Orexins stimulate corticosterone secretion of rat adrenocortical cells, through the activation of the adenylate cyclase-dependent signaling cascade

Orexins-A and B are two novel hypothalamic peptides, which, like leptin and neuropeptide-Y (NPY), are involved in the central regulation of feeding. Since leptin and NPY were found to modulate adrenal function, we have examined whether orexins are able to directly affect rat adrenal steroid secretion. Both orexin-A and orexin-B raised basal corticosterone secretion of dispersed rat zona fasciculata–reticularis (ZF/R) cells, their maximal effective concentration being 10⁻⁸ M. In contrast, orexins did not affect either maximally ACTH (10⁻⁹ M)-stimulated corticosterone production by ZF/R cells or the basal and agonist-stimulated aldosterone secretion of dispersed zona glomerulosa cells. The ACTH-receptor antagonist corticotropin-inhibiting peptide (10⁻⁶ M) annulled corticosterone response of ZF/R cells to ACTH (10⁻⁹ M), but not to orexins (10⁻⁸ M). Orexins (10⁻⁸ M) enhanced cyclic-AMP release by ZF/R cells, and the selective inhibitor of protein-kinase A (PKA) H-89 (10⁻⁵ M) abolished corticosterone responses to both ACTH (10⁻⁹ M) and orexins (10⁻⁸ M). A subcutaneous injection of both orexins (5 or 10 nmol/kg) evoked a clear-cut increase in the plasma concentration of corticosterone (but not aldosterone), the effect of orexin-A being significantly more intense than that of orexin-B. Collectively, these findings suggest that orexins exert a selective and direct glucocorticoid secretagogue action on the rat adrenals, acting through a receptor-mediated activation of the adenylate cyclase/PKA-dependent signaling pathway.     

Angiotensin II and angiotensin-(1-7) inhibit the inner cortex Na+ -ATPase activity through AT2 receptor

In the present paper, the modulation of the basolateral membrane (BLM) Na⁺-ATPase activity of inner cortex from pig kidney by angiotensin II (Ang II) and angiotensin-(1–7) (Ang-(1–7)) was evaluated. Ang II and Ang-(1–7) inhibit the Na⁺-ATPase activity in a dose-dependent manner (from 10⁻¹¹ to 10⁻⁵ M), with maximal effect obtained at 10⁻⁷ M for both peptides. Pharmacological evidences demonstrate that the inhibitory effects of Ang II and Ang-(1–7) are mediated by AT₂ receptor: The effect of both polypeptides is completely reversed by 10⁻⁸ M PD 123319, a selective AT₂ receptor antagonist, but is not affected by either (10⁻¹²–10⁻⁵ M) losartan or (10⁻¹⁰–10⁻⁷ M) A779, selective antagonists for AT₁ and AT_(1–7) receptors, respectively. The following results suggest that a PTX-insensitive, cholera toxin (CTX)-sensitive G protein/adenosine 3′,5′-cyclic monophosphate (cAMP)/PKA pathway is involved in this process: (1) the inhibitory effect of both peptides is completely reversed by 10⁻⁹ M guanosine 5′-O-(2-thiodiphosphate) (GDPβS; an inhibitor of the G protein activity), and mimicked by 10⁻¹⁰ M guanosine 5′-O-(3-thiotriphosphate) (GTPγS; an activator of the G protein activity); (2) the effects of both peptides are mimicked by CTX but are not affected by PTX; (3) Western blot analysis reveals the presence of the G_s protein in the isolated basolateral membrane fraction; (4) (10⁻¹⁰–10⁻⁶ M) cAMP has a similar and non-additive effect to Ang II and Ang-(1–7); (5) PKA inhibitory peptide abolishes the effects of Ang II and Ang-(1–7); and (6) both angiotensins stimulate PKA activity.     

