The age-related decline in testosterone biosynthesis in testicular Leydig cells has been well documented, but the mechanisms involved in the decline are not clear. Recent studies have described a cyclooxygenase-2 (COX2)-dependent tonic inhibition of Leydig cell steroidogenesis and expression of the steroidogenic acute regulatory protein (StAR). The present study was conducted to determine whether COX2 protein increases with age in rat Leydig cells and whether COX2 plays a role in the age-related decline in testosterone biosynthesis. Our results indicate that from 3 months of age to 30 months, COX2 protein in aged rat Leydig cells increased by 346% over that of young Leydig cells, StAR protein decreased to 33%, and blood testosterone concentration and testosterone biosynthesis in Leydig cells decreased to 41 and 33%, respectively. Further experiments demonstrated that overexpressing COX2 in MA-10 mouse Leydig cells inhibited StAR gene expression and steroidogenesis and that the inhibitory effects of COX2 could be reversed by blocking COX2 activity. Notably, incubation of aged Leydig cells with the COX2 inhibitor NS398 enhanced their testosterone biosynthesis. Blood testosterone concentrations in aged rats fed the COX2 inhibitor DFU, at doses of 5, 10, 15, and 20 mg/kg body weight per day were increased by 15, 23, 56, and 120%, respectively, over the levels in the rats receiving no DFU. The present study suggests a novel mechanism in male aging involving COX2 and a potential application of the mechanism to delay the age-related decline in testosterone biosynthesis.

Steroidogenic acute regulatory protein (StAR) is a nuclear encoded mitochondrial protein that enhances steroid synthesis by facilitating the transfer of cholesterol to the inner membranes of mitochondria in hormonally regulated steroidogenic cells. It is currently assumed that StAR activity commences before or during StAR import into the mitochondrial matrix. The present study was designed to demonstrate that, once imported and becoming physiologically irrelevant, exhaustive accumulation of StAR must be limited by a rapid degradation of the protein to prevent potential damage to the organelles. The use of uncouplers and manipulation of the interior mitochondrial pH in hormone-induced ovarian granulosa cells and StAR-expressing COS cells suggests that StAR degradation is biphasic and involves two classes of proteases. During phase I, which normally lasts for the first approximately 2 h following import, StAR is rapidly degraded by a protease, or proteases, that can be arrested by a nonclassical action of proteasome inhibitors such as MG132. StAR molecules that evade phase I are subjected to a second class of protease(s), which is slower and MG132 resistant. A third proteolytic entity was revealed in studies with C-28 StAR, a loss-of-function mutant of StAR. Upon initiation of its import, C-28 StAR dissipates the inner membrane potential and causes swelling of the mitochondria. Degradation of C-28 StAR, probably by an intermembrane space protease, is extremely rapid and MG132 insensitive. Collectively, this study defines StAR as the first naturally occurring mitochondrial protein that can serve as a substrate to probe multiple proteolytic activities in mammalian mitochondria.

The present study sought to characterize the concerted action of FSH and insulin-like growth factor-1 (IGF-1) on functional differentiation of prepubertal rat ovarian granulosa cells in culture. To this end, we examined the regulation of three key genes encoding pivotal proteins required for progesterone biosynthesis, namely, side-chain cleavage cytochrome P450 (P450(scc)), steroidogenic acute regulatory (StAR) protein, and 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD). Time-dependent expression profiles showed that P450(scc), StAR, and 3beta-HSD gene products accumulate in chronic, acute, and constitutive patterns, respectively. Each of these genes responded to FSH and/or IGF-1 in a characteristic manner: A synergistic action of IGF-1 was indispensable for FSH induction of P450(scc) mRNA and protein; IGF-1 did not affect FSH-mediated upregulation of StAR products; and IGF-1 alone was enough to promote expression of 3beta-HSD. The responsiveness of the genes to IGF1 correlated well with their apparent susceptibility to the inhibitory impact of tyrphostin AG18, a potent inhibitor of protein tyrosine kinase receptors. Thus, IGF-1-dependent P450(scc) and 3beta-HSD expression was completely arrested in the presence of AG18, whereas StAR expression was unaffected in the presence of tyrphostin. These findings suggest that FSH/cAMP signaling and IGF-1/tyrosine phosphorylation events are interwoven in rat ovarian cells undergoing functional differentiation. We also sought the mechanism of IGF-1 synergy with FSH. In this regard, our studies were unable to demonstrate a stabilizing effect of IGF-1 on P450(scc) mRNA, nor could IGF-1 augment FSH-induced transcription examined using a proximal region of the P450(scc) promoter (-379/+6). Thus, the mechanism of IGF-1 and FSH synergy remains enigmatic and provides a major challenge for future studies.

