Regulation of presynaptic calcium in a mammalian synaptic terminal.

Ca(2+) signaling in synaptic terminals plays a critical role in neurotransmitter release and short-term synaptic plasticity. In the present study, we examined the role of synaptic Ca(2+) handling mechanisms in the synaptic terminals of mammalian rod bipolar cells, neurons that play a pivotal role in the high-sensitivity vision pathway. We found that mitochondria sequester Ca(2+) under conditions of high Ca(2+) load, maintaining intraterminal Ca(2+) near resting levels. Indeed, the effect of the mitochondria was so powerful that the ability to clamp intraterminal Ca(2+) with a somatically positioned whole cell patch pipette was compromised. The plasma membrane Ca(2+)-ATPase (PMCA), but not the Na(+)/Ca(2+) exchanger (NCX) or the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), was an important regulator of resting Ca(2+). Furthermore, PMCA activity, but not NCX or SERCA activity, was essential for the recovery of Ca(2+) levels following depolarization-evoked Ca(2+) entry. Loss of PMCA function was also associated with impaired restoration of membrane surface area following depolarization-evoked exocytosis. Given its roles in the regulation of intraterminal Ca(2+) at rest and after a stimulus-evoked Ca(2+) rise, the PMCA is poised to modulate luminance coding and adaptation to background illumination in the mammalian rod bipolar cell.

Interaction between mGluR8 and calcium channels in photoreceptors is sensitive to pertussis toxin and occurs via G protein betagamma subunit signaling.

PURPOSE: The most recently identified metabotropic glutamate receptor (mGluR), type 8 mGluR (mGluR8), has been identified functionally as a presynaptic autoreceptor in rod photoreceptors. This study analyzed the mechanism of action underlying mGluR8 activity and modulation of the cytosolic Ca2+ concentration in mouse photoreceptors. METHODS: The cytosolic Ca2+ concentration of acutely isolated rod photoreceptors was monitored optically with microspectrofluorimetry and in the presence of modulators of G protein activity. RESULTS: mGluR8 activation by the group III mGluR agonists l-2-amino-4-phosphonobutyrate and l-serine-O-phosphate or the physiological ligand l-glutamate produced a decrease in influx of extracellular Ca2+ into the cytosol. Pretreatment of isolated rod photoreceptors with the G protein uncoupler suramin or pertussis toxin, which inactivates Gi/o/z proteins and Gt protein/transducin, or a G protein betagamma subunit-inhibiting peptide abolished this activity. Preincubation of cells with cholera toxin (CTX), an activator of Gs protein, had no effect. CONCLUSIONS: These results suggest that the function of mGluR8 of modulating the cytosolic Ca2+ concentration and thereby potentially the release of neurotransmitter from rod spherules, the axon terminal systems of rod photoreceptors, is mediated by a pertussis toxin-sensitive G protein potentially via the betagamma subunit. The absence of Go and Gz proteins, as reported previously, implies a novel potential interaction between Gi2 and/or Gt protein/transducin and mGluR8 in photoreceptors. These results have potential implications for the regulatory function and pharmacologic targeting of mGluR8 in photoreceptors.

Differentially distributed IP3 receptors and Ca2+ signaling in rod bipolar cells.

PURPOSE: Inositol (1,4,5)-trisphosphate receptors (IP3Rs) contribute substantially to cytosolic free calcium ion (Ca2+) concentration transients and thereby modulate neuronal function. The present study was undertaken to determine the contribution of IP3Rs to the function of rod bipolar cells in the retina. METHODS: Immunoreactivity for IP3Rs in rod bipolar cells from mouse retinas was detected by immunocytochemical methods. Intracellular Ca2+ concentrations were optically recorded in acutely isolated rod bipolar cells, and biophysical properties of IP3Rs were analyzed with single channel electrophysiology. RESULTS: The distribution of IP3R isoforms was correlated with cytosolic Ca2+ transients induced by activation of group I metabotropic glutamate receptors (mGluRs) and with biophysical properties of differentially expressed IP3Rs. CONCLUSIONS: The differential distribution of IP3Rs is used by rod bipolar cells to convey Ca2+ signals that are distinct in their duration, amplitude, and kinetics at the subcellular level, and that serve the functions of individual subcellular compartments. IP3R-mediated Ca2+ signaling indicates a potential mechanism for the adaptation of the ON-pathway of vision and for coincidence and threshold detection in retinal neurons.

Uterine molecular responses to bisphenol A treatment before and after decidual induction in pseudopregnant rats.

The results of the study demonstrate that the weak estrogenic action of bisphenol A (a daily subcutaneous dose of 200 mg/kg on 4 consecutive days, administered before or after decidual induction that occurs on day 4 of pseudopregnancy) on deciduoma growth in pseudopregnant Sprague-Dawley rats, was functionally associated with the hormonal status of the uterus. Whereas bisphenol A displayed uterotrophic action during the pre-decidual, estrogen related period, it inhibited decidual growth and progesterone secretion during the post-decidual, progesterone-dominated period. The estrogenic action of bisphenol A on uterine decidual growth was not correlated with the reduced levels of estrogen receptor binding sites and mRNA expression, nor the unchanged serum estradiol concentrations. BPA action appeared to be antagonized by progesterone.