Interactions between vasotocin and other corticotropic factors on the frog adrenal gland

The adrenocortical cells of the amphibian interrenal (adrenal) gland are controlled by multiple factors including neuropeptides and classical neurotransmitters. In particular, it has recently been shown that vasotocin (AVT), the amphibian counterpart of vasopressin, is a potent stimulator of frog corticosteroidogenesis. In the present study, we have investigated the possible interactions between AVT and other regulatory factors on frog interrenal tissue. When AVT (10⁻⁹ M) and serotonin (10⁻⁶ M) were infused together, a strict addition of the individual effects was observed. Similar results were obtained with concomitant infusion of AVT and vasoactive intestinal peptide or AVT and ACTH. In contrast, when AVT (10⁻⁹ M) and acetylcholine (5 × 10⁻⁵ M) were added together, the increase in corticosteroid secretion was less than additive. Dopamine induced a significant reduction of AVT-evoked stimulation of corticosterone production. These results indicate that regulatory peptides or classical neurotransmitters which participate in the control of adrenal steroidogenesis may interact on their target cell to modulate the activity of their congeners.     

Cortisol and testosterone: Inhibitory agents of the mineralocorticoid pathway. Is there a physiopathological meaning in adrenal tumors?

The authors incubated adrenal mitochondria to study the in vitro action of cortisol and testosterone on the transformation of corticosterone and 18-hydroxycorticosterone into aldosterone. The results show that cortisol at concentrations of 5 × 10⁻⁶ and 10⁻⁴ M inhibit the conversion of corticosterone into aldosterone by 23.6 to 90%; testosterone 5 × 10⁻⁵ and 10⁻⁴ M inhibit the reaction by 78.4 and 87.2%, respectively. The inhibition of the conversion of 18-hydroxycorticosterone into aldosterone is 12.5 to 91% by cortisol with concentrations ranging from 5 × 10⁻⁷ to 5 × 10⁻⁵ M and testosterone 5 × 10⁻⁵ and 10⁻⁴ M inhibits the reaction by 87.3 and 91%, respectively. Aldosterone (10⁻⁸ and 10⁻⁶ M) does not inhibit aldosterone biosynthesis from corticosterone or 18-hydroxycorticosterone. It thus appears that cortisol and testosterone have an effect on the aldosterone biosynthesis pathways in mitochondria. This action may be located at the binding site of the cytochrome P450 11β, which catalyzes all hydroxylation steps in the mineralocorticoid biosynthesis pathway. Because cortisol and testosterone may interfere with aldosterone biosynthesis, and since functional zonation is expected in adrenal carcinomas, the presence of these steroids in substantial amounts could explain the very low plasma aldosterone level usually observed, in adrenal carcinomas studies in our laboratory.     

Gastric inhibitory polypeptide stimulates glucocorticoid secretion in rats, acting through specific receptors coupled with the adenylate cyclase-dependent signaling pathway.

Gastric inhibitory polypeptide (GIP) is a 42-amino acid peptide, belonging to the VIP-secretin-glucagon superfamily, some members of this group are able to regulate adrenocortical function. GIP-receptor mRNA has been detected in the rat adrenal cortex, but investigations on the effect of GIP on steroid-hormone secretion in this species are lacking. Hence, we have investigated the distribution of GIP binding sites in the rat adrenal gland and the effect of their activation in vivo and in vitro. Autoradiography evidenced abundant [125I]GIP binding sites exclusively in the inner adrenocortical layers, and the computer-assisted densitometric analysis of autoradiograms demonstrated that binding was displaced by cold GIP, but not by either ACTH or the selective ACTH-receptor antagonist corticotropin-inhibiting peptide (CIP). The intraperitoneal (IP) injection of GIP dose-dependently raised corticosterone, but not aldosterone plasma concentration: the maximal effective dose (10 nmol/rat) elicited a twofold increase. GIP did not affect aldosterone and cyclic-AMP release by dispersed zona glomerulosa cells. In contrast, GIP enhanced basal corticosterone secretion and cyclic-AMP release by dispersed inner adrenocortical cells in a concentration-dependent manner, and the maximal effective concentration (10(-7) M) evoked 1.5- and 2.4-fold rises in corticosterone and cyclic-AMP production, respectively. GIP (10(-7) M) did not display any additive or potentiating effect on corticosterone and cyclic-AMP responses to submaximal or maximal effective concentrations of ACTH. The corticosterone secretagogue action of 10(-7) M GIP was abolished by the protein kinase A (PKA) inhibitor H-89 (10(-5)M), and unaffected by CIP (10(-6)M). Collectively, these findings indicate that GIP exerts a moderate but statistically significant stimulatory effect on basal glucocorticoid secretion in rats, acting through specific receptors coupled with the adenylate cyclase/PKA-dependent signaling pathway.     