The Steroidogenic Acute Regulatory (StAR) protein is a mitochondrial protein required for the transport of cholesterol substrate to the P450scc enzyme located in the inner mitochondrial membranes of steroid producing cells. This study suggests that the acute regulation of the rodent StAR gene in the ovary is mediated by two factors, C/EBPbeta and GATA-4. Once translated, the StAR precursor protein is either imported into the mitochondria, or it is rapidly degraded in the cytosol. We predicted that in order to perpetuate StAR activity cycles, imported StAR should turn over rapidly to avoid a potentially harmful accumulation of the protein in sub-mitochondrial compartments. Pulse-chase experiments in metabolically labeled cells showed that: (a) the turnover rate of mature mitochondrial StAR protein (30kDa) is much faster (t(1/2)=4-5h) than that of other mitochondrial proteins; (b) dissipation of the inner membrane, potential (-Deltapsi) by carbonyl cyanide m-chlorophenylhydrazone (mCCCP) accelerates the mitochondrial degradation of StAR; (c) unexpectedly, the mitochondrial degradation of StAR is inhibited by MG132 and lactacystin, but not by epoxomicin. Furthermore, StAR degradation becomes inhibitor-resistant two hours after import. Therefore, these studies suggest a bi-phasic route of StAR turnover in the mitochondria. Shortly after import, StAR is degraded by inhibitor-sensitive protease(s) (phase I), whereas at later times, StAR turnover proceeds to completion through an MG132-resistant proteolytic activity (phase II). Collectively, this study defines StAR as a unique protein that can authentically be used to probe multiple proteolytic activities in mammalian mitochondria.

The steroidogenic acute regulatory (StAR) protein, which mediates cholesterol delivery to the inner mitochondrial membrane and the P450scc enzyme, has been shown to require a mitochondrial electrochemical gradient for its activity in vitro. To characterize the role of this gradient in cholesterol transfer, investigations were conducted in whole cells, utilizing the protonophore carbonyl cyanide m-chlorophenylhydrazone (m-CCCP) and the potassium ionophore valinomycin. These reagents, respectively, dissipate the mitochondrial electrochemical gradient and inner mitochondrial membrane potential. Both MA-10 Leydig tumor cell steroidogenesis and mitochondrial import of StAR were inhibited by m-CCCP or valinomycin at concentrations which had only minimal effects on P450scc activity. m-CCCP also inhibited import and processing of both StAR and the truncated StAR mutants, N-19 and C-28, in transfected COS-1 cells. Steroidogenesis induced by StAR and N-47, an active N-terminally truncated StAR mutant, was reduced in transfected COS-1 cells when treated with m-CCCP. This study shows that StAR action requires a membrane potential, which may reflect a functional requirement for import of StAR into the mitochondria, or more likely, an unidentified factor which is sensitive to ionophore treatment. Furthermore, the ability of N-47 to stimulate steroidogenesis in nonsteroidogenic HepG2 liver tumor cells, suggests that the mechanism by which StAR acts may be common to many cell types.