Neuroprotective effects of nonfeminizing estrogens in retinal photoreceptor neurons.

PURPOSE: Retinal diseases such as macular degeneration and glaucoma are disorders that target specific retinal neurons that can ultimately lead to vision loss. Under these conditions and pathologies, retinal neurons can die via apoptosis that may be due to increased oxidative stress. The neuroprotective effects of 17ß-estradiol (E2) and three synthetic nonfeminizing estrogen analogs (ZYC-26, ZYC-23, and ZYC-3) were investigated to examine their abilities to protect retinal neurons against glutamate toxicity. METHODS: Using an in vitro model of glutamate-induced cell death in 661W cells, a mouse cone photoreceptor cell line, shown to express both estrogen receptors (ERs) via immunoblotting, was pretreated with E2 and its analogs and cell viability were assessed. RESULTS: It was observed that E2 and estrogen analogs, ZYC-26 and ZYC-3, were protective against a 5 mM glutamate insult in 661W cells. The neuroprotective abilities of ZYC-26 and ZYC-3 were autonomous of estrogen receptor-α (ERα) and ERß demonstrated by their ability to protect in the presence of ICI 182780, a pan-ER antagonist with a high affinity for the estrogen receptor. Treatment with PPT and DPN, ERα- and ERß-specific agonists, respectively, did not protect the 661W cells from the glutamate insult. Studying the membrane ER (mER) or GPR30 did show that activation of the receptor by G1 protected the retinal neuron from insult, whereas G15, an antagonist of the mER was not able to antagonize the protection previously seen. CONCLUSIONS: These data demonstrate that nonfeminizing estrogens may emerge as useful compounds for neuroprotection of retinal cells.

Progesterone potentiates IP(3)-mediated calcium signaling through Akt/PKB.

The activity of cells critically depends on the control of their cytosolic free calcium ion (Ca(2+)) concentration. The objective of the present study was to identify mechanisms of action underlying the control of the gain of intracellular Ca(2+) release by circulating gonadal steroid hormones. Acute stimulation of isolated neurons with progesterone led to IP(3)R-mediated Ca(2+) transients that depend on the activation of the PI3 kinase/Akt/PKB signaling pathway. These results were confirmed at the molecular level and phosphorylation of IP(3)R type 1 by Akt/PKB was identified as the mechanism of action. Hence, it is likely that circulating gonadal steroid hormones control neuronal activity including phosporylation status through receptor- and kinase-mediated signaling. With a direct control of the gain of the Ca(2+) second messenger system as a signaling gatekeeper for neuronal activity the present study identifies a novel pathway for interaction of the endocrine and central nervous system.

Uterine-ovarian biochemical and developmental interactions to the postimplantation treatment with a butadiene metabolite, diepoxybutane, in pregnant rats.

An industrial chemical used in synthetic rubber production, 1,3-butadiene, is toxic to reproduction in rats and mice. Bioactivation of butadiene to reactive intermediates, i.e. diepoxybutane and other metabolites, is responsible for this toxicity. The present study examines the biochemical and developmental mechanisms of diepoxybutane at the feto-maternal placental axis during gestation. Female Sprague-Dawley rats were administered four daily intraperitoneal doses of diepoxybutane in groups (0.25, 0.30, 0.35, or 0.40 mmol in sesame oil per kg body weight, n = 6/group) during postimplantation (gestation days 5-8) and euthanized on gestation day 9 or 12 for retrieval of uterine and ovarian tissues, and serum for assays. The results demonstrate that this timely diepoxybutane treatment significantly decreased placental levels of pituitary adenylate cyclase-activating polypeptide mRNA expression that was measured by reverse transcription-polymerase chain reaction and of matrix metalloproteinase-9 activity that was determined by gelatin zymography, and serum progesterone levels on gestation days 9 and 12. From a developmental standpoint, fetal growth and viability were reduced in correlation with treatment-related effects of diepoxybutane on implantation losses and fetal resorptions on gestation day 9. Additionally, fetal mortality was maximally increased due to significantly pronounced, dose-independent effects on these parameters on gestation day 12. This trend towards more severe embryolethal treatment effects from gestation day 9 to 12 suggests that fetal metabolism in the gravid uteri of rats may be more sensitive to diepoxybutane exposure as pregnancy progresses. The inhibitory actions of diepoxybutane on placental pituitary adenylate cyclase-activating polypeptide expression and matrix metalloproteinase activity may contribute towards altering placental molecular support for fetal development and viability. Moreover, the reproductive toxicity of diepoxybutane in rats appears to be linked to progesterone action.