In eukaryotic cells, hormones and neurotransmitters that engage the phosphoinositide pathway evoke a biphasic increase in intracellular free Ca 2+ concentration: an initial transient release of Ca 2+ from intracellular stores is followed by a sustained phase of Ca 2+ in¯ux. This in¯ux is generally store dependent. Most attention has focused on the link between the endoplasmic reticulum and store-operated Ca 2+ channels in the plasma membrane. Here, we describe that respiring mitochondria are also essential for the activation of macroscopic store-operated Ca 2+ currents under physiological conditions of weak intracellular Ca 2+ buffering. We further show that Ca 2+ -dependent slow inactivation of Ca 2+ in¯ux, a widespread but poorly understood phenomenon, is regulated by mitochondrial buffering of cytosolic Ca 2+ . Thus, by enabling macroscopic store-operated Ca 2+ current to activate, and then by controlling its extent and duration, mitochondria play a crucial role in all stages of store-operated Ca 2+ in¯ux. Store-operated Ca 2+ entry re¯ects a dynamic interplay between endoplasmic reticulum, mitochondria and plasma membrane.

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In eukaryotic cells, activation of cell surface receptors that couple to the phosphoinositide pathway evokes a biphasic increase in intracellular free Ca 2+ concentration: an initial transient phase re¯ecting Ca 2+ release from intracellular stores, followed by a plateau phase due to Ca 2+ in¯ux. A major component of this Ca 2+ in¯ux is store-dependent and often can be measured directly as the Ca 2+ release-activated Ca 2+ current (I CRAC ). Under physiological conditions of weak intracellular Ca 2+ buffering, respiring mitochondria play a central role in store-operated Ca 2+ in¯ux. They determine whether macroscopic I CRAC activates or not, to what extent and for how long. Here we describe an additional role for energized mitochondria: they reduce the amount of inositol 1,4,5-trisphosphate (InsP 3 ) that is required to activate I CRAC . By increasing the sensitivity of store-operated in¯ux to InsP 3 , respiring mitochondria will determine whether modest levels of stimulation are capable of evoking Ca 2+ entry or not. Mitochondrial Ca 2+ buffering therefore increases the dynamic range of concentrations over which the InsP 3 is able to function as the physiological messenger that triggers the activation of store-operated Ca 2+ in¯ux.

This work was supported by research grants from ISCIII-FEDER (PI11/0091, Red RIC RD12/0042/0029), Consellería de Educación-Generalitat Valenciana (PROMETEO/2011/027), Beca de Investigación Fundación Dexeus para la Salud de la Mujer (2011/0469), and by Fundación Investigación Hospital La Fe (2011/211). A.B-B. has a Contrato Posdoctoral de Perfeccionamiento Sara Borrell-ISCIII (CD13/00005). J.M-A. has a predoctoral grant PFIS-ISCIII (FI12/00012). The authors have no conflicts of interest to declare.

Endometrium and peritoneal fluid from women with endometriosis have increased levels of VEGF, uPA and MMP-3 levels. Therefore, the development of endometriotic implants at ectopic sites may be facilitated, promoting the progress of the endometriosis.

The increase in antigenic levels of uPA and MMP-3 in endometrium of women with endometriosis might contribute to the invasive potential of endometrial cells. Once the ovarian endometriotic cyst is developed, an increase in PAI-1 and TIMP-1 is detected and significant proteolytic activity is no longer observed. This increase in inhibitors and decrease in proteolytic activity could explain the frequent clinical finding of isolated endometriotic cyst without invasion of the surrounding ovarian tissue.

