Corticotropin-releasing factor (CRF) and the urocortins differentially regulate catecholamine secretion in human and rat adrenals, in a CRF receptor type-specific manner

Corticotropin-releasing factor (CRF) affects catecholamine production both centrally and peripherally. The aim of the present work was to examine the presence of CRF, its related peptides, and their receptors in the medulla of human and rat adrenals and their direct effect on catecholamine synthesis and secretion. CRF, urocortin I (UCN1), urocortin II (UCN2), and CRF receptor type 1 (CRF1) and 2 (CRF2) were present in human and rat adrenal medulla as well as the PC12 pheochromocytoma cells by immunocytochemistry, immunofluorescence, and RT-PCR. Exposure of dispersed human and rat adrenal chromaffin cells to CRF1 receptor agonists induced catecholamine secretion in a dose-dependent manner, an effect peaking at 30 min, whereas CRF2 receptor agonists suppressed catecholamine secretion. The respective effects were blocked by CRF1 and CRF2 antagonists. CRF peptides affected catecholamine secretion via changes of subplasmaliminal actin filament polymerization. CRF peptides also affected catecholamine synthesis. In rat chromaffin and PC12 cells, CRF1 and CRF2 agonists induced catecholamine synthesis via tyrosine hydroxylase. However, in human chromaffin cells, activation of CRF1 receptors induced tyrosine hydroxylase, whereas activation of CRF2 suppressed it. In conclusion, it appears that a complex intraadrenal CRF-UCN/CRF-receptor system exists in both human and rat adrenals controlling catecholamine secretion and synthesis.     

mRNA expression of type I and type II receptors for activin, transforming growth factor-beta, and bone morphogenetic protein in the murine erythroleukemic cell line, F5-5.fl

OBJECTIVE: Intracellular signaling of activin and transforming growth factor-beta (TGF-beta) is thought to be mediated by the same molecules (Smad2/3 and Smad4). Although differentiation of murine erythroleukemia F5-5.fl cells is induced by activin, it is not induced by TGF-beta, suggesting that at some point TGF-beta signaling is defective. The aim of this study was to investigate the unresponsiveness of F5-5.fl cells to TGF-beta. DESIGN: mRNA expression of ligands, receptors, and signal mediators for the TGF-beta family was examined in F5-5.fl cells using RT-PCR. RESULTS: Activin induced erythrodifferentiation of F5-5.fl cells in a dose-dependent manner. Neither TGF-beta1 nor bone morphogenetic protein (BMP)-4 affected the differentiation of F5-5.fl cells in the presence or absence of activin. Although mRNAs of TGF-betas (TGF-beta1, TGF-beta2 and TGF-beta3) were detected, those of inhibin/activin (alpha-, betaA- and betaB-subunits) and BMPs (BMP-2, BMP-4 and BMP-7) could not be detected in the cells, suggesting that neither activins nor BMPs are produced in F5-5.fl cells. The expression of both type I (ALK-4/ActRIB) and type II (ActRII) receptors for activin was detected in F5-5.fl cells. In contrast, while the expression of type I receptor for TGF-beta (ALK-5/TbetaRI) was detected, that of type II receptor (TbetaRII) was not. The mRNA of all Smads examined was detected in F5-5.fl cells. CONCLUSIONS: A defect in the type II receptor might cause unresponsiveness to TGF-beta in F5-5.fl cells. An erythrodifferentiation assay using F5-5.fl cells would be useful for measuring net activin activity because it would not be necessary to consider endogenous activins and BMPs.     

Inhibins differentially antagonize activin and bone morphogenetic protein action in a mouse adrenocortical cell line

Inhibin, a member of the TGF-beta superfamily, has been proposed to act as an inhibitor of activin and bone morphogenetic protein (BMP) by sequestering their type II receptors in nonsignaling complexes with betaglycan. This mechanism of inhibin action was tested in a mouse adrenocortical (AC) cell line by examining the effects of inhibins A and B on cytochrome P450 17alpha-hydroxylase 17,20-lyase (Cyp17) expression and 17alpha-hydroxylase activity, measured by progesterone 17alpha-hydroxylation, in the absence and presence of activin or BMP isoforms. Cyp17 mRNA endogenously expressed by AC cells was suppressed by activins A and B and BMP-2, -6, and -7, and each ligand accordingly inhibited 17alpha-hydroxyprogesterone production (IC(50) of 0.24, 0.27, 0.4, 0.51, and 2.2 nm, respectively). Neither inhibin A nor inhibin B alone affected Cyp17 expression or 17alpha-hydroxyprogesterone production. Both inhibin A and inhibin B blocked the inhibitory actions of activins A and B in AC cells, supporting the antiactivin model of inhibin action. Inhibin A provided more potent and effective antagonism of both activins than did inhibin B, and activin A was less subject to antagonism by either inhibin than was activin B. In contrast to the major antagonism of activin by both inhibins, only inhibin A antagonized the actions of BMP-2, BMP-6, and BMP-7, whereas inhibin B was ineffective against all tested BMP isoforms except BMP-7 at high concentrations. These results provide limited support for the anti-BMP model of inhibin action and reveal that, relative to inhibin A, inhibin B essentially behaves as a selective activin antagonist in AC cells. In conclusion, inhibins A and B differentially antagonize the actions of activins and BMPs to control adrenocortical C(19) steroid production.     