Steroidogenic acute regulatory protein (StAR) is a vital accessory protein required for biosynthesis of steroid hormones from cholesterol. The present study shows that in primary granulosa cells from prepubertal rat ovary, StAR transcript and protein are acutely induced by gonadotropin (FSH). To determine the sequence elements required for hormone inducibility of the StAR promoter, truncated regions of the -1002/+6 sequence of the mouse gene were ligated to pCAT-Basic plasmid and transfected by electroporation to freshly prepared cells. FSH inducibility determined over a 6-h incubation was 10-40-fold above basal levels of chloramphenicol acetyltransferase activity. These functional studies, supported by electrophoretic mobility shift assays indicated that two sites were sufficient for transcription of the StAR promoter constructs: a non-consensus binding sequence (-81/-72) for CCAAT enhancer-binding protein beta (C/EBP beta) and a consensus motif for GATA-4 binding (-61/-66). Western analyses showed that GATA-4 is constitutively expressed in the granulosa cells, while all isoforms of C/EBP beta were markedly inducible by FSH. Site-directed mutations of both binding sequences practically ablated both basal and hormone-driven chloramphenicol acetyltransferase activities to less than 5% of the parental -96/+6 construct. Unlike earlier notions, elimination of potential binding sites for steroidogenic factor-1, a well known tissue-specific transcription factor, did not impair StAR transcription. Consequently, we propose that C/EBP beta and GATA-4 represent a novel combination of transcription factors capable of conferring an acute response to hormones upon their concomitant binding to the StAR promoter.

Abstract: Cultured cerebellar granule cells were subjected to toxic activation of the NMDA receptor that was terminated by MK‐801. Subsequent resuscitation experiments were mostly conducted in the presence of a physiological concentration of Ca2+. Addition of pyruvate and inorganic phosphate, in addition to glucose, which was always present, rescued ∼40% of the dying neurons. La3+ and ruthenium red were also effective resuscitating agents. The combination of pyruvate, inorganic phosphate, and ruthenium red rescued 65% of the dying neurons. Parallel studies with 45Ca indicated that La3+ and ruthenium red facilitated the decrease of 45Ca in the neurons, whereas inorganic phosphate, supported by energy‐yielding pyruvate, formed perhaps, a less harmful Ca complex inside the neurons.

Excessive entry of Ca2+ through the NMDA receptor is thought to be the major cause of glutamate toxicity in brain neurons. However, actual quantitation of the calcium overload has not been achieved. Here we show that the absolute amount of Ca-45(2+) taken up via the NMDA receptor correlates quantitatively with the amount of acute cell death in cultured cerebellar granule cells of the rat. Analysis of 9- and 16-day cultures reveals that the NMDA-induced Ca2+ uptake is about the same at these ages, whereas the Ca-dependent lethal process is more developed in the older neurons. The calculated lethal concentration of Ca-45 taken up exceeds by similar to 10,000 times the maximal concentration of [Ca2+] that can be measured by fluorescence imaging. It is suggested that the Ca2+ taken up induces the lethal process in a subcellular structure in which it has been segregated.

Abstract: The activities mediated by the N‐methyl‐D‐aspartate (NMDA) receptor were studied in cultured rat cerebellar granule cells. Micromolar concentrations of the metal binding compounds, EDTA, cysteine, and histidine, as well as serum albumin strongly potentiated receptor activity in the presence of millimolar concentrations of Ca2+ and Mg2+. The findings indicated that these agents remove an endogenous metal, probably Zn2+, which attenuates NMDA receptor‐mediated 45Ca uptake and toxicity. Several added metal ions were therefore tested at low micromolar concentrations. Zn2+ was found to be the most potent inhibitor of NMDA‐induced 45Ca uptake, followed by Cu2+ and Fe2+. Co2+, Cd2+, Fe3+, and AI3+ had no significant effect, whereas Ni2+ potentiated the 45Ca uptake but inhibited at much higher concentrations. The potentiating agents that remove the endogenous metal had a particularly dramatic effect in the presence of Mg2+, the voltage‐dependent suppressor of the NMDA receptor. Mg2+ also played an important role in the inhibitory effect of added Zn2+. Much lower concentrations of Zn2+ were needed to achieve inhibition of NMDA‐induced 45Ca uptake in the presence of Mg2+. Under a variety of conditions, a very good correlation was found between NMDA receptor‐mediated 45Ca uptake and the magnitude of acute neurotoxicity.

