Glucocorticoid interactions with ethanol effects on depolarization-induced calcium influx in brain synaptosomes.

Depolarization-induced Ca2+ influx in brain synaptosomes is known to be inhibited by ethanol and stimulated by glucocorticoids. The present study was undertaken to characterize the interactions of corticosterone (CORT) with ethanol effects on 45Ca2+ uptake in synaptosomes depolarized by high K+ (70 mM). CORT was shown to antagonize the inhibitory effects of ethanol on the fast-phase component of the K(+)-induced 45Ca2+ uptake (the initial 3 s following depolarization). Glucocorticoid antagonism of ethanol inhibition of the 45Ca2+ uptake exhibited a high degree of steroid specificity; steroids with glucocorticoid activity including cortisol, dexamethasone and triamcinolone were effective, whereas gonadal steroids and excitatory neuroactive steroid metabolites were ineffective. From the shift of concentration-response relationships when CORT and ethanol were present in combination, the interaction between steroid stimulation and ethanol inhibition of 45Ca2+ uptake occurred in an additive manner over the range of their effective concentrations. Parallel to 45Ca2+ uptake, the binding sites for [3H]PN 200-110 were reduced by ethanol and increased by CORT. These opposite effects on [3H]dihydropyridine labeled sites were found also to antagonize each other, and the antagonism again occurred in an additive relationship. These results demonstrate that glucocorticoids antagonized ethanol inhibition of voltage-dependent Ca2+ channel activity in brain synaptosomes, and support the notion that these steroids may be among the endogenous factors that modulate neuronal sensitivity to ethanol.

Glucocorticoid interactions with ethanol effects on synaptic plasma membranes: influence on [125I]calmodulin binding.

Ca(++)-dependent binding of calmodulin (CaM) to brain synaptic plasma membranes is known to be inhibited by ethanol and stimulated by glucocorticoids. These opposite neurochemical actions between ethanol and the steroids in vitro are consistent with glucocorticoid antagonism of ethanol-induced sedation reported to occur in vivo. The present study was undertaken to characterize the interactions of corticosterone with ethanol effects on [125I]CaM binding in synaptic plasma membranes. From the shift of concentration-response curves when corticosterone and ethanol were present in combination, the interaction between steroid stimulation and ethanol inhibition occurred in an additive relationship over the range of their effective concentrations. From Scatchard analyses, ethanol-induced decrease in membrane affinity for [125I]CaM was antagonized by steroid-induced increase in the membrane affinity, indicating that the convergent event in their interaction was the alteration of membrane affinity for CaM. Glucocorticoid antagonism of ethanol inhibition of [125I]CaM binding exhibited a high degree of steroid specificity; steroids with glucocorticoid activity including cortisol, dexamethasone and triamcinolone were effective, whereas gonadal steroids and excitatory neuroactive steroid metabolites were ineffective. The demonstration that glucocorticoids antagonized the inhibition of CaM binding by ethanol provides support for the hypothesis that these steroids are among the endogenous factors that modulate neuronal sensitivity to ethanol.

Biochemical characterization of ethanol actions on dihydropyridine-sensitive calcium channels in brain synaptosomes.

This study was undertaken to investigate the biochemical events underlying the inhibitory action of ethanol on dihydropyridine-sensitive voltage-dependent Ca2+ channels in brain synaptosomes. The binding of radiolabeled dihydropyridine was used to determine functional Ca2+ channels in synaptosomes following exposure to ethanol. No effect on [3H]PN 200-110 binding was found when disrupted synaptosomal membranes were incubated with ethanol concentrations as high as 300 mM, suggesting that ethanol did not interact directly with sites on or near the Ca2+ channels. However, when intact synaptosomes were first incubated with ethanol (100 mM) at 37 degrees and then disrupted, a significant reduction in membrane binding of [3H]PN 200-110 was found. Ethanol incubation of synaptosomes at 0 degree was ineffective. It appears that metabolic processes involving intracellular factors were required in the ethanol action. In examining this possibility, [3H]PN 200-110 binding was activated by incubation of disrupted membranes with MgATP and Ca(2+)-calmodulin, and ethanol was found to inhibit the activation in a concentration-dependent manner (50-200 mM). [3H]PN 200-110 binding to membranes was also activated by incubation with MgATP and cyclic AMP-dependent protein kinase, but this activation was not inhibited by ethanol. These findings are consistent with the interpretation that ethanol acts on Ca2+ channels by inhibiting calmodulin-dependent activation of the channels.