Vasopressin and oxytocin modulation of melatonin secretion from rat pineal glands

The rat pineal gland is known to release melatonin in response to noradrenergic stimulation. Since vasopressin (VP)- and oxytocin (OT)-containing fibers innervate the pineal gland, the effects of VP and OT on melatonin release from perifused rat pineal glands were investigated. VP (10⁻⁷ M) and OT (10⁻⁶ M) decreased the basal melatonin secretion. No dose-dependent effect was observed. At high concentrations (10⁻⁵) these peptides potentiated the isoproterenol-induced increase of melatonin secretion. Below 10⁻⁵ M no potentiation was observed. Fragments of VP {[pGlu⁴,Cys⁶]VP(4–9)} and OT {[pGlu⁴,Cys⁶]OT(4–9)} did not display any effect on the isoproterenol-induced melatonin secretion.     

Bradykinin counteracts the stimulatory effect of angiotensin-(1-7) on the proximal tubule Na+ -ATPase activity through B2 receptor

Recently, we demonstrated that angiotensin-(1–7) (Ang-(1–7)) stimulates the Na⁺-ATPase activity through a losartan-sensitive angiotensin receptor, whereas bradykinin inhibits the enzyme activity through the B₂ receptor [Regul. Pept. 91 (2000) 45; Pharmacol. Rev. 32 (1980) 1]. In the present paper, the effect of bradykinin (BK) on Ang-(1–7)-stimulated Na⁺-ATPase activity was evaluated. Preincubation of Na⁺-ATPase with 10⁻⁹ M Ang-(1–7) increases enzyme activity from 7.9±0.9 to 14.1±1.5 nmol Pi mg⁻¹ min⁻¹, corresponding to an increase of 79% (p<0.05). This effect is reverted by bradykinin in a dose-dependent manner (10⁻¹⁴–10⁻⁸ M), reaching maximal inhibitory effect at 10⁻⁹ M. Des-Arg⁹ bradykinin (DABK), an agonist of B₁ receptor, at the concentrations of 10⁻⁹–10⁻⁷ M, does not mimic the BK inhibitory effect, and des-Arg⁹-[Leu⁸]-BK (DALBK), a B₁ receptor antagonist, at the concentrations of 10⁻¹⁰–10⁻⁷ M, does not prevent the inhibitory effect of BK on Ang-(1–7)-stimulated enzyme. On the other hand, HOE 140, an antagonist of B₂ receptor, abolishes the inhibitory effect of BK on the Ang-(1–7)-stimulated enzyme in a dose-dependent manner, reaching maximal effect at 10⁻⁷ M. Taken together, these data indicate that stimulation of B₂ receptors by BK can counteract the stimulatory effect of Ang-(1–7) on the proximal tubule Na⁺-ATPase activity.     

Biphasic regulation by N, 2′-O-dibutyryl adenosine 3′, 5′-cyclic monophosphate (dbcAMP) of steroid 21-hydroxylase activity in rat hepatocytes

Steroid 21-hydroxylase activity has been identified in many tissues, including liver. But it is possible that the enzyme found in the liver is different from adrenal 21-hydroxylase. In the adrenal cortex, steroid 21-hydroxylase activity is increased by corticotropin (ACTH); the effect of ACTH is mediated by cyclic AMP (cAMP), and presumably involves a cAMP-dependent protein kinase (PKA). It is not yet clear, however, how extra-adrenal steroid 21-hydroxylase activity is regulated. In the present study, we examined the effect of N⁶, 2′-O-dibutyryl adenosine 3′,5′-cyclic monophosphate (dbcAMP), forskolin, N-[2-(methylamino)ethyl]5-isoquinolinesulfonamide (H-8) and 12-O-tetradecanoylphorbol-13-acetate (TPA) on steroid 21-hydroxylase activity in primary cultures of rat hepatocytes to determine the nature of regulation of extra-adrenal steroid 21-hydroxylase activity. Steroid 21-hydroxylase activity in hepatocytes incubated with 10⁻¹¹M dbcAMP for 24 h was 1.6 times higher than that in control hepatocytes untreated with dbcAMP. On the other hand, steroid 21-hydroxylase activity decreased by 20 and 50% when the cells were incubated with 10⁻⁵ and 10⁻³ M dbcAMP, respectively. The stimulatory effect of 10⁻¹¹ M dbcAMP was not blocked by 10⁻⁵ M H-8 (PKA inhibitor), but the inhibitory effect of 10⁻⁵ or 10⁻³ M cAMP was. TPA did not alter the activity of steroid 21-hydroxylase. These findings indicate that the steroid 21-hydroxylase in rat liver is regulated by mechanisms different from those in the adrenal glands.     