Activities of bone morphogenetic proteins in prolactin regulation by somatostatin analogs in rat pituitary GH3 cells

Involvement of the pituitary BMP system in the modulation of prolactin (PRL) secretion regulated by somatostatin analogs, including octreotide (OCT) and pasireotide (SOM230), and a dopamine agonist, bromocriptine (BRC), was examined in GH3 cells. GH3 cells are rat pituitary somato-lactotrope tumor cells that express somatostatin receptors (SSTRs) and BMP system molecules including BMP-4 and -6. Treatment with BMP-4 and -6 increased PRL and cAMP secretion by GH3 cells. The BMP-4 effects were neutralized by adding a BMP-binding protein Noggin. These findings suggest the activity of endogenous BMPs in augmenting PRL secretion by GH3 cells. BRC and SOM230 reduced PRL secretion, but OCT failed to reduce the PRL level. In GH3 cells activated by forskolin, BRC suppressed forskolin-induced PRL secretion with reduction in cAMP levels. OCT did not affect forskolin-induced PRL level, while SOM230 reduced PRL secretion and PRL mRNA expression induced by forskolin. BMP-4 treatment enhanced the reducing effect of SOM230 on forskolin-induced PRL level while BMP-4 did not affect the effects of OCT or BRC. Noggin treatment had no significant effect on the BRC actions reducing PRL levels by GH3 cells. However, in the presence of Noggin, OCT elicited an inhibitory effect on forskolin-induced PRL secretion and PRL mRNA expression, whereas the SOM230 effect on PRL reduction was in turn impaired. It was further found that BMP-4 and -6 suppressed SSTR-2 but increased SSTR-5 mRNA expression of GH3 cells. These findings indicate that Noggin rescues SSTR-2 but downregulates SSTR-5 by neutralizing endogenous BMP actions, leading to an increase in OCT sensitivity and a decrease in SOM230 sensitivity of GH3 cells. In addition, BMP signaling was facilitated in GH3 cells treated with forskolin. Collectively, these findings suggest that BMPs elicit differential actions in the regulation of PRL release dependent on cellular cAMP-PKA activity. BMPs may play a key role in the modulation of SSTR sensitivity of somato-lactotrope cells in an autocrine/paracrine manner.     

Activation by cyclic 3'':5''-adenosine monophosphate of tyrosine hydroxylase in the rat brain.

Membrane-permeable derivatives of cyclic AMP (cAMP) produced concentration-dependent increases in activity of tyrosine hydroxylase (L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2) in membrane-limited nerve endings (synaptosomes) prepared from three regions of rat brain. Increased hydroxylation occurred even after preincubation and removal of dibutyryl cyclic AMP. In all brain regions, the hydroxylation of phenylalanine and tyrosine was increased, but dibutyryl cAMP had little effect on activity of tryptophan hydroxylase, no effect on aromatic amino-acid decarboxylase, on uptake of tyrosine or phenylalanine, uptake or efflux of dopamine, or distribution of hydroxylase between cytoplasmic and particulate components of the synaptosomes. Dibutyryl cAMP decreased inhibition of catecholamine synthesis in synaptosomes by dopamine and apomorphine. In a soluble preparation of striatal tyrosine hydroxylase, activity was increased by addition of lower concentrations of cAMP or dibutyryl cAMP than with unbroken nerve endings, when subsaturating concentrations of tyrosine and cofactor were employed, while butyrate, chloride, 5'-AMP, ADP, ATP, and cyclic GMP had no activating effect. Increased activity of soluble tyrosine hydroxylase was reflected in increased affinity (Km) for substrate and cofactor and decreased affinity (Ki) for inhibitory end-product (dopamine), suggesting a change in the physical-chemical state of the enzyme or an activator molecule. Cyclic AMP may activate tyrosine hydroxylase during periods of increased neuronal activity.     