Glutamate induced 45Ca influx and toxicity were enhanced 2-10 fold by EDTA. A chelator concentration of 10 microM, which was equivalent to less than 1% of the Mg2+ and Ca2+ concentration in the medium, was effective. The chelator revealed no activity on its own and caused potentiation only when present simultaneously with the agonist of the NMDA receptor. Cysteine, which is known to bind certain metals tightly through its sulfhydryl group, and another chelator, O-phenanthroline, produced the same effect as EDTA. The findings indicate that when the N-methyl-D-aspartate receptor is activated, an endogenous metal can become bound to a chelator or to a physiological metal binding agent, such as cysteine, leading to enhanced Ca2+ influx into the neuron and toxicity.

Cell death was due to activation of the receptor responsive to N-methyl-D-aspartate (NMDA). Even during a 30-min incubation, glutamate toxicity was unaffected by 5 microM CNQX and was greater than 90% blocked by 160 nM MK-801, a specific antagonist of the NMDA receptor. The extent of toxicity was dependent on the age of the cultured cells, the concentration of KCl, which determined membrane polarization, the duration of exposure to, and the concentration of, glutamate. Toxicity was critically pH-dependent, increasing from a minimal effect at pH 7.0 to an extremely high level at pH 8.0. Among a number of acidic amino acids tested, glutamate showed the highest potency. Aspartate, cysteine sulfinate and homocysteate reached the same maximal toxicity, but at a 25 times higher concentration. Cysteate and quinolinate were of very low potency. Zn2+, at 0.5 microM, attenuated glutamate toxicity considerably. At concentrations above 5 microM the metal ion showed strong toxicity on its own. Dialysed serum and serum albumin strongly potentiated glutamate and NMDA toxicity in presence and absence of Mg2+. A concentration of 2 mg/ml bovine serum albumin caused maximal potentiation; fatty acid-free bovine serum albumin and human serum albumin were all about equipotent. To be effective, the serum albumin had to act simultaneously with glutamate. Among a number of proteins and other polymers, only casein showed an ability to potentiate glutamate toxicity, similar to serum albumin. It is concluded that minor effects of glutamate toxicity could be considerably aggravated by the potentiating activity of serum albumin, also in pathological situations in vivo.

Two different processes, mediated by the N-methyl-D-aspartate receptor, appear to cause acute cell death in cultured cerebellar granule cells. A Ca2+-independent process takes place at zero and very low concentration of the added cation. Under these conditions, the known destabilization of excitable membranes at low extracellular Ca2+ probably plays a major role. A Ca2+-dependent process becomes dominant as its concentration is increased above 1.0 mM. The remarkable potentiation of glutamate toxicity by serum albumin is a calcium-dependent reaction.

The life span of neonatal rat cerebellar granule cells, grown in basal minimal Eagle's medium containing 10% (vol/vol) fetal calf serum, was extended to 21-30 days by weekly supplementation with glucose. Addition of 1% fetal calf serum to the culture at 14 days killed 85% of the cells within 1 hr. This lethal effect could be prevented by the N-methyl-D-aspartate (NMDA) receptor antagonists dibenzocyclohepteneimine (MK-801) and 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate (CPP). These findings suggested that the glutamate in the serum caused the dramatic neuronal death through action on the NMDA receptor. Indeed, a 5-min incubation in a Locke physiological salt solution containing 20 microM glutamate and 5 microM glycine killed 55-90% of the cells. This acute toxicity could be prevented by a lyso-GM1 ganglioside with N-acetylated sphingosine. The relatively low glutamate content of the sera analyzed suggests that factors in addition to glycine potentiate serum neurotoxicity. The above noted antagonists of the NMDA receptor also greatly reduced the lethal effect of depolarization by 90 mM KCl or 10 microM veratridine. Therefore, it is likely that the toxicity of the depolarizing agents is mediated by glutamate released from the cells. It is concluded that survival of cerebellar neurons in primary culture may be strongly affected by unsuspected neurotoxic phenomena elicited by brief action of a rather low glutamate concentration.