Effects of glucagon and glucagon-like peptide-1 on glucocorticoid secretion of dispersed rat adrenocortical cells.

The effects of glucagon and glucagon-like peptide-1 (GLP-1) on the secretory activity of rat adrenocortical cells have been investigated in vitro. Neither hormones affected basal or agonist-stimulated aldosterone secretion of dispersed rat zona glomerulosa cells or basal corticosterone production of zona fasciculata-reticularis (inner) cells. In contrast, glucagon and GLP-1 partially (40%) inhibited ACTH (10(-9) M)-enhanced corticosterone secretion of inner cells, maximal effective concentration being 10(-7) M. The effect of 10(-7) M glucagon or GPL-1 was suppressed by 10(-6) M Des-His1-[Glu9]-glucagon amide (glucagon-A) and exendin-4(3-39) (GPL-1-A), which are selective antagonists of glucagon and GLP-1 receptors, respectively. Glucagon and GLP-1 (10(-7) M) decreased by about 45-50% cyclic-AMP production by dispersed inner adrenocortical cells in response to ACTH (10(-9) M), but not to the adenylate cyclase activator forskolin (10(-5) M). Again this effect was blocked by 10(-6) M glucagon-A or GLP-1-A. The exposure of dispersed inner cells to 10(-7) M glucagon plus GLP-1 completely suppressed corticosterone response to ACTH (10(-9) M). However, they only partially inhibited (by about 65-70%) both corticosterone response to forskolin (10(-5) M) or dibutyryl-cyclic-AMP (10(-5) M) and ACTH (10(-9) M)-enhanced cyclic-AMP production. Quantitative HPLC showed that 10(-7) M glucagon or GLP-1 did not affect ACTH-stimulated pregnenolone production, evoked a slight rise in progesterone and 11-deoxycorticosterone release, and markedly reduced (by about 55%) corticosterone secretion of dispersed inner adrenocortical cells. In light of these findings the following conclusion are drawn: (i) glucagon and GLP-1, via the activation of specific receptors, inhibit glucocorticoid response of rat adrenal cortex to ACTH; and (ii) the mechanism underlying the effect of glucagon and GLP-1 is probably two-fold, and involves both the inhibition of the ACTH-induced activation of adenylate cyclase and the impairment of the late steps of glucocorticoid synthesis.     

Properties of amino-terminal parathyroid hormone-related peptides modified at positions 11–13

Biological properties of amino-terminal PTHrP analogues modified in the region 11–13 were examined using ROS 17/2.8 cells. [Leu¹¹,D-Trp¹²,Arg¹³,Tyr³⁶]PTHrP(1–36)amide had a 17-fold lower binding affinity for the receptor (apparent K_d: 5 × 10⁻⁸ M) than [Tyr³⁶]PTHrP(1–36)amide or [Arg^11,13,Tyr³⁶]PTHrP(1–36)amide (apparent K_d for both: 2 × 10⁻⁹ M). Moreover, it is only a weak partial agonist despite completely inhibiting radioligand binding. [Leu¹¹,D-Trp¹²,Arg¹³,Tyr³⁶,Cys³⁸]PTHrP(7–38) and PTHrP(7–34)amide had similar receptor affinities (apparent K_ds: 5 × 10⁻⁸ M and 8 × 10⁻⁸ M), while that of [Nle^8,18,Tyr³⁴]bPTH(7–34)amide was more than 10-fold lower (apparent K_d: 2 × 10⁻⁶ M). These changes in biological properties suggest that high affinity receptor binding requires both amino- and carboxyl-terminal domains of the PTHrP(1–36) sequence and/or intramolecular interactions which are impaired by the D-Trp substitution for Gly¹².     

