Calcium-dependent regulation of calcium efflux by the cardiac sodium/calcium exchanger

Allosteric regulation by cytosolic Ca²⁺ of Na⁺/Ca²⁺ exchange activity in the Ca²⁺ efflux mode has received little attention because it has been technically difficult to distinguish between the roles of Ca²⁺ as allosteric activator and transport substrate. In this study, we used transfected Chinese hamster ovary cells to compare the Ca²⁺ efflux activities in nontransfected cells and in cells expressing either the wild-type exchanger or a mutant, (241–680), that operates constitutively; i.e., its activity does not require allosteric Ca²⁺ activation. Expression of the wild-type exchanger did not significantly lower the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) compared with nontransfected cells. During Ca²⁺ entry through store-operated Ca²⁺ channels, Ca²⁺ efflux by the wild-type exchanger became evident only after [Ca²⁺]_i approached 100–200 nM. A subsequent decline in [Ca²⁺]_i was observed, suggesting that the activation process was time dependent. In contrast, Ca²⁺ efflux activity was evident under all experimental conditions in cells expressing the constitutive exchanger mutant. After transient exposure to elevated [Ca²⁺]_i, the wild-type exchanger behaved similarly to the constitutive mutant for tens of seconds after [Ca²⁺]_i had returned to resting levels. We conclude that Ca²⁺ efflux activity by the wild-type exchanger is allosterically activated by Ca²⁺, perhaps in a time-dependent manner, and that the activated state is briefly retained after the return of [Ca²⁺]_i to resting levels. persistent calcium activation; store-operated channels; calcium transient     

Uterine epithelial and eosinophil estrogen receptors in rats during the estrous cycle

Summary In the uterus of the adult female rats, the luminal epithelial cells and the eosinophil leukocytes are rich in cytoplasmic estrogen receptors. During the estrous cycle, the epithelial estrogen receptor concentration reaches its peak level, in proestrus, drops precipitously in estrus, and hits the trough, at metestrus. Repopulation of the cytoplasm with estrogen binding sites occurs during diestrus. This pattern of cyclic change is indicative of a rapid turnover of estrogen receptors in the epithelial cells and its regulation by endogenous estrogens. The concentration of estrogen receptors in the cytoplasm of the eosinophils does not appear to fluctuate during the cycle. But the intrauterine, distribution of these leukocytes is clearly cyclic in pattern, ostensibly influenced by estrogens. While progesterone binding activity is consistently demonstrated in tandem with estrogen receptors in the cytoplasm of the epithelial cells, it has not been observed in the eosinophil leukocytes. These findings support the claim that there are two estrogen receptor systems in the rat uterus, one mediating the intracellular events of the genomic response to estrogens, and the other being concerned with non-genomic responses.Abbreviations BSA Bovine serum albumin - FITC fluorescein isothiocyanate - TMRITC tetramethylrhodamine isothiocyanate - CMO O-carboxymethyl oxime     

Lanthanum is transported by the sodium/calcium exchanger and regulates its activity

La³⁺ uptake was measured in fura 2-loaded Chinese hamster ovary cells expressing the bovine cardiac Na⁺/Ca²⁺ exchanger (NCX1.1). La³⁺ was taken up by the cells after an initial lag phase of 50-60 s and achieved a steady state within 5-6 min. Neonatal cardiac myocytes accumulated La³⁺ in a similar manner. La³⁺ uptake was due to the activity of the exchanger, because no uptake was seen in nontransfected cells or in transfected cells that had been treated with gramicidin to remove cytosolic Na⁺. The low rate of La³⁺ uptake during the lag period resulted from insufficient cytosolic Ca²⁺ to activate the exchanger at its regulatory sites, as shown by the following observations. La³⁺ uptake occurred without a lag period in cells expressing a mutant of NCX1.1 that does not exhibit regulatory activation by cytosolic Ca²⁺. The rate of La³⁺ uptake by wild-type cells was increased, and the lag phase was reduced or eliminated, when the cytosolic Ca²⁺ concentration was increased before initiating La³⁺ uptake. La³⁺ could substitute for Ca²⁺ at very low concentrations to activate exchange activity. Thus preloading cells expressing NCX1.1 with a small quantity of La³⁺ increased the rate of exchange-mediated Ca²⁺ influx by 20-fold; in contrast, cytosolic La³⁺ partially inhibited Ca²⁺ uptake by the regulation-deficient mutant. With an estimated K_D of 30 pM for the binding of La³⁺ to fura 2, we conclude that cytosolic La³⁺ activates exchange activity at picomolar concentrations. We speculatively suggest that endogenous trace metals might activate exchange activity under physiological conditions. fura 2; NCX1.1; myocyte     

