Katsuhiko Doi

Aging‐related changes in calcium oscillations in fertilized mouse oocytes

Aging of oocytes, being not fertilized after ovulation for a prolonged time, considerably affects normal development of the fertilized oocyte. We examined effects of the aging on a series of highly repetitive Ca2+ transients commonly seen in fertilized mouse oocytes (Ca2+ oscillations). Frequency of Ca2+ oscillations in the aged oocyte [20 hrs after induction of superovulation by i.p. human chorionic gonadotropin (hCG)] was significantly higher (34.1 ± 5.8 1/hr) than the fresh oocyte (14 hr post‐hCG, 21.8 ± 7.9 1/hr). Rates of rise and fall of individual Ca2+ transient in the aged oocyte were significantly slower than the fresh oocyte, whereas durations of individual Ca2+ transients were similar. When extracellular Ca2+ was raised from 2.04 mM to 5.00 mM, aged oocytes showed significant prolongation of the duration of individual Ca2+ transient, that resulted in a sustained elevation of intracellular Ca2+ ([Ca2+]i) in 33% of the aged oocyte. Transient increase in [Ca2+]i by photolysis of a caged Ca2+, Nitr‐5, injected into cytoplasm was completely restored in the fresh oocyte [fluorescence intensity of [Ca2+]i indicator dye Fluo‐3 (F480) returned to 97 ± 2% of the control level, time constant = 37 ± 9 sec]. In contrast, in the aged oocyte, restoration of F480 following Nitr‐5 photolysis was incomplete (115 ± 12% of the control) and slow (time constant = 64 ± 23 sec). Because inhibition of the Ca2+ pump of the endoplasmic reticulum (ER) by 5 μM thapsigargin almost completely inhibited restoration of F480 following Nitr‐5 photolysis in the fresh oocyte, we conclude that the aging‐related changes in Ca2+ oscillations may be accounted for by dysfunction of intracellular Ca2+ regulation, presumably of the Ca2+ pump of the ER. Mol. Reprod. Dev. 48:383–390, 1997.

Effects of aging on inositol 1,4,5-triphosphate-induced Ca2+ release in unfertilized mouse oocytes.

We previously demonstrated in the mouse oocyte that in vivo postovulatory aging significantly suppresses activity of the endoplasmic reticulum (ER) Ca2+‐ATPase (Igarashi et al. 1997. Mol Reprod Dev 48:383–390). We undertook the present study to further examine the effects of oocyte aging on Ca2+ release from the inositol 1,4,5‐triphosphate (InsP3)‐sensitive Ca2+ channels of the ER membrane, because not only Ca2+ reuptake, but also Ca2+ release from the ER, substantially affect Ca2+ oscillations in fertilized oocytes. A transient increase in cytosolic free Ca2+ concentration ([Ca2+]i) was induced by photolysis of caged InsP3 microinjected into the cytoplasm in both fresh (14 hr post hCG) and aged (20 hr or 24 hr post hCG) oocytes, where the maximum rate of increase in [Ca2+]i significantly decreased in the aged oocytes. Reduced ER Ca2+ release in the aged oocyte may not be attributable to aging‐related desensitization of the InsP3‐sensitive Ca2+ channels in the ER because concentrations of caged InsP3 for half maximal [Ca2+]i increase were identical for fresh and aged oocytes. The peak [Ca2+]i response following administration of 5 μM thapsigargin, a specific ER Ca2+‐ATPase inhibitor, was significantly reduced in the aged oocyte, suggesting reduction of the ER Ca2+ stores. We conclude from these results that reduction of Ca2+ release from the InsP3‐sensitive Ca2+ stores in the aged oocyte arises from depletion of the ER Ca2+ stores with aging. These aging‐related changes in Ca2+ release and reuptake may account for alterations in Ca2+ oscillations in aged fertilized oocytes. Mol. Reprod. Dev. 55:299–306, 2000.

Direct observation of radial intracellular PO2 gradients in a single cardiomyocyte of the rat.