Glucocorticoid actions on synaptic plasma membranes: modulation of dihydropyridine-sensitive calcium channels.

We have previously shown that glucocorticoids accelerate depolarization-induced 45Ca2+ influx in synaptosomes isolated from rat cerebral cortex, indicating that the steroids may modulate voltage-dependent Ca2+ channels. The present study was undertaken to characterize the biochemical action of glucocorticoids on dihydropyridine-sensitive voltage-dependent Ca2+ channels known to be present in brain synaptosomes. The [3H]dihydropyridine labeled site was used as a marker to determine the levels of functional Ca2+ channels. No effect on equilibrium binding of [3H]PN 200-110 was found when membranes from disrupted synaptosomes were incubated with corticosterone as high as 1 microM. However, when intact synaptosomes were first incubated with corticosterone at 37 degrees C and then disrupted, a significant increase in [3H]PN 200-110 binding was found. Steroid incubation of synaptosomes at 0 degree C was ineffective. It appears that metabolic processes requiring intracellular factors were involved in the steroid action. In examining this possibility, [3H]PN 200-110 binding was activated in disrupted membranes by MgATP and Ca(2+)-calmodulin, and corticosterone was found to enhance the activation in a concentration-dependent manner. [3H]PN 200-110 binding to membranes was also activated by incubation with MgATP and cAMP-dependent protein kinase, but this activation was not enhanced by the steroid. These findings are consistent with the interpretation that the steroid promotes Ca2+ channel activity by enhancing calmodulin-dependent activation of the channels. The action on voltage-dependent Ca2+ channels in synaptic terminals may well be a mechanism whereby glucocorticoids modulate neuronal activity.

Acute and chronic actions of ethanol on endogenous calmodulin content in synaptic plasma membranes from rat brain.

The present study was designed to demonstrate that endogenous calmodulin (CaM) content in synaptic plasma membranes (SPM) is altered by acute and chronic administration of ethanol and is a sequel to the kinetic characterization of ethanol inhibition of [125I]CaM binding to SPM reported in our previous study. In rats, an acute ethanol injection (2 g/kg, i.p.) rapidly reduced CaM content in SPM from cerebral cortex, whereas chronic ethanol treatment [6% (w/v) in a liquid diet for 3 weeks] led to an up-regulation of the CaM content. In both cases, the alteration of CaM content in SPM occurred in the EGTA-dissociable pool of CaM (77% of total membrane CaM); the EGTA-nondissociable pool (23% of total CaM) was not affected. In animals receiving chronic ethanol treatment, CaM content in SPM was not altered significantly by the acute ethanol dose that produced rapid reduction of CaM content in control animals, indicating that resistance to ethanol develops. This resistance to ethanol can be attributed to alterations of membrane properties. In control SPM, ethanol at 50 mM markedly accelerated the temperature-dependent dissociation of endogenous CaM, whereas in SPM from animals chronically treated with ethanol, significant acceleration of CaM dissociation required ethanol concentrations as high as 150-200 mM. These findings on SPM in vitro were consistent with the data on CaM content obtained in vivo. Since CaM mediates a variety of biochemical processes in synaptic membranes, we hypothesize that the effects of ethanol in altering the content of membrane-bound CaM may lead to a cascade of consequences in synaptic membrane function.

Correlation between [125I]calmodulin binding and lipid fluidity in synaptic plasma membranes: effects of ethanol and other short-chain alcohols.

Ethanol inhibits [125I]calmodulin binding to synaptic plasma membranes from rat brain, and this inhibition is correlated in a concentration-dependent manner with the increase of membrane fluidity, as determined by diphenylhexatriene fluorescence polarization. Moreover, several short-chain alcohols that increase membrane fluidity are also effective inhibitors of [125I]calmodulin binding. These data support the notion that ethanol inhibits calmodulin binding by increasing lipid fluidity of the synaptic membranes.

Regulation of calmodulin content in synaptic plasma membranes by glucocorticoids.