This report defines the nature of the molecules responsible for the increased plasma plasminogen activator inhibitor (PAI) activity in preeclamptic patients and the relationship of these inhibitors to the severity of placental damage in preeclampsia. Clinical groups consisting of pregnant women with either severe preeclampsia or chronic hypertension with superimposed severe preeclampsia, as well as normal pregnant and nonpregnant women, were analyzed in a panel of functional and immunologic assays for PAI-1 and PAI-2. Pure severe preeclamptic patients in their third trimester showed a significant increase in both antigenic (136 ng/mL) and functional (5.76 U/mL) type 1 PAI (PAI-1) as compared with normal third-trimester pregnant women (34.8 ng/mL and 2.57 U/mL, respectively). In contrast, antigenic (186 ng/mL) and functional (5.76 U/mL) levels of type 2 PAI (PAI-2) were significantly lower in the pure severe preeclampsia group as compared with the values of the normal pregnant group (269 ng/mL and 9.58 U/mL, respectively). The patients with chronic hypertension and superimposed severe preeclampsia exhibited PAI-2 levels comparable to those of the pure preeclamptic group, whereas their antigenic and functional PAI-1 levels were intermediate (94 ng/mL and 3.25 U/mL, respectively) between the normal pregnant and the pure preeclamptic groups. During early puerperium of both normal pregnant women and patients, plasma PAI-1 antigen and activity decreased within one day to approximately the levels detected in normal nonpregnant women, while PAI-2 levels remained elevated for over 11 days. Similar results were obtained in plasma samples obtained from citrated blood and blood collected with an anticoagulant/antiplatelet mixture, suggesting that increased PAI-1 levels in preeclamptic patients were not due to platelet activation in vitro. In preeclamptic patients, a positive correlation between birth weight and PAI-2 values was observed (r = .64, P less than .05), whereas birth weight was inversely correlated with both PAI-1 levels and total PAI activity (r = -.6, P less than .005 and r = -.76, P less than .005 respectively). Preeclamptic patients with extensive placental infarction exhibited higher plasma PAI activity (24.1 U/mL v 11.6 U/mL) and PAI-1 values (305 ng/mL v 80.9 ng/mL) than preeclamptic patients without extensive placental infarction. In contrast, PAI-2 levels were reduced in preeclamptic patients with infarction in comparison with those of patients without infarction (141 ng/mL v 212.9 ng/mL). Our data indicate that increases in the level of PAI-1 accounts for the high plasma PAI activity in severe preeclampsia as measured using single-chain t-PA.

1. This study was planned to clarify the mechanism of Ca2+ channel facilitation by depolarizing prepulses given to voltage-clamped bovine chromaffin cells. The hypothesis for an autocrine modulation of such channels was tested by studying the effects of a soluble vesicle lysate (SVL) on whole-cell Ba2+ currents (IBa).2. SVL was prepared from a bovine adrenal medullary homogenate. The ATP content in this concentrated SVL amounted to 3a 18 + 0a12 mm (n = 4). The concentration of noradrenaline and adrenaline present in the SVL was 11 2 + 0 97 and 15X2 + 2 mm, respectively (n = 5). A 1:1000 dilution of SVL in the external solution halved the magnitude of IBa and produced a 7-fold slowing of its activation kinetics. The blocking effects of SVL were concentration dependent and quickly reversed upon washout. 3. Inhibition and slowing of the kinetics of IBa by SVL could be partially reversed by strong depolarizing prepulses (+90 mV, 45 ms). This reversal of inhibition, called Ca2P channel facilitation, persisted in the presence of 3 uM nifedipine.4. Intracellular dialysis of GDP-/l-S (0 5 mM) or pretreatment of the cells with pertussis toxin (100 ng ml-' for 18-24 h) prevented the reduction in peak current caused by a 1 :100 dilution of SVL; no prepulse facilitation could be observed under these conditions. 5. The receptor blockers naloxone (10 uM) or suramin (100 uM) and PPADS (100 uM) largely antagonized the effects of SVL. Treatment of SVL with alkaline phosphatase or dialysis against a saline buffer to remove low molecular mass materials (<1O kDa) considerably reduced the activity of SVL.6. Stopping the flow of the external solution (10 mm Ba2+) gradually reduced the size, and slowed down the activation phase, of the current. Prepulse facilitation of IBa was absent or weak in a superfused cell, but was massive upon flow-stop conditions in the presence or absence of 3 uM nifedipine. 7. Our experiments suggest that facilitation by prepulses of whole-cell current through Ca2+ channels is due to the suppression of an autoinhibitory autocrine loop present in bovine chromaffin cells. By acting at least on purinergic and opiate receptors, the exocytotic release of ATP and opiates will cause a tonic inhibition of the current through a G-proteinmediated mechanism. Such a mechanism will be removed by strong depolarizing prepulses, and will involve preferentially non-L-type channels. In the light of these and other recent results, previously held views on the selective recruitment by prepulses of dihydropyridinesensitive Ca2+ channels are not tenable.t To whom correspondence should be addressed.