Effects of neuropeptides on growth of cultivated rat molar pulp fibroblasts

The effect of the neuropeptides substance P (SP), neurokinin A (NKA), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) on DNA synthesis of dental pulp cells was investigated in cells grown from molar tooth bud explants from 4–6 days old rat pups. A concentration response-assay of the proliferative response of pulpal cells was performed with SP, NPY, NKA, CGRP and VIP (0.01 to 1 nM) in the presence of EGF (10 ng/ml), hydrocortisone (0.4 μg/ml) and 3% FCS, using [³H]thymidine incorporation. The results showed that SP, NKA and CGRP, but not NPY and VIP, increased the cell number in a concentration-dependent manner, with maxima at 10⁻¹⁰ – 10⁻⁹ M (SP, NKA) and 10⁻⁷ M (CGRP). No potentiating effect was noted when cells are simultaneously stimulated with SP and CGRP. The finding that SP, NKA and CGRP have growth regulatory properties on pulpal cells in vitro suggests that sensory neuropeptides may be involved during pulpal development or in wound healing after pulpal injury.     

In vitro stimulation of prolactin release by vasoactive intestinal peptide

The effect of vasoactive intestinal peptide (VIP) on anterior pituitary hormone release was examined in a variety of in vitro preparations. Synthetic VIP was capable of stimulating increased prolactin (PRL) release from male rat hemipituitaries in doses as low as 10⁻⁹ M only when the enzyme inhibitor bacitracin was present in the incubation medium. Natural porcine VIP was similarly capable of stimulating PRL release, but only at higher doses (10⁻⁶ M). Additionally, synthetic VIP was capable of stimulating PRL release from dispersed anterior pituitary cells harvested from adult male and lactating female rats and from an enriched population of lactotrophs obtained by unit gravity sedimentation of similar dispersed cells from infantile female rats. No effect of VIP on luteinizing hormone, growth hormone or thyroid stimulating hormone release was seen. These findings taken in concert with the presence of VIP in the hypothalamus, pituitary and hypophyseal portal plasma of the rat suggest a physiological role for VIP in the control of PRL secretion.     

ACTH(1–24) stimulates the migration of human monocytes in vitro

In view of the increasing evidence that a variety of stresses can influence immune responses, the direct effect of adrenocorticotropic hormone on the migration of human monocytes was studied in vitro. ACTH(1–24) significantly increased the number of migrating cells when placed in the same or the opposite compartment of the chemotaxis chamber, maximum activity being obtained at 10⁻¹⁴ and 10⁻⁸ M. The results indicate that ACTH(1–24) directly and potently stimulates the migration of human monocytes by means of a chemokinetic effect.     

Nitric oxide stimulates prostaglandin synthesis in cultured rabbit gastric cells

Both prostaglandins (PGs) and nitric oxide (NO) have cytoprotective and hyperemic effects in the stomach. However, the effect of NO on PG synthesis in gastric mucosal cells is unclear. We examined whether sodium nitroprusside (SNP), a releaser of NO, stimulates PG synthesis in cultured rabbit gastric mucus-producing cells. These cells did not release NO themselves. Co-incubation with SNP (2 × 10⁻⁴, 5 × 10⁻⁴, 10⁻³ M) increased PGE₂ synthesis, and SNP (10⁻³ M) increased PGI₂ synthesis in these cells. Hemoglobin, a scavenger of NO, (10⁻⁵ M) eliminated the increase in PGE₂ synthesis by SNP, but methylene blue, an inhibitor of soluble guanylate cyclase, (5 × 10⁻⁵ M) did not affect the increase in PGE₂ synthesis by SNP. 8-bromo guanosine 3′ : 5′-cyclic monophosphate (8-bromo cGMP), a cGMP analogue, (10⁻⁶, 10⁻⁵, 10⁻⁴, 10⁻³ M) did not affect PGE₂ synthesis. These findings suggest that NO increased PGE₂ and PGI₂ synthesis via a cGMP-independent pathway in cultured rabbit gastric cells.     