Synaptic plasma membranes (SPM) from the brain are known to have specific binding sites for several steroid hormones, but the mechanisms of membrane transduction of steroid signals is not understood. In this study, corticosterone was found to prevent temperature-dependent dissociation of endogenous calmodulin (CaM) from highly purified SPM from rat cerebral cortex. The steroid stabilizes Ca(2+)-dependent membrane binding of endogenous CaM (78% of total CaM), whereas Ca(2+)-independent binding of CaM (the other 22%) is not affected. The stabilization of membrane binding of endogenous CaM by corticosterone is concentration-dependent, with the maximal effect occurring at steroid concentration of 1 microM. The EC50 is estimated as 130 nM, which is almost identical to the Kd of specific binding of the steroid to SPM (120 nM) reported previously. The effect in stabilizing membrane binding of CaM is specific to corticosterone and other glucocorticoids (cortisol, dexamethasone and triamcinolone); gonadal steroids (17 (17 beta-estradiol, progesterone and testosterone) are ineffective. Furthermore, corticosterone administration in vivo (2 mg/kg, i.p.) produced a rapid increase of CaM content in SPM, occurring within 5 min after steroid injection and persisting for at least 20 min. Since CaM mediates a variety of biochemical processes in synaptic membranes, we hypothesize that the effect of glucocorticoids in promoting membrane binding of CaM may lead to a cascade of consequences in synaptic membrane function.

Glucocorticoid action on depolarization-dependent calcium influx in brain synaptosomes.

Synaptic plasma membranes from the brain are known to have specific binding sites for several steroid hormones, but the mechanism of membrane transduction of steroid signals is not understood. In this study, corticosterone was found to stimulate 45Ca2+ uptake in brain synaptosomes upon depolarization of the synaptosomes by high K+ (70 mM). The stimulation of the depolarization-dependent 45Ca2+ uptake by corticosterone is concentration dependent, with the maximal effect occurring at steroid concentration of 5-10 x 10(-7) M (70-80% above control). The EC50 is estimated as 1.3 x 10(-7) M, which is almost identical to the Kd of the specific binding of the steroid to synaptic membranes (1.2 x 10(-7) M) reported previously. The stimulation of 45Ca2+ uptake in brain synaptosomes is specific to corticosterone and other glucocorticoids (cortisol, dexamethasone and triamcinolone); gonadal steroids (17 beta-estradiol, progesterone and testosterone) are ineffective. [3H]Nitrendipine binding was used to examine the effect of corticosterone on voltage-dependent Ca2+ channels. No effect on [3H]nitrendipine binding was found when disrupted synaptic membranes were preincubated with the steroid. However, a significant increase of membrane binding of [3H]nitrendipine was found when intact synaptosomes were first preincubated with the steroid at 37 degrees C and then disrupted. Steroid preincubation of synaptosomes at 0 degrees C was ineffective. Since preincubation at 37 degrees C is required, it appears that metabolic processes modulating Ca2+ channel activity are involved in the steroid action.

Ethanol modulates calmodulin-dependent Ca(2+)-activated ATPase in synaptic plasma membranes.

The effects of ethanol in vitro on calmodulin-dependent Ca(2+)-activated ATPase (CaM-Ca(2+)-ATPase) activity were studied in synaptic plasma membranes (SPM) prepared from the brain of normal and chronically ethanol-treated rats. In SPM from normal animals, ethanol at 50-200 mM inhibited the Ca(2+)-ATPase activity. Lineweaver-Burk analysis indicates that the inhibition was the result of a decreased affinity of the enzyme for calmodulin, whereas the maximum activity of the enzyme was not changed. Arrhenius analysis indicates that the enzyme activity was influenced by lipid transition of the membranes, and ethanol in vitro resulted in a shift of the transition temperature toward a lower value. From animals receiving chronic ethanol treatment (3 weeks), the SPM were resistant to the inhibitory effect of ethanol on the enzyme activity. The resistance to ethanol inhibition was correlated with a higher enzyme affinity for calmodulin and a higher transition temperature, as compared with normal SPM. Since the calmodulin-dependent Ca(2+)-ATPase in synaptic plasma membranes is believed to be the Ca2+ pump controlling free Ca2+ levels in synaptic terminals, its inhibition by ethanol could therefore lead to altered synaptic activity.