Uterine and ovarian blood flow during the estrous cycle in mares

Uterine and ovarian blood flow was investigated in four mares during two consecutive estrous cycles using transrectal color Doppler sonography. The uterine and ovarian arteries of both sides were scanned to obtain waves of blood flow velocity. The pulsatility index (PI) reflected blood flow. There were significant time trends in PI values of all uterine and ovarian blood vessels during the estrous cycle (P < 0.05). PI values did not differ between the uterine arteries ipsi- and contralateral to the corpus luteum or the ovulatory follicle. PI values of the uterine arteries showed a wave shaped profile throughout the estrous cycle. The highest PI values occurred on Days 0 and 1 (Day 0 = ovulation) and around Day 11, and the lowest PI values were measured around Days 5 and -2 of the estrous cycle. During diestrus (Days 0-15) PI values of the ovarian artery ipsilateral to the corpus luteum were significantly lower than PI values of the contralateral ovarian artery (P < 0.0001). No differences (P > 0.05) in resistance to ovarian blood flow occurred between sides during estrus (Days -6 to -1). In this cycle stage PI values decreased in both ovarian vessels (P < 0.05). During diestrus, high PI values of the ovarian artery ipsilateral to the corpus luteum were measured between Days 0 and 2, followed by a decline until Day 6 (P < 0.05). From this time on, the resistance to blood flow increased continuously until Day 15 (P < 0.05). The cyclic blood flow pattern in the contralateral ovarian artery was similar to that in the uterine arteries (r = 0.68; P < 0.0001). No correlations occurred between the diameter of the corpus luteum and the PI values of the ipsilateral ovarian artery (P > 0.05) during diestrus. During estrus, there was a negative relationship between growth of the diameter of the ovulatory follicle and changes in PI values of the dominant ovarian artery (r = -0.41; P < 0.05). PI values of the uterine arteries and of the ovarian artery ipsilateral to the ovulatory follicle were negatively related to estrogen (E) levels in plasma during estrus (uterine arteries: r = -0.21; P < 0.05; dominant ovarian artery: r = -0.35; P < 0.05). In diestrus, PI values of the dominant ovarian artery were negatively related to plasma progesterone levels (r = -0.38; P < 0.0001), but not the PI values of the uterine arteries (P > 0.05). The findings of this study show that there are characteristic changes in blood supply of the uterus and the ovaries throughout the equine estrous cycle. There are negative correlations between resistance to blood flow in the uterine and ovarian arteries and the plasma estrogen levels during estrus. In diestrus, there is a negative relationship between the resistance to ovarian blood flow and the progesterone levels.     

Transmembrane regulation of intracellular calcium by a plasma membrane sodium/calcium exchanger in mouse ova

Regulation of cytoplasmic free calcium concentration ([Ca2+)]i) is a key factor for maintenance of viability of cells, including oocytes. Indeed, during fertilization of an ovum, [Ca2+]i is known to undergo oscillations, but it is unknown how basal [Ca2+]i or calcium oscillations are regulated. In the present study we investigated the role of the plasma membrane in regulating [Ca2+]i of metaphase II-arrested mouse oocytes (ova). Ova were collected from B6C3F1 mice treated with eCG (10 IU) and hCG (5 IU), and intracellular calcium was determined by means of fura-2. Extracellular calcium flux across the zona pellucida was detected noninvasively by a calcium ion-selective, self-referencing microelectrode that was positioned by a computer-controlled micromanipulator. Under basal conditions ova exhibited a calcium net efflux of 20.6 +/- 5.2 fmol/cm2 per sec (n = 69). Treatment of ova with ethanol (7%) or thapsigargin (25 nM-2.5 microM) transiently increased intracellular calcium and stimulated calcium efflux that paralleled levels of [Ca2+]i. The presence of a Na+/Ca2+ exchanger was indicated by experiments employing both bepridil, an inhibitor of Na+/Ca2+ exchange, and sodium-depleted media. In the presence of bepridil, a net influx of calcium was revealed across the zona pellucida, which was reflected by an increase in the [Ca2+]i. In addition, replenishment of extracellular sodium to ova that had been incubated in sodium-depleted media induced a large calcium efflux, consistent with the actions of Na+/Ca2+ exchange. Sodium/calcium exchange in mouse ova may be an important mechanism that regulates [Ca2+]i.     

