element in Apoptosis Triggered by GM-CSF Deprivation in TF1 Cells

BACKGROUND: Hemopoietic cells require the constant presence of growth factors for survival in vitro and in vivo. Caspases have been known as central executors of apoptotic cell death. We have, therefore, investigated the pathways that regulate caspase activity and apoptosis using the CD34+ cell line, TF-1 which requires GM-CSF for survival. METHODS: Apoptosis was measured by annexin V staining and mitochondrial membrane potential was measured by DiOC6 labelling. Intracellular pH was measured using pH sensitive fluorochrome, BCECF or SNARF-1, followed by flow cytometry analysis. Caspase activation was analyzed by PARP cleavage using anti-PARP antibody. RESULTS: Removal of GM-CSF induceed PARP cleavage, a hallmark of caspase activity, concomitant with pHi acidification and a drop in mitochondrial potential. Treatment with ZVAD, a competitive inhibitor of caspases, partially rescued cell death without affecting pHi acidification and the reduction of mitochondrial potential, suggesting that both these events act upstream of caspases. Overexpression of Bcl-2 prevented cell death induced by GM-CSF deprivation as well as pHi acidification and the reduction in mitochondrial membrane potential. In parental cells maintained with GM-CSF, EIPA, a competitive inhibitor of Na+/H+ antiporter induced apoptosis, accompanied by a drastic reduction in mitochondrial potential. In contrast, EIPA induced apoptosis in Bcl-2 transfectants without causing mitochondrial membrane depolarization. CONCLUSION: Taken together, our results suggest that the regulation of H fluxes, either through a mitochondrion- dependent or independent pathway, is central to caspase activation and apoptosis.

Modulation of Inwardly Rectifying K+ Channel by Intracellular and Extracellular pH in Bovine Aortic Endothelial Cells

The effects of intracellular and extracellular pH on the inwardly rectifying K+ (IRK) channel of the bovine aortic endothelial cells (BAECs) were examined using whole-cell patch-clamp technique. The IRK current, efficiently blocked by Ba2+ (200microM), is the most prominent membrane current in BAECs, which mainly determines the resting membrane potential. The expression of Kir2.1 was observed in BAECs using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Intracellular alkalinization, elicited by the extracellular substitution of NaCl with NH4Cl (30 mM), significantly augmented the amplitude of IRK current. On the contrary, the amplitude of IRK current was attenuated by the Na-acetate (30 mM)-induced intracellular acidification. The changes in extracellular pH also closely modulated the amplitude of IRK current, which was decreased to 40.2+/-1.3% of control upon switching the extracellular pH to 4.0 from 7.4. The extracellular pH value for half-maximal inhibition (pK) of IRK current was 5.11. These results demonstrate that the activity of IRK channel in BAECs, probably Kir2.1, was suppressed by proton at both sides of plasma membrane.

Functional roles of Na+/H+ exchanger isoforms in saliva secretion

No abstract available.

Functional roles of Na+/H+ exchanger isoforms in saliva secretion

No abstract available.

Relationships of intracellular pH to oxygenated hemoglobin/myoglobin and to phosphate compounds in active muscle during forearm exercise / 体力科学

The purpose of this study is to find a key to clarifying the mechanism of lactic acid production during exercise. Five healthy men performed the grip and wrist flexion exercises at different occasions. Exercise intensities were increased by 5% MVC (maximum voluntary contraction force) per minute from 10% MVC. Intracellular pH, oxygenated hemoglobin/myoglobin (Oxy-Hb/Mb), inorganic phosphate (Pi), and phosphocreatin (PCr) in forearm flexor muscles were measured by <SUP>31</SUP>P-MRS and NIRS. The lowest Oxy-Hb/Mb concentrations during the grip and wrist flexion exercises were 40.7± 8.86% (average±SE) and 15.4 ± 2.26%, respectively. These results suggest that oxygen remain sufficient in the muscles at least during the grip exercise. Intracellular pH dropped as exercise intensity rose above 25% MVC for the grip and above 10% MVC for the wrist flexion exercise. These results support the idea that oxygen deficiency is not the only cause for lactic acid production during exercise. On the other hand, intracellular pH fell with either negative or positive relations to Pi/PCr ratio, Pi, and PCr in each exercise. These results support the suggestion that the main causes of lactic acid production during exercise are the changes in ADP, Pi, and PCr.

