The survival of rat Purkinje cell (PCs) cerebellar cultures was used to test the hypothesis that progesterone is protective againstoxygen-glucose deprivation through potentiation of GABA A receptor activity. Electrophysiological recordings confirm that PCs develop robust excitatory and inhibitory synapses in culture. Exposure of cultured PCs to increasing concentrations of progesterone during oxygen-glucose deprivation revealed a concentration-dependent protection by progesterone, with significant protection observed at physiological concentrations, as low as 10 nm. The concurrent application of the GABA A receptor antagonist picrotoxin (100 µm) completely abolished the neuroprotection afforded by progesterone, indicating that progesterone is neuroprotective through activation of GABA A receptors. Progesterone potentiates GABA A receptor activity indirectly through its metabolites, such as allopregnanolone (ALLO). Therefore, ALLO was applied to PC cultures and was observed to produce significant protection at all concentrations tested, from 10 to 1000 nm. Finally, the inhibition of progesterone metabolism with finasteride abolished the protection afforded by progesterone without having any effect on the neuroprotection caused by ALLO. These data indicate that progesterone protects cerebellar PCs at physiological concentrations through a GABA-active metabolite.
Many studies have reported ischemia protection using various preconditioning techniques, including single dose 3-nitropropionic acid (3-NPA), a mitochondrial toxin. However, the cellular signal transduction cascades resulting in ischemic tolerance and the mechanisms involved in neuronal survival in the tolerant state still remain unclear. The current study investigated the mRNA and protein expression of the antiapoptotic bcl-2 and the proapoptotic bax. two antagonistic members of the bcl-2 gene family, in response to a single dose of 3-NPA, to global cerebral ischemia-reperfusion. and to the combination of both 3-NPA-pretreatment and subsequent global cerebral ischemia-reperfusion. Brain homogenates of adult Wistar rats (n = 25) were analyzed for bcl-2 and bax mRNA expression using a new highly sensitive and quantitative polymerase chain reaction (PCR) technique that allows real-time fluorescence measurements of the PCR product (LightCycler; Roche Diagnostics, Mannheim, Germany). Animals for mRNA analysis received 3-NPA (20 mg/kg, intraperitoneal; "chemical preconditioning") or vehicle (normal saline), and were either observed for 24 plus 3 hours or were subjected to 15 minutes of global cerebral ischemia 24 hours after the pretreatment and observed for 3 hours of reperfusion. Immunohistochemistry was applied to serial brain sections of additional rats (n = 68) to determine amount and localization of the respective Bcl-2 and Bax protein expression in various brain areas. One set of animals was injected with 3-NPA and observed for 3, 12, 24, and 96 hours; a second set was exposed to 15 minutes global cerebral ischemia, 3, 12, and 24 hours reperfusion; and a third set was pretreated with 3-NPA or saline 24 hours before the ischemic brain insult and observed for 96 hours of reperfusion. The authors found single dose 3-NPA treatment to be associated with an elevated bcl-2:bax ratio (increased bcl-2 expression, decreased bax expression), both on the transcriptional (mRNA) and the translational (protein) level. The differential influence of 3-NPA was maintained during early recovery from global cerebral ischemia (3 hours), when 3-NPA pretreated animals showed higher bcl-2 and lower bax mRNA levels compared with rats with saline treatment. Respective changes in protein expression were localized predominately in neurons vulnerable to ischemic damage. Compared with baseline, Bcl-2 protein was significantly higher in surviving neurons at 96 hours after the insult, whereas Bax protein remained unchanged. However, at this late time of postischemic recovery (96 hours), the protein expression pattern of surviving neurons was not different between animals with and without 3-NPA pretreatment. To the authors' knowledge, the current study is the first report on the differential expression of pro- and antiapoptotic genes after a single, nonlethal dose of 3-NPA. The current results suggest alterations in the balance between pro- and antiapoptotic proteins as a potential explanation for the reported protection provided by chemical pr...
The bcl-2 gene is an important antagonist of apoptosis, the programmed cell death. Bcl-2 is highly expressed in a variety of lymphomas. Lymphocytes of patients with chronic lymphocytic leukemia (CLL) express high amounts of bcl-2 even in the absence of the t(14; 18) translocation, resulting in a strong resistance towards corticosteroid induced apoptosis. Within the 5'-untranslated region of the bcl-2 gene a p53 dependent negative response element has been described. Genetic alterations within this element could lead to uncontrolled overexpression of bcl-2 and subsequent resistance towards apoptosis. We therefore analyzed the mRNA from the 5'-untranslated region -279 to -85 bp of the bcl-2 gene by direct PCR sequencing from peripheral blood derived lymphocytes from patients with CLL and normal healthy donors. Compared to published sequences (Tsujimoto and Croce (1986) Proc. Natl. Acad. Sci. USA 83, 5214), we consistently found an exchange at position 1271 from A to G and at position 1284 from G to A in all CLL as well as normal donor derived samples analyzed. Thus, CLL specific alterations compared to normal cells could not be found and deregulated expression of bcl-2 in CLL cells does not appear to be due to alterations in the p53 dependent negative response element of the bcl-2 gene. However, our data add information to published sequence data of this region.
The time course of the behaviour of GABAA receptors (affinity of muscimol for GABA binding sites and their number) in membranes obtained from five distinct rat brain areas was evaluated in dependence of the decline of the serum concentrations of estradiol and progesterone following hormone deprivation by either ovariectomy (OVX) or ovary- plus adrenalectomy (OVX-ADX) after 1, 2, 3, 5, 14 and 30 days. Diestrus rats served as control. The effect of OVX on the GABA binding sites was due to a decrease of affinity rather than alterations in their density. Conversely, the combined surgery of OVX plus ADX affected both the affinity and the density. Also, the OVX-linked changes of muscimol binding were observed only after five days as compared to control animals, while the initial OVX-ADX associated changes occurred already after one day. OVX-ADX produced significant decreases in the number of binding sites at the shortest times investigated, in frontal cortex (FC) at day one and two and in the medulla oblongata (MED) after three days, which disappeared in the subsequent times studied. Concomitant with the major decrease of steroid hormone serum levels one day after OVX-ADX the FC displayed an increase in affinity by 45%. From the time when steroid serum levels are low the alterations in affinity elicited by both OVX-ADX and OVX only take a similar course, reflected by the following decrease in affinity. In MED an additional adrenalectomy did not significantly modify the OVX linked decrease of affinity, except that the initial effect was already evident two days after OVX-ADX.(ABSTRACT TRUNCATED AT 250 WORDS)
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.