Ethanol modulates [125I]calmodulin binding to synaptic plasma membranes from rat brain.

The effects of ethanol in vitro on Ca(++)-dependent binding of [125I] calmodulin to brain synaptic plasma membranes (SPM) from control and chronically ethanol-treated rats were studied. In SPM from control animals, ethanol at 50 to 200 mM inhibited [125I] calmodulin binding; the inhibition was correlated with a decreased membrane affinity for [125I]calmodulin as shown by Scatchard analysis, and an increased dissociation of [125I]calmodulin-membrane complexes as shown by kinetic analysis. Arrhenius analysis indicates that [125I]calmodulin binding was influenced by lipid transition of the membrane, and that ethanol in vitro resulted in a shift of the transition temperature toward a lower value. From animals receiving chronic ethanol treatment (3 weeks), the SPM were found to be resistant to the inhibitory effect of ethanol on binding. The resistance to ethanol inhibition was correlated with a higher membrane affinity for [125I]calmodulin and a higher transition temperature, as compared with control SPM. Because a variety of membrane-bound processes are regulated by calmodulin or calmodulin-dependent processes, the inhibitory effect of ethanol on membrane binding of calmodulin could lead to a cascade of consequences in synaptic function. Moreover, the resistance of the membranes to ethanol inhibition after chronic ethanol treatment implies that membrane binding of calmodulin is part of the mechanism underlying alcohol tolerance and dependence.

Glucocorticoid actions on synaptic plasma membranes: modulation of [125I]calmodulin binding.

The effects of corticosterone on Ca(2+)-dependent binding of [125I]calmodulin to purified synaptic plasma membranes (SPM) from rats brain were characterized. The enhancement of [125I]calmodulin binding was a sigmoidal function of steroid concentration, with the maximal increase (> 55% above control) occurring at a steroid concentration of 1 x 10(-6) M and EC50 estimated at 1-2 x 10(-7) M. Other glucocorticoids including hydrocortisone, dexamethasone and triamcinolone produced similar effects, whereas steroids without glucocorticoid activity such as 11-deoxycortisol, 11-deoxycorticosterone and cholesterol were ineffective. The steroid-induced increase of binding was correlated with an increase of membrane affinity for [125I]calmodulin as shown by Scatchard analysis, and a decrease of the rate of dissociation of [125I]calmodulin from the membranes as shown by kinetic analysis. Arrhenius analysis indicates that [125I]calmodulin binding was influenced by lipid transition of the membranes and that corticosterone resulted in a shift of membrane transition toward a higher temperature. Since a variety of biochemical processes associated with synaptic membranes are dependent upon calmodulin for their regulation, we hypothesize that the effects of glucocorticoids in promoting membrane binding of calmodulin may lead to a cascade of alterations in synaptic function.

[125I]calmodulin binding to synaptic plasma membrane from rat brain: kinetic and Arrhenius analysis.

Binding of [125I]calmodulin was characterized in highly purified synaptic plasma membrane (SPM) prepared from rat brain. By Scatchard analysis, the Ca(2+)-dependent membrane binding of [125I]calmodulin was found to have a Bmax of 284 pmol/mg protein and an apparent affinity with a Kd of 131 nM. Kinetic analysis indicates that at 37 degrees C, the dissociation of [125I]calmodulin-membrane complexes follows first-order reaction and consists of two components: a dissociation constant (k) of 3.7 x 10(-1) min-1 and a half-time (t1/2) of 1.8 min for the fast component, and a k of 4.8 x 10(-2) min-1 and a t1/2 of 14.5 min for the slow component. At 0 degrees C, substantial dissociation still occurred, with a k of 4.5 x 10(-2) min-1 and a t1/2 of 15.3 min for the fast component, and a k of 5.5 x 10(-3) min-1 and a t1/2 of 125.5 min for the slow component. These data on binding affinity and dissociation kinetics are consistent with the notion that SPM can readily and rapidly associate and dissociate calmodulin. In Arrhenius analysis of temperature effects, [125I]calmodulin binding to SPM exhibits a biphasic function, with the transition temperature (Td) estimated to be 23.8 degrees C, suggesting that binding is influenced by lipid phase transition of the membrane. The binding of [125I]calmodulin to the synaptic membrane was found to be increased by corticosterone (10(-7)-10(-6) M), a steroid hormone, and decreased by ethanol (50-200 mM), a centrally acting drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucocorticoids antagonize the sedative action of ethanol in mice.