Cysteine sulfinic acid, a putative transmitter in the brain induces release ofd-[3H]aspartate and [14C]GABA without the help of any general depolarizing agent. Tetrodotoxin partially blocks the release ofd-[3H]aspartate and completely blocks the induced release of [14C]GABA. Withdrawal of Ca2+ from the medium does not affect thed-[3H]aspartate release, but increases the extent of inhibition by tetrodotoxin. In contrast, removal of Ca2+ increases the cysteine sulfinic acid-induced [14C]GABA release, which remains totally blocked by the toxin.Anemonia sulcata toxin type II, which slows down Na+ channel inactivation, acts in synergism with cysteine sulfinic acid to increase the rate of release of both of the labeled amino acids. Comparison of glutamate with cysteine sulfinic acid in the same experiments indicates a different action pattern of the two acidic amino acids. Forskolin plus isobutyl methyl xanthine, which are known to raise intracellular cyclic adenosine monophosphate (cyclic AMP) levels, caused little release of the labeled amino acids on their own, but strongly enhanced the cysteine sulfinic acid-induced release. The experiments conducted by double labeling withd-[3H]aspartate and [14C]GABA, revealed several characteristic differences between the glutamatergic and the GABAergic neurons. It is tentatively concluded that cysteine sulfinic acid brings about excitation of the glutamatergic as well as the GABAergic neurons, leading to opening of Na+ channels which play a role in the release in both systems. Cyclic AMP, presumably by initiating phosphorylation of a specific component, has a remarkable potentiating effect on the release.

Extended incubation of hippocampus slices in the presence of fatty acid-free bovine serum albumin (FAF-BSA) strongly enhanced the release of d-[3H]aspartate and [14C]GABA induced by veratridine. Saturation of the FAF-BSA with oleic acid abolished the enhancing effect. Spontaneous release and K+-induced release were not significantly changed by the addition of FAF-BSA. Amino-oxyacetic acid in the medium enhanced the veratridine-induced release of d-[3H]aspartate. The spontaneous release of [14C]GABA was greatly increased by Ca2+ withdrawal. With the further addition of EGTA the spontaneous release in the absence of Ca2+ increased more than 8-fold over that measured in the presence of 1.5 mM Ca2+. The enhanced release caused by Ca2+ withdrawal was totally blocked by tetrodotoxin. The toxin was effective even when added after the spontaneous release in the absence of Ca2+ was already proceeding at a high rate. The veratridine-induced release of [14C]GABA was also considerably augmented by Ca2+ withdrawal. d-[3H]aspartate release, studied simultaneously with [14C]GABA by double labeling, did not show enhanced spontaneous release upon Ca2+ withdrawal. The findings provide evidence that the enhanced [14C]GABA release caused by Ca2+ withdrawal is mediated by voltage-dependent Na+ channels.

Detergent-solubilized preparations of the beta-adrenergic receptor (R) and of the guanyl nucleotide binding proteins (Gs) were extensively treated to remove phospholipids and cholesterol. Reconstitution of an R-Gs system was subsequently performed in the presence of a mixture of natural phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine or the synthetic dioleoyl derivatives of the same phospholipids. In both cases, an additional lipid was required for the agonist-dependent activation of Gs. The requirement could be fulfilled by alpha-tocopherol, or by unsaturated fatty acids such as oleic acid. Inclusion of this non-phosphorylated lipid in the reconstituted system enhanced the isoproterenol-dependent activation of Gs by guanosine 5'-O-[gamma-thio]triphosphate 16-33-fold. The rate of activation was largely dependent on the addition of the agonist. Efficient functional reconstitution of R-Gs was thus achieved in a totally defined lipid system. Additional studies of the reconstituted system and of the native membrane led to the notion that the non-phosphorylated lipid plays a role in the function of the hormone-R complex.