The purpose of the present study was to directly visualize radial gradients of intracellular [Formula: see text] in a single individual cardiomyocyte isolated from the rat ventricle. Microspectrophotometry with the use of cytosolic myoglobin as an oxygen probe was conducted at 410 nm. When the quiescent cell was incubated with 1 μM carbonyl cyanide m-chlorophenylhydrazone to increase oxygen consumption approximately eightfold, gradual decreases in myoglobin oxygen saturation (SMb) were demonstrated toward the core of the cell, whereas these decreases disappeared when the cell was treated with 2 mM NaCN. These results highlighted the importance of diffusional oxygen transport in determining intracellular oxygenation in cardiac cells. From the measured SMb, we assessed the profile of radial changes in intracellular [Formula: see text]at the mean SMb comparable to that in vivo (∼0.5). Quite steep [Formula: see text]gradients were demonstrated in the vicinity of the sarcolemma that were rapidly attenuated toward the cell core. These radial profiles of intracellular [Formula: see text] demonstrate the significance of myoglobin-facilitated diffusion of oxygen. Furthermore, the shallow gradients of [Formula: see text] near the center of the cell might arise from partial depression of oxygen consumption near the cell core.The purpose of the present study was to directly visualize radial gradients of intracellular PO2 in a single individual cardiomyocyte isolated from the rat ventricle. Microspectrophotometry with the use of cytosolic myoglobin as an oxygen probe was conducted at 410 nm. When the quiescent cell was incubated with 1 microM carbonyl cyanide m-chlorophenylhydrazone to increase oxygen consumption approximately eightfold, gradual decreases in myoglobin oxygen saturation (SMb) were demonstrated toward the core of the cell, whereas these decreases disappeared when the cell was treated with 2 mM NaCN. These results highlighted the importance of diffusional oxygen transport in determining intracellular oxygenation in cardiac cells. From the measured SMb, we assessed the profile of radial changes in intracellular PO2 at the mean SMb comparable to that in vivo ( approximately 0.5). Quite steep PO2 gradients were demonstrated in the vicinity of the sarcolemma that were rapidly attenuated toward the cell core. These radial profiles of intracellular PO2 demonstrate the significance of myoglobin-facilitated diffusion of oxygen. Furthermore, the shallow gradients of PO2 near the center of the cell might arise from partial depression of oxygen consumption near the cell core.

Activation of chloride current by P2‐purinoceptors in rat ventricular myocytes

1 Rat ventricular myocytes were dissociated and their responses to extracellularly applied ATP were recorded using patch pipettes under the whole cell configuration. 2 ATP initially induced an inward current followed by an outward current at − 50 mV. With a Cs‐rich pipette solution the late outward current was blocked, leaving a sustained inward current (IATPs) suggesting that a K+conductance underlies the late response. 3 When the extracellular Cl−concentration was changed, the reversal potential of IATPs corresponded well to the shift of the Cl−equilibrium potential. IATPs was reversibly blocked by the chloride channel blocker, 4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid (DIDS). 4 The concentration‐response curve of IATPs had a Hill coefficient of 0.98 and an EC50 value of 5.2 × 10−6m. 5 ATP was more potent than ADP, while AMP and adenosine were ineffective, suggesting that P2‐purinoceptor activation induced IATPs. 6 The activation of IATPs was depressed by depleting the extracellular Mg2+and increased by adding Mg2+. 7 Our results strongly suggest that P2‐purinoceptor activation by ATP induces both a Cl−‐conductance (IATPs) and a K+‐conductance in rat ventricular myocytes.

Visualization of Myoglobin-Facilitated Mitochondrial O 2 Delivery in a Single Isolated Cardiomyocyte

The purpose of the present study was to visualize myoglobin-facilitated oxygen delivery to mitochondria at a critical mitochondrial oxygen supply in single isolated cardiomyocytes of rats. Using the autofluorescence of mitochondrial reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), the mitochondrial oxygen supply was imaged from approximately 1.4 microm inside the cell surface at a subcellular spatial resolution. Significant radial gradients of intracellular oxygenation were produced by superfusing the cell suspension with a mixed gas containing 2-4% oxygen while stimulating mitochondrial respiration with an uncoupler of oxidative phosphorylation. Augmentation of the NAD(P)H fluorescence started from the core of the cell (anoxic core) and progressively expanded toward the plasma membrane, as the extracellular Po(2) was lowered. Inactivation of cytosolic myoglobin by 5 mM NaNO(2) significantly enlarged such anoxic regions. Nitrite affected neither mitochondrial respiration in uncoupled cells nor the relationship between Po(2) and the NAD(P)H fluorescence in coupled cells. Thus we conclude that myoglobin significantly facilitates intracellular oxygen transport at a critical level of mitochondrial oxygen supply in single cardiomyocytes.