Changes in regulation of sodium/calcium exchanger of avian ventricular heart cells during embryonic development

It has been suggested that the sodium/calcium exchanger NCX1 may have a more important physiological role in embryonic and neonatal hearts than in adult hearts. However, in chick heart sarcolemmal vesicles, sodium-dependent calcium transport is reported to be small and, moreover, to be 3–12 times smaller in hearts at embryonic day (ED) 4–5 than at ED18, the opposite of what would be expected of a transporter that is more important in early development. To better assess the role of NCX1 in calcium regulation in the chick embryonic heart, we measured the activity of NCX1 in chick embryonic hearts as extracellular calcium-activated exchanger current (I_NCX) under controlled ionic conditions. With intracellular calcium concentration ([Ca²⁺]_i) = 47 nM, I_NCX density increased from 1.34 ± 0.28 pA/pF at ED2 to 3.22 ± 0.55 pA/pF at ED11 (P = 0.006); however, with [Ca²⁺]_i = 481 nM, the increase was small and statistically insignificant, from 4.54 ± 0.77 to 5.88 ± 0.73 pA/pF (P = 0.20, membrane potential = 0 mV, extracellular calcium concentration = 2 mM). Plots of I_NCX density against [Ca²⁺]_i were well fitted by the Michaelis-Menton equation and extrapolated to identical maximal currents for ED2 and ED11 cells (extracellular calcium concentration = 1, 2, or 4 mM). Thus the increase in I_NCX at low [Ca²⁺]_i appeared to reflect a developmental change in allosteric regulation of the exchanger by intracellular calcium rather than an increase in the membrane density of NCX1. Supporting this conclusion, RT-PCR demonstrated little change in the amount of mRNA encoding NCX1 expression from ED2 through ED18. NCX1; chick embryo; allosteric regulation; sodium/calcium exchange current     

Uterine contractile activity in mares during the estrous cycle and early pregnancy

Transrectal ultrasonography was used to quantitate uterine contractile activity during the estrous cycle and early pregnancy in pony mares (nonbred, n = 9; pregnant, n = 16). Continuous 1-min scans of longitudinal sections of the uterine body were videotaped, and uterine activity scores (1=minimal activity, 5=maximal activity) were assigned to each tape segment. There was a tendency (P<0.06) for a main effect of reproductive status (nonbred versus pregnant), a main effect of day (P<0.0001), and a reproductive status by day interaction (P<0.006). Uterine activity scores were higher (P<0.05) in pregnant mares on Days 1, 11, 12, and 17 (Day 0=day of ovulation) than in nonbred mares. Maximal activity in pregnant mares occurred on Days 11 to 14 during the reported period of maximal embryo mobility. Activity scores decreased (P<0.05) between the day prior to and the day of fixation (mean = Day 15) of the embryonic vesicle. Activity scores were maintained at an intermediate level for several days following fixation before declining to minimal levels by 7 d postfixation. A postovulatory decrease (P<0.04) in activity scores was observed in nonbred mares, but not in pregnant mares, between Days 0 and 1 followed by a progressive increase (P<0.03) between Days 2 and 4. Maximal activity in nonbred mares occurred during the late luteal phase (Days 13 to 14), corresponding temporally to the reported onset of luteolysis.     

Prolactin secretion and its response to stress during the estrous cycle of the rats

Serum and pituitary prolactin (PRL) concentrations were measured during the estrous cycle of the rat with particular attention to the afternoons of the days of proestrus and estrus. Homogenizing machines, a Polytron and Sonifier, were used to extract PRL from the pituitary gland. The effects of ether anesthesia and restraint were also examined on the afternoons of both proestrus and estrus. The occurrence of a surge in PRL secretion during proestrus was confirmed with a peak at 1500 h, and this was accompanied by a decline in pituitary PRL content. A relatively high level of serum PRL was observed in the afternoon of estrus, during which time pituitary PRL content increased progressively. Ether anesthesia had no effect on the proestrus PRL surge, while restraint enhanced it. On the afternoon of estrus, restraint completely suppressed the rise in serum PRL, but ether anesthesia failed to suppress it completely. From these results, the following conclusions were drawn: 1) the PRL surge on the afternoon of proestrus occurs without synthesis of the hormone in the pituitary; 2) PRL secretion on the afternoon of estrus is accompanied by its synthesis in the gland; 3) the PRL response is distinct for each type of stress applied; and 4) PRL secretion is thus regulated by different mechanisms in proestrus and estrus.     

cDNA cloning of rat prolyl oligopeptidase and its expression in the ovary during the estrous cycle

A cDNA for rat prolyl oligopeptidase was cloned which contained an open reading frame of 2,130 nucleotides encoding a protein of 710 amino acids. The deduced amino acid sequence is around 95% homologous to other mammalian prolyl oligopeptidases and about 40% to bacterial prolyl oligopeptidases. The recombinant prolyl oligopeptidase generated in E. coli was purified and its properties were examined. The substrate specificity and the susceptibility to proteinase inhibitors were similar to those of the native enzyme. Northern blot analysis showed wide expression of the prolyl oligopeptidase gene. Using ovaries from hormone-treated rats, it was found that both the mRNA expression and enzyme activity increased in the luteal phase. These findings suggest the involvement of prolyl oligopeptidase in events associated with corpus luteum formation and/or luteal regression.     