The effect of corticosterone on sleep time in mice following a hypnotic dose of ethanol (3 g/kg) was determined. An acute dose of the steroid (10 mg/kg) administered 15 min prior to ethanol injection significantly shortened the sleep time (by 55%). Brain levels of ethanol were not affected by the steroid treatment. The effect was specific to glucocorticoids because steroids without glucocorticoid activity including testosterone and 17 beta-estradiol were ineffective. These results indicate that glucocorticoids have an antagonistic effect to the acute action of ethanol in the brain. The rapid onset of the corticosterone action in antagonizing ethanol-induced sedation suggests that the action is mediated by a membrane mechanism rather than the classical steroid mechanism involving an intracellular receptor and gene expression.

Developmental profile of glucocorticoid binding to synaptic plasma membrane from rat brain.

The plasma membranes of several mammalian tissues including the brain are known to have specific binding sites for glucocorticoids. The developmental changes in specific glucocorticoid binding to synaptic plasma membrane (SPM) from rat brain were determined at various postnatal ages, using [3H]triamcinolone acetonide (TA) as the steroid ligand. The specific binding of the labeled glucocorticoid to SPM during the first 2 postnatal weeks was only 40% of the adult level. An increase of the specific binding occurred after day 15, and this developmental rise of binding reached the adult level approximately by the end of the fourth week. Methodologically, these developmental data are detailed in the present article to include nonspecific binding as well as specific binding. Scatchard analysis indicates that the developmental rise of the specific glucocorticoid binding was due to an increase in the membrane binding sites. The ontogenetic increase of membrane binding sites during postnatal brain development provides additional evidence that these binding sites have physiological significance in brain function.

Pharmacological effects of phenylalanine on seizure susceptibility: an overview.

The effects of excessive doses of phenylalanine on seizure susceptibility were examined in animal models in the past, primarily because of their relevance to phenylketonuria. It was thought that such effects might involve brain monoaminergic mechanisms. Recently, this issue has been pursued with a renewed interest but for a different reason. The dipeptide sweetener, aspartame, contains a phenylalanine residue. In the last three years, a number of studies involving as many as nine animal models of seizures have reexamined the effects of phenylalanine (and aspartame) on seizure thresholds. Data from these studies are in general agreement that aspartame at dosage levels below 1,000 mg/kg, or phenylalanine at equimolar doses, is without an effect on seizure susceptibility in animals. When the dosage level of aspartame reaches 1,000 mg/kg, the findings between various laboratories and from different animal models of seizures are inconsistent, showing either no effect or a proconvulsant effect. The Acceptable Daily Intake of aspartame in humans set by the Food and Drug Administration is 50 mg/kg/day. Thus, the data from the excessive bolus doses in rodents do not appear to be relevant to human use. This article provides a detailed review of the data from both early and recent studies and points out the methodological problems apparent at such high doses.

Purification and characterization of a soluble cyclic nucleotide-independent Ca2+-calmodulin-sensitive protein kinase from rat brain.

Following partial purification, the characteristics of a cytosol protein kinase were investigated. The protein kinase was purified by ammonium sulfate precipitation and diethylaminoethyl-cellulose, ATP-agarose, and hydroxyapatite chromatography. Analysis of the purified protein kinase preparation by polyacrylamide gel electrophoresis revealed three major protein bands. The cytosol protein kinase was purified approximately 442-fold, as calculated from the cyclic nucleotide independent protein kinase activity in the 40,000 g supernatant. The activity of the kinase was found to be independent of either cyclic AMP or cyclic GMP. Moreover, the kinase activity was unaffected by the addition of the endogenous protein kinase inhibitor, or the regulatory subunit from the type II cyclic AMP-dependent protein kinase from bovine heart. The molecular weight of the enzyme was determined to be 95,000 by Sephadex G-200 gel filtration. The activity of the kinase was increased approximately twofold in the presence of 10 microM Ca+2 and calmodulin. This increase was reversed by the addition of EGTA. The subcellular distribution of the protein kinase was also examined. The soluble fraction from nerve terminal was found to have the highest concentration of the kinase activity.