Intracellular gradients of O2supply to mitochondria in actively respiring single cardiomyocyte of rats

We demonstrated in a previous study [Takahashi, E., K. Sato, H. Endoh, Z.-L. Xu, and K. Doi. Am. J. Physiol. 275 (Heart Circ. Physiol. 44): H225-H233, 1998] that significant radial gradients of intracellular PO2 may be produced in an uncoupled actively respiring, single isolated cardiomyocyte of the rat. The present study was designed to further determine whether such intracellular PO2 gradients can be a limiting factor of oxidative metabolism in uncoupled cardiomyocytes. The NAD(P)H fluorescence of a single cardiomyocyte was captured by a digital charge-coupled device camera and quantitated with a subcellular spatial resolution by a ratio-imaging technique. In the conditions that we demonstrated significant radial PO2 gradients (cells treated with 1 microM carbonyl cyanide m-chlorophenylhydrazone and superfused with 2.09% or 3.14% O2 gas at 27 degreesC), we demonstrated significant augmentation of NAD(P)H fluorescence near the core of an individual cell. The heterogeneous fluorescence pattern was not found in the control cell, whereas fluorescence intensity averaged over the cell was increased by hypoxia. These results suggest the possibility that oxidative phosphorylation near the core of actively respiring, uncoupled cardiomyocytes may be severely suppressed due to insufficient diffusional oxygen supply (hypoxic core) even if regions near the sarcolemma are adequately oxygenated.We demonstrated in a previous study [Takahashi, E., K. Sato, H. Endoh, Z.-L. Xu, and K. Doi. Am. J. Physiol. 275 ( Heart Circ. Physiol. 44): H225-H233, 1998] that significant radial gradients of intracellular [Formula: see text] may be produced in an uncoupled actively respiring, single isolated cardiomyocyte of the rat. The present study was designed to further determine whether such intracellular [Formula: see text] gradients can be a limiting factor of oxidative metabolism in uncoupled cardiomyocytes. The NAD(P)H fluorescence of a single cardiomyocyte was captured by a digital charge-coupled device camera and quantitated with a subcellular spatial resolution by a ratio-imaging technique. In the conditions that we demonstrated significant radial[Formula: see text] gradients (cells treated with 1 μM carbonyl cyanide m-chlorophenylhydrazone and superfused with 2.09% or 3.14% O2 gas at 27°C), we demonstrated significant augmentation of NAD(P)H fluorescence near the core of an individual cell. The heterogeneous fluorescence pattern was not found in the control cell, whereas fluorescence intensity averaged over the cell was increased by hypoxia. These results suggest the possibility that oxidative phosphorylation near the core of actively respiring, uncoupled cardiomyocytes may be severely suppressed due to insufficient diffusional oxygen supply (hypoxic core) even if regions near the sarcolemma are adequately oxygenated.

Diffusion coefficient for O2 in plasma and mitochondrial membranes of rat cardiomyocytes

Oxygen diffusion in plasma membranes and mitochondrial (Mt) membranes of ventricular myocytes isolated from adult rat hearts was measured using O2-induced fluorescence quenching of pyrene-butyric acid. The diffusion coefficient for oxygen (DO2 in 10(-6).cm2 x s-1) of the plasma membrane was 2.91 +/- 0.05 (37 degrees C) in the control group, which was significantly higher than that of Mt membrane (2.23 +/- 0.11) (P < 0.001). The DO2 of the plasma membrane was reduced to 2.50 +/- 0.08 (P < 0.001) in myocytes isolated from heart after 15 min reperfusion following no-flow ischemia for 30 min, while that of the Mt membrane remained almost unchanged. Administration of hydrogen peroxide and hypochlorous acid to the isolated myocytes also reduced the plasma membrane DO2. The decrease in the plasma membrane DO2 correlated with that in the fraction of rod-shaped myocytes. We conclude that the plasma membrane is more susceptible to reperfusion injury than the Mt membrane, but these membranes do not limit O2 uptake in the heart because of the high absolute values of DO2.