Pattern of follicular development during the estrous cycle of aged rats

Summary The pattern of follicular development during the estrous cycles of aged rats was examined and compared with that of mature rats. In both, preovulatory follicles are derived from a select group of small pre-Graafian follicles which begin to develop at estrus and reach the preovulatory size by the morning of proestrus, but the rate of growth, as judged by an increase in the percentage of granulosa cells incorporating ³H-thymidine, is accelerated in the follicles of aged rats. A second mechanism, which accounts for preovulatory follicles in aged rats, involves the rescue from atresia of pre-Graafian and preovulatory follicles. The existence of this mechanism is supported by the observation that at metestrus in aged rats virtually all follicles, regardless of their state of atresia, possess a high percentage of granulosa cells incorporating ³H-thymidine, indicating that the follicles are growing rapidly. However, some of these rapidly growing follicles show signs of atresia such as pyknotic nuclei within their granulosa cell layers. Since follicles in the initial stage of atresia contain defective oocytes (Peluso et al. 1979b), their rescue and development into preovulatory follicles would result in the ovulation of defective oocytes, a fact which accounts in part of the lower fertility in these older animals.     

The antiepileptic effect of sodium valproate during different phases of the estrous cycle in PTZ-induced seizures in rats

Catamenial epilepsy is a form of epilepsy which is related to the menstrual cycle. Cyclic variation in the levels of ovarian hormones plays a pivotal role in its pathogenesis. Sodium valproate (VPA) is one of the oldest antiepileptic drugs (AEDs) which inhibits hepatic metabolizing enzymes. The aim of this study was to evaluate the antiepileptic effects of VPA during different phases of the estrous cycle in rats. 72 adult female Wistar rats in three groups (control, 75 and 100 mg/kg VPA), each with four subgroups (proestrous, estrous, metestrous and diestrous) were used (n = 6). Initially, puberty was assessed using vaginal smears and rats with two regular cycles were selected. VPA with doses 75 and 100 mg/kg was administered intraperitoneally (i.p) in the treatment groups followed by i.p. injection of 80 mg/kg pentylentetrazol (PTZ) in the treatment and control groups. After induction of seizure by PTZ, initiation time of myoclonic seizures (ITMS), initiation time of tonic–clonic seizures (ITTS), seizures duration (SD) and mortality rate (MR) were recorded for 30 min. Data were presented as mean±SD, one-way ANOVA followed by Tukey–Kramer multiple comparison post hoc test were used for analysis of data (P < 0.05). The results of this study showed that VPA significantly improved antiepileptic parameters including ITMS, ITTS, SD, and MR, in which they were significantly more prominent during the luteal phase than the follicular phase (P < 0.05). In addition, there was no significant difference neither between proestrous and estrous nor between metestrous and diestrous in each separately group of rats (P > 0.05).     

Differential expression of estradiol receptors alpha and beta by gonadotropes during the estrous cycle.

This study focused on expression of estradiol receptors (ER) during the estrous cycle. Labeling for ERalpha or beta antigens and luteinizing hormone (LH) or follicle-stimulating hormone (FSH) beta-subunits was done on freshly dispersed pituitary cells. The lowest expression of ERalpha and beta was seen in estrus (23% and 12%, respectively). Expression increased to 42-54% of pituitary cells by diestrus. In males, cells with ERalpha or beta were 37% or 20% of the population, respectively. ERalpha or beta and gonadotropin antigens were in 6-9% of pituitary cells from male rats. Early in the cycle (estrus and metestrus), less than 5% of pituitary cells expressed ERalpha or beta with gonadotropins. These values doubled to reach a peak of 10% during proestrus (just before ovulation). These data show that a rise in expression of both ERalpha and ERbeta is a part of preovulatory differentiation of pituitary gonadotropes.(J Histochem Cytochem 49:665-666, 2001)     

Ionophore-resistant mutant of Toxoplasma gondii reveals involvement of a sodium/hydrogen exchanger in calcium regulation

Calcium is a critical mediator of many intracellular processes in eukaryotic cells. In the obligate intracellular parasite Toxoplasma gondii, for example, a rise in [Ca2+] is associated with significant morphological changes and rapid egress from host cells. To understand the mechanisms behind such dramatic effects, we isolated a mutant that is altered in its responses to the Ca2+ ionophore A23187 and found the affected gene encodes a homologue of Na+/H+ exchangers (NHEs) located on the parasite's plasma membrane. We show that in the absence of TgNHE1, Toxoplasma is resistant to ionophore-induced egress and extracellular death and amiloride-induced proton efflux inhibition. In addition, the mutant has increased levels of intracellular Ca2+, which explains its decreased sensitivity to A23187. These results provide direct genetic evidence of a role for NHE1 in Ca2+ homeostasis and important insight into how this ubiquitous pathogen senses and responds to changes in its environment.