Role of c-fos gene in vasoactive intestinal peptide promoted synthesis of pulmonary surfactant phospholipids

We previously reported that vasoactive intestinal peptide (VIP) promoted synthesis of phosphatidylcholine (PC) in alveolar type II (ATII) cells. But the intracellular mechanism for this effect was unknown. In this work, we investigated the intracellular signal transduction pathway for VIP promoted synthesis of PC, the major lipid component of pulmonary surfactant (PS), by using an antagonist of VIP receptors, inhibitor of protein kinase C (PKC) and antisense oligonucleotides (AS-ODN) for c-fos oncogene. Our results showed that: ① [D-P-Cl-Phe(6)-Leu(17)]-VIP (10^− 6 mol/l), an antagonist of VIP receptors, could decrease the quantity of [³H] choline incorporation, microsomal choline-phosphate cytidylyltransferase (CCT) mRNA expression and CCT activity induced by VIP (10^− 8 mol/l) in cultured lung explants to the control levels; ② VIP (10^− 8 mol/l) upregulated c-Fos protein expression in ATII cells. AS-ODN for c-fos oncogene (9 × 10^− 6 mol/l) could block the elevation of [³H] choline incorporation, microsomal CCT mRNA expression and CCT activity induced by VIP in cultured lung explants and in ATII cells; ③ H7 (10^− 5 mol/l), a PKC inhibitor could also reduce VIP induced [³H] choline incorporation, microsomal CCT mRNA expression and CCT activity in cultured lung explants and in ATII cells. These results demonstrated that VIP receptors, PKC and c-Fos protein played important roles in the signaling pathway through which VIP promoted the synthesis of PC.     

Glutathione-proline activation of feeding behavior in the zoanthid Palythoa psammophilia Walsh & Bowers

Palythoa psammophilia Walsh & Bowers has a well coordinated, stereotyped feeding response, the culminating step of which is ingestion; this may be elicited by the synergistic effect of the tripeptide glutathione and the -imino acid, proline. Either activator acting separately causes responses only at high concentrations (above 10⁻⁵ M for glutathione; above 10⁻⁴ M for proline) in a reduced number of animals and at a low rate (5.00 ± 1.73 min in 5 × 10⁻³ M solutions of glutathione; 11.10±3.74 min in 5 × 10⁻³ M solutions of proline). Highest percentages of response were obtained in combinations where glutathione was at a concentration of 5 × 10⁻³ M and proline at 5 × 10⁻⁴ M or in combinations of glutathione at concentrations 5 × 10⁻⁶ M and proline at 5 × 10⁻⁵ M. The speed of ingestion is considerably enhanced when these activators are combined (1.17±1.18 min).     

Effect of nomegestrol acetate on estrone-sulfatase and 17β-hydroxysteroid dehydrogenase activities in human breast cancer cells

It is well recognized that estradiol (E₂) is one of the most important hormones supporting the growth and evolution of breast cancer. Consequently, to block this hormone before it enters the cancer cell or in the cell itself, has been one of the main targets in recent years. In the present study we explored the effect of the progestin, nomegestrol acetate, on the estrone sulfatase and 17β-hydroxy-steroid dehydrogenase (17β-HSD) activities of MCF-7 and T-47D human breast cancer cells. Using physiological doses of estrone sulfate (E₁S: 5 × 10⁻⁹ M), nomegestrol acetate blocked very significantly the conversion of E₁S to E₂. In the MCF-7 cells, using concentrations of 5 × 10⁻⁶ M and 5 × 10⁻⁵ M of nomegestrol acetate, the decrease of E₁S to E₂ was, respectively, −43% and −77%. The values were, respectively, −60% and −71% for the T-47D cells. Using E₁S at 2 × 10⁻⁶ M and nomegestrol acetate at 10⁻⁵ M, a direct inhibitory effect on the enzyme of −36% and −18% was obtained with the cell homogenate of the MCF-7 and T-47D cells, respectively. In another series of studies, it was observed that after 24 h incubation of a physiological concentration of estrone (E₁: 5 × 10⁻⁹ M) this estrogen is converted in a great proportion to E₂. Nomegestrol acetate inhibits this transformation by −35% and −85% at 5 × 10⁻⁷ M and 5 × 10⁻⁵ M, respectively in T-47D cells; whereas in the MCF-7 cells the inhibitory effect is only significant, −48%, at 5 × 10⁻⁵ M concentration of nomegestrol acetate. It is concluded that nomegestrol acetate in the hormone-dependent MCF-7 and T-47D breast cancer cells significantly inhibits the estrone sulfatase and 17β-HSD activities which converts E₁S to the biologically active estrogen estradiol. This inhibition provoked by this progestin on the enzymes involved in the biosynthesis of E₂ can open new clinical possibilities in breast cancer therapy.     