Studies on brain monoamine neurotransmitters in mice after prenatal exposure to barbiturate.

Pregnant HS/Ibg mice received 3 g/kg phenobarbital in their milled food; control dams received unadulterated milled food. Their offspring were tested on ages 22 and 50 days for norepinephrine (NE) and dopamine (DA) levels and DA turnover in the hypothalamus and striatum. Additional groups were tested on days 8, 22, and 50 for brain stem tryptophan hydroxylase (TPH) activity. Hypothalamic DA level among offspring with prenatal PhB exposure (B offspring) was 37% below control level on age 22 days and 61% below control on day 50 (p less than 0.001). Hypothalamic NE level of B offspring was reduced on age 50 days (52%, p less than 0.05) and not on day 22. DOPAC level and DA turnover did not differ among B and control groups. Prenatal exposure to PhB did not have a significant effect on TPH activity. The changes in catecholamines may mediate, at least in part, some of the early barbiturate induced neuromorphological and behavioral changes previously found in our laboratory.

Influence of ACTH on tyrosine hydroxylase activity in the locus coeruleus of mouse brain.

Tyrosine hydroxylase activity in noradrenergic neurons of the locus coeruleus was found to rise in bilaterally adrenalectomized adult mice, with maximum increase of the enzyme activity (52% above control) occurring 7 days after the surgery. No such increase of tyrosine hydroxylase activity was found in dopaminergic neurons of the substantia nigra. The increase of enzyme activity in the locus coeruleus following adrenalectomy was totally prevented by corticosterone replacement. Moreover, the adrenalectomy effect was abolished by hypophysectomy, indicating the involvement of the pituitary rather than the adrenocortical function. Chronic administration of ACTH (20 IU/kg, i.p., daily) resulted in an increase of tyrosine hydroxylase activity in the locus coeruleus, with a time course and magnitude similar to those found after adrenalectomy. Additionally, the effects of four ACTH analogs were determined. ACTH 1-24 and ACTH 4-10 were as effective as the whole ACTH molecule, whereas ACTH 4-10,7-D-Phe and ACTH 11-24 were ineffective. The effect of ACTH 4-10, a peptide fragment with no adrenocorticotrophic activity, further indicates that glucocorticoids are not involved. From these data, it appears that tyrosine hydroxylase in the locus coeruleus neurons is under the regulatory influence of pituitary ACTH. It remains to be determined whether the hormone can be transported from its pituitary origin to the locus coeruleus and exerts a direct action on the noradrenergic neurons. Regardless of the mechanism, the response of the noradrenergic neurons to pituitary activity may be an important component in physiological adaptation of the central nervous system to chronic stress.

Increased uptake of [3H]choline by rat superior cervical ganglion: an effect of dexamethasone.

The high affinity uptake of [3H]choline by the superior cervical ganglion, isolated from the rat, was found to be increased by dexamethasone. Maximal increase (60-65% above control values) occurred at the steroid concentration of 5 X 10(-5) M. Other glucocorticoids (triamcinolone, corticosterone and hydrocortisone) were without an effect on the [3H]choline uptake. Following administration of dexamethasone (25 mg/kg, i.p.), there was a marked increase in the level of choline in the ganglion. The increase was 3-fold at 1 hr and 10-fold at 6 hr, and by 24 hr the choline levels still remained higher in the steroid-treated animals than in the controls. Levels of acetylcholine in the ganglion were also increased, beginning at 1 hr after the injection of steroid. The increase was 85% by 3 hr and 60% by 6 hr. Triamcinolone, a glucocorticoid that was without an effect on [3H]choline uptake in vitro, was also ineffective in altering the levels of choline and acetylcholine in vivo. It seems probable that the increase of choline uptake in the ganglion induced by dexamethasone may, at least in part, occur in the preganglionic cholinergic terminals, leading to increased synthesis of acetylcholine. Such an effect of dexamethasone provides another case of a selective steroid acting directly on nerve terminals by altering a transport mechanism.