Effect of Parathyroid Hormone on the intracellular pH Regulation of Osteoblast-like Cells / 대한정형외과학회잡지

Intracellular pH regulation of osteoblasts is of a great importance in the process of bone formation and resorption, and has been suggested to be mediated via intracellular Ca2+ and cAMP messenger systems. To elucidate the mechanism of modulation of intracellular pH by parathyroid hormone and PMA(Phorbo1-12-myristate-13-acetate), effects of these agonists on the individual transporter system, Na+-H+ antiporter and Cl−-HCO3-(−OH−) exchanger, were investigated. Intracellular pH and Ca2+ were measured by using the fluorescent dye BCECF and fura-2, respectively, in UMR-106 cell monolayer grown on glass coverslip. Addition of tumor promotor, PMA(luM) caused 0.14 unit pH rise of resting intracellular pH(pHi) and 38% increase of the initial rate of pHi recovery after cytosolic acid load. Perfusion of Cl−-free solution resulted in rapid cytosolic alkalinization of which the rate was increased 26% by preincubation of PMA. Ca2+ ionophore, ionomycin (1uM) decreased resting pHi by 0.17 unit, but had no effect on the initial rate of pHi recovery after cytosolic acid load. However, the addition of ionomycin augmented the initial rate of pHi increase after Cl−-depletion outside the cells by 34% over the control. Stimulation of cells with parathyroid hormone(10-8M) caused an initial acidification (0.27 unit) followed by cytosolic alkalinization, with inhibiting effect on the initial rate of pHi recovery after acid load (42%). But parathyroid hormone did not have any significant effect on the rate of pHi increase after Cl−-depletion. PMA caused a sustained increase of intracellular Ca2+, of which the peak depended on the concentration of Ca2+ in extracellular medium. Ionomycin caused a transient increase of Ca2+ but PTH had no significant increase of intracellular Ca2+ in the concentration range of 10-6M to 10-12M tested. 10-8M PTH increased cAMP levels by about 10-fold and 10-10M PTH did by 1.6-fold. PMA, which increased cytosolic Ca2+ concentration, also had an stimulatory effect on cAMP production in the concentration range of 10-5M to 10-6M by 2-fold. These findings suggest that in UMR-106 cells Ca2+ and cAMP can influence pHi by altering the activity of pHi regulatory transporter system, and parathyroid hormones modulate pHi by inhibiting Na+-H+ antiporter via intracellular increase of cAMP, which is probably accounts for the inhibitory effect of parathyroid hormone on the proliferation of osteoblasts.

Effect of Increased Intracellular pH by HMA on the Cytotoxicity of Combined Treatment of Hyperthermia and Chemotherapy / 대한치료방사선과학회지

PURPOSE: The enhanced cytotoxic effect of combined treatement of hyperthermia and chemotherapy by increasing intracellular acidify with HMA was investigated. MATERIALS AND METHODS: Fsall tumor cells were injected on the hindlegs of female C3H mice. When the tumor volume reached about 200mm3 , experiments were performed on the groups classified as follows : Group I : Control Group II : Melphalan alone (2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg). Group III : Heat alone (42.5degree C for 1 hour) Group IV : Melphalan + Heat (42.5degree C for 1 hour) Group V : HMA(10 mg/kg) + Melphalan (5.0 mg/kg) + Heat (42.5degree C for 1 hour) Each group included 8-12 mice on each experiment. HMA (3-amino-6-chloro-5(1-homopiperidyl)-N-(diaminomethylene)-c-pyrazinecarboxamide), an analog of amiloride which increases intracellular pH(pHi) was dissolved in dimethyl sulfoxide(DMS) and injected into the tumor-bearing mice through the tail vein. 10mg/kg of HMA and each dose of melphalan were injected into peritoneum of the tumor-bearing mice 30 minutes before heating. Tumor growth delay was calculated when the tumor vlme reached at 1500mm3 . Excision assay was performed on each group and repeated 2-4 times. RESULTS: Tumor growth delay of each experimental groups at 1500 mm3 were 9, 10, 13 and 19 days respectively. In vivo-in vitro excision assay using Fsall tumor cells, the cytotoxicity of each experimental groups was 1.2 X 107 , 1 X 107 , 6 X 106 , 1.7 X 106 and 1 X 105 clonogenic cells/gm respectively. When HMA was added to the combined treatment of heat and chemotherapy, the tumor growth ws delayed more than combined treatment without HMA i.e., 6 days tumor growth delay at 1500 mm3 of tumor volume. CONCLUSION: he combined effect of cytotoxicity by heat and chemotherapy can be much more enhanced by HMA.