Uptake of corticosterone into isolated rat liver cells: Possible involvement of Na/K-ATPase

Isolated rat hepatocytes posses a saturable glucocorticoid uptake system with high affinity (K_d value = 2.8 ± 0.7 × 10⁻⁸ M; 318,000 ± 80,000 binding sites per cell; 317 fmol/mg protein). The initial rates of uptake decrease by about 30–40% if the cells are incubated simultaneously with [³H]corticosterone and either SH-reagents (N-ethylmaleimide and p-chloromercuriphenylsulphonate, 1 mM), metabolic inhibitors (2,4-dinitrophenol, 1 mM; and antimycin, 0.1 mM) or the Na⁺/K⁺-ATPase-inhibitors, ouabain and quercetine. These Na⁺/K⁺-ATPase-blockers exert half-maximal inhibition at 3 × 10⁻⁷ and 3 × 10⁻⁶ M, respectively. A slight increase in K⁺ concentration and a corresponding decrease in Na⁺ in the medium leads to a significant reduction in the initial uptake rate. The uptake system from the rat hepatocytes shows a clear steroid specificity, being different from the intracellular receptor. Corticosterone and progesterone are the strongest competitors, cortisol, 5- and 5β-dihydrocorticosterone, 11-deoxycorticosterone, cortisone and testosterone have an intermediate effect and only weak competition is exerted by dexamethasone and by the mineralocorticoid, aldosterone. Estradiol and estrone sulphate as well as the synthetic glucocorticoid triamcinolone acetonide are unable to inhibit initial corticosterone uptake.     

Possible interaction of [H]glutamate binding sites with anion channels in rat neural tissues

The effect of various ions on [³H] -glutamic acid (Glu) binding was examined using crude synaptic membrane preparations from the rat brain. In vitro addition of sodium acetate (1–100 mM) exhibited a significant enhancement of the binding in a concentration dependent manner. Ammonium chloride (20 mM) prevented the potentiation by sodium acetate at 2°C, whereas sodium acetate exerted an inhibitory action on the ammonium chloride-induced augmentation of the binding at 30°C. Ammonium chloride (1–100 mM) itself elicited a temperature dependent stimulation of the binding, which was invariably attenuated by an antagonist for the anion channels such as picrotoxinin (10⁻³ M) as well as by inhibitors of anion transport including ethacrynic acid (10⁻³ M) and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (10⁻⁴−10⁻³ M), respectively. The later two inhibitors also caused a significant additional raise of the sodium acetate-induced enhancement of the binding. A significant augmentation of the binding resulted from the addition (20 mM) of various anions known to penetrate the anion channels such as bromide, iodide, nitrate, bicarbonate and thiocyanate in a permeability related manner, while that of non-permeable anions including fluoride, sulfate, acetate, formate, phosphate, oxalate, lactate, succinate and tartarate had no such a profound effect on the binding. Addition of -aspartic acid resulted in the complete abolition of the Na⁺-dependent binding while sparing the Cl⁻-dependent binding. Scatchard analysis revealed that Cl⁻ ions induced a two-fold increase in the number of the binding sites without affecting their affinity, whereas Na⁺ ions reduced the affinity with a concomitant increase of the number of the binding sites. Addition of quisqualic acid (10⁻⁵−10⁻³ M) inhibited the Cl⁻-dependent binding of [³H]Glu to a significantly greater extent than the inhibition on Na⁺-dependent binding. acid and kainic acid exerted no preventive action on the basal, Cl⁻-dependent and Na⁺-dependent binding. respectively. The highest basal binding activity was found in the retina among various central structures examined. A significant basal binding activity of [³H]Glu was also detected in the pituitary and adrenal but not in the kidney. Chloride ions exhibited a significant facilitation of [³H]Glu binding to central regions without altering that to peripheral tissues such as pituitary and adrenal. In contrast, Na⁺ ions induced significant attenuation of the binding to the pituitary, adrenal and retina despite the occurrence of augmentation of the binding to other central structures.

These results suggest the Glu binding sites may be linked to the anion channels in the rat central nervous system and that this linkage may be absent from the pituitary, adrenal and retina.