Glycolysis vs. respiration as ATP source for the shape of quiescent cardiomyocytes

ATP concentration ([ATP]) and the fraction of rod-shaped myocytes (Rrod) were measured for quiescent ventricular cells isolated from the rat heart. The myocytes were gently shaken with metabolic inhibitors for 30 min at 37 degrees C, and then [ATP] was assayed enzymatically. [ATP] (mean +/- SE in nmol.(10(6) cells)-1) of the control group (80.2 +/- 6.3) was significantly reduced to 32.3 +/- 3.6 and 21.9 +/- 4.5 by inhibiting glycolysis with 2-deoxyglucose (DG, 10 mM) and iodoacetic acid (IAA, 2 mM), respectively. Inhibition of the oxidative phosphorylation by sodium cyanide (CN, 2 mM) or 2,4-dinitrophenol (DNP, 40 microM) did not induce significant [ATP] reduction (56.5 +/- 5.6 or 53.2 +/- 7.5). Rrod were 54.4 +/- 1.9, 47.3 +/- 5.1, 43.3 +/- 3.3, 28.5 +/- 3.6 and 11.6 +/- 1.8% for the control, CN, DNP, DG and IAA groups, respectively. Under glycolytic inhibition with DG, addition of pyruvate (5 mM) with a pyruvate dehydrogenase activator (dichloroacetate (DCA, 1 mM) or (-)isoproterenol (ISO, 10 microM)) restored [ATP] to 52.5 +/- 8.3 and 67.7 +/- 7.2, respectively, but Rrod still remained depressed (34.6 +/- 2.1 and 35.4 +/- 1.3%). We conclude that the normal shape of quiescent myocytes is maintained primarily by glycolytic ATP.

In Vivo sup 31 P Magnetic Resonance Spectroscopy for Evaluation of Testicular Function in Cryptorchid Rats

PURPOSE To assess in vivo metabolism of rat testicles in experimental cryptorchidism, we used 31P magnetic resonance (MR) spectroscopy and compared testicular MR spectroscopic parameters with flow cytometric DNA analysis. MATERIALS AND METHODS In vivo 31P MR spectroscopy and flow cytometric DNA analysis of rat testis were performed before and during 14 days of experimental cryptorchidism. RESULTS The testicular phosphomonoester (PM)/ATP ratio showed a transient increase when multinuclear giant cells appeared in the seminiferous tubules. However, the ratio returned to the preoperative level when these cells disappeared. The phosphodiester (PD)/ATP ratio gradually decreased and the inorganic phosphate (Pi)/ATP ratio slowly increased. DNA flow cytometry showed a decrease in the percentage of haploid cells and an increase in the percentage of diploid cells from 7 days after cryptorchidism. The percentage of tetraploid cells did not change before and during cryptorchidism. CONCLUSION This study indicates that in vivo 31P MR spectroscopy in combination with flow cytometric DNA analysis provides useful biochemical and histological information for evaluation of testicular function.

Direct Estimation of Intracellular Po2 Gradients in a Single Cardiomyocyte of the Rat

In a previous study, we have demonstrated that microspectrophotometry coupled with a digital image processing is able to determine fractional binding of oxygen to myoglobin in an individual single cardiac myocyte isolated from the ventricles of the rat (Takahashi and Doi, 1995). Using this technique, we have determined the oxygen pressure (Po2) gradients from the extracellular space to cytosol in a quiescent cardiomyocyte (Takahashi and Doi, 1996). We also found that the Po2 gradients diminish as the cell becomes hypoxic (Takahashi and Doi, 1996). This reduction of Po2 gradients may partially restore the oxygenation of the intracellular space so that oxygen supply to mitochondria required for minimal electron flux through the respiratory chain is maintained even in severe hypoxia. We, however, were not able to quantitate the relative contributions of the extracellular (from extracellular medium to sarcolemma) and intracellular (from the sarcolemma to cytosol) Po2 gradients in the control of intracellular Po2. Thus, we undertook the present study to directly assess the magnitude of intracellular Po2 gradients with a subcellular spatial resolution in the single isolated cardiomyocyte.