Intracellular acidosis decreases the outward Na(+)-Ca2+ exchange current in guinea pig ventricular myocytes

The Na(+)-Ca2+ exchange transport operating in outward mode has been suggested to cause Ca2+ entry during reperfusion or reoxygenation, exchanging extracellular Ca2+ for intracellular Na+ that has accumulated during ischemia or cardioplegia. During cardioplegia, however, an increase in Ca2+ entry via this mechanism can be decreased due to increased intracellular H+ activity and a decrease in cellular ATP content. In this study giant excised cardiac sarcolemmal membrane patch clamp technique was employed to investigate the effect of cytosolic pH change on the Na(+)-Ca2+ exchanger, excluding the effect of ATP, in guinea pig cardiac myocytes. The outward Na(+)-dependent current, which has a characteristics of Hill equation, was decreased as pH was decreased in the range of 7.5-6.5. The current density generated by the Na(+)-Ca2+ exchange transport was 56.6 +/- 4.4 pA/pF (Mean +/- S.E.M.) at pH 7.2 and decreased to 42.9 +/- 3.0 pA/pF at pH 6.9. These results imply that Na(+)-Ca2+ exchange transport, operating in a reverse mode during cardioplegia, decreases due to increased intracellular H+, and further suggest that consequent intracellular Na+ accumulation is one of aggravating factors for Ca2+ influx during reoxygenation or reperfusion.

Changes in cytoplasmic pH upon heat shock in embryonic and adult rat liver ceils.

All living cells, when exposed to elevated temperatures, undergo physiological changes which result in the expression of a specific set of heat shock proteins. Study of the possible physiological changes in adult and embryonic rat liver cells indicated a change in intracellular pH upon heat shock. Using 2', 7'-bis (2-carboxyethyl)-5 (and -6) carboxyfluorescein acetoxymethyl ester, we demonstrate here that the intracellular pH of adult and embryonic liver cells is different and that there is an increase in relative fluorescence intensity in both adult and embryonic cells upon heat shock, which corresponds to about 0·2 to 0·3 pH units. We also show that in addition to heat, some of the inducers of heat shock like response in many systems also induce a change in intracellular pH and induce heat shock proteins at 37°C in fetal liver cells. The possible mechanisms of induction of heat shock proteins during heat shock and in the presence of inducers at normal temperature are discussed.

Studies on the effect of NaHCO3 intake on intracellular pH and PCr concentration during exercise by 31P NMR / 体力科学

To evaluate the changes in muscle energetics following NaHCO<SUB>3</SUB> intake, we measured the phosphorus-31 nuclear magnetic resonance (<SUP>31</SUP>P NMR) spectra of human muscle <I>in vivo</I> during exercise. Seven male subjects performed two trials, a NaHCO<SUB>3</SUB> (Alka, Tr.) and a NaCI trial (Cont. Tr.), on two occasions. <SUP>31</SUP>P NMR spectra were obtained serially during leg-elevating exercises. Before and during exercise, the intracellular phosphocreatine (PCr), inorganic phosphate (Pi) and pH were determined from the NMR spectra. The decrease of intracellular pH during exercise showed a tendency to be inhibited by NaHCO<SUB>3</SUB> intake, and the intracellular pH at the end of the exercise was 6.69 for Alka. Tr, and 6.51 for Cont. Tr. The decline of the PCr/ (PCr+Pi) ratio during exercise was not influenced by NaHCO<SUB>3</SUB> intake. The PCr/ (PCr+Pi) ratio was related exponentially to the intracellular pH. A remarkable decline of PCr/ (PCr+Pi) ratio occurred until the intracellular pH fell to about 6.7, but did not decrease below that. It was suggested that the intake of NaHCO<SUB>3</SUB> could decrease the rate of fall in the intracellular pH during exercise, and that the PCr store could be influenced by the intracellular pH when the pH was above 6.7, but not below that level.

The regulation of muscarinic receptor activated Kir3.1/3.4 currents by intracellular pH / 中国药理学通报

Kir2 1. Moreover, drop of pHi reduced the M 1 induced inhibition of Kir3 1/3 4 currents, and enhanced the desensitization of M 2 induced Kir3 1/3 4 activation. CONCLUSION The basal currents and M receptor induced currents of Kir3 1/3 4 can be regulated by intracellular pH. These changes may play some important roles in pathophysiological conditions like cardiac ischemia.

~31P-NMR STUDIES ON INTRACELLULAR pH AND ENERGY METABOLISM OF HEART, KIDNEY AND LIVER IN BURN SHOCK: AN ANIMAL EXPERIMENT / 第二军医大学学报

Cellular energy metabolism and intracellular pH of intact heart, kidney and liver of small animals were studied before and after burn with an effective technique of phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy. Advantages of this technique are that the measurement can be performed constantly without any complicated and destructive process, that it is possible to use this technique in clinical examination in the near future and that a lot of information can be obtained from only one spectrum.It was demonstrated that there were a short cellular energy source and a cellular acidosis in' heart, kidney and liver during burn shock. The high-energy phosphate compounds-lowered wavily but not straight, which could be divided into stress stage, compensation stage and decompensation stage. Intracellular pH decreased first in the heart, and then in the kidney and liver. In contrast to the results obtained by way of direct blood flow determination, the early and significant intracellular acidosis indicated the insufficiency of blood and oxygen supply in myocardium.