Dehydroepiandrosterone sulfate and allopregnanolone directly stimulate catecholamine production via induction of tyrosine hydroxylase and secretion by affecting actin polymerization

Adrenal cortical cells of zona reticularis produce the neuroactive steroids dehydroepiandrosterone (DHEA), its sulfate ester dehydroepiandrosterone sulfate (DHEAS), and allopregnanolone (ALLO). An interaction between zona reticularis and adrenal medulla has been postulated based on their close proximity and their interwoven borders. The aim of this paper was to examine in vitro the possible paracrine effects of these steroids on catecholamine production from adrenomedullary chromaffin cells, using an established in vitro model of chromaffin cells, the PC12 rat pheochromocytoma cell line. We have found the following: 1) DHEA, DHEAS, and ALLO increased acutely (peak effect between 10-30 min) and dose-dependently (EC50 in the nanomolar range) catecholamine levels (norepinephrine and dopamine). 2) It appears that the acute effect of these steroids involved actin depolymerization/actin filament disassembly, a fast-response cellular system regulating trafficking of catecholamine vesicles. Specifically, 10(-6) m phallacidin, an actin filament stabilizer, completely prevented steroid-induced catecholamine secretion. 3) DHEAS and ALLO, but not DHEA, also affected catecholamine synthesis. Indeed, DHEAS and ALLO increased catecholamine levels at 24 h, an effect blocked by L-2-methyl-3-(-4-hydroxyphenyl)alanine and 3-(hydrazinomethyl)phenol hydrochloride, inhibitors of tyrosine hydroxylase and L-aromatic amino acid decarboxylase, respectively, suggesting that this effect involved catecholamine synthesis. The latter hypothesis was confirmed by finding that DHEAS and ALLO increased both the mRNA and protein levels of tyrosine hydroxylase. In conclusion, our findings suggest that neuroactive steroids exert a direct tonic effect on adrenal catecholamine synthesis and secretion. These data associate the adrenomedullary malfunction observed in old age and neuroactive steroids.     

Role of renal catecholamines in the control of sodium and water excretion: lack of natriuresis by endogenous dopamine in the rat kidney

The physiological role of dopamine in renal Na and water excretion was studied in rats treated with L-3,4-dihydroxyphenylalanine (L-DOPA), haloperidol or 6-hydroxydopamine for 1 week. Daily-pooled urine samples, plasma, and kidneys were obtained from the experimental groups and untreated controls. Urinary dopamine was markedly elevated in L-DOPA-treated and transiently decreased in haloperidol-treated rats. Urinary epinephrine, but not norepinephrine was increased in the L-DOPA and haloperidol groups. However, there was no significant difference in urine volume, urinary Na, urinary K, plasma Na and K levels between the experimental groups and controls, except for 6-hydroxydopamine-treated rats. They showed a definite increase in urinary volume, Na, K and aldosterone excretion, whereas the renal norepinephrine and epinephrine content was reduced. Urinary cAMP and Na-K ATPase activity were also similar in all groups, whereas renal cAMP content and aromatic amino acid decarboxylase activity were significantly increased in the L-DOPA and haloperidol groups, suggesting an activated catecholamine synthesis in the kidney. [3H]haloperidol binding was also increased by the L-DOPA and 6-hydroxydopamine administration, but the half maximal displacement dose was similar in all groups (6.4 x 10(-9)mol/l), indicating an increase in the number of dopamine binding sites. These results indicate that 1) in contrast to the pharmacological effect of dopamine, locally-formed dopamine has no direct natriuretic action in the kidney; 2) an enhanced production of dopamine in the kidney does not suppress aldosterone secretion.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel role for bone morphogenetic proteins in the synthesis of follicle-stimulating hormone

FSH is produced in pituitary gonadotropes as an alpha/beta heterodimer, and synthesis of the beta-subunit is the rate-limiting step in overall FSH production. Synthesis of FSHbeta can be regulated by activin and inhibin, both of which are members of the transforming growth factor-beta superfamily. Bone morphogenetic proteins (BMPs) also belong to the transforming growth factor-beta family and are multifunctional growth factors involved in many aspects of tissue development and morphogenesis, including regulation of FSH action in the ovary. Here we report a novel function for BMP-7 and BMP-6 in regulating FSH synthesis in the pituitary. Using primary pituitary cell cultures derived from transgenic mice that carry the ovine FSHbeta promoter linked to a luciferase reporter gene (oFSHbetaLuc), BMP-7 or BMP-6 was found to stimulate oFSHbetaLuc expression by 6-fold. Transient expression of the oFSHbetaLuc in a transformed gonadotrope cell line, LbetaT2, was induced 4-fold by BMP-7 or BMP-6 treatment. More importantly, BMP-7 and BMP-6 increased endogenous FSH secretion by 10- and 14-fold, respectively, from LbetaT2 cells, demonstrating for the first time that a functional signaling BMP system is present in gonadotropes. Two bioneutralizing antibodies to BMP-7, which cross-react with BMP-6, but not with activin A, decreased basal oFSHbetaLuc expression and FSH secretion from transgenic mouse pituitary cultures by 83-88% and 47-48%, respectively, suggesting an autocrine or paracrine role for BMP-7 or BMP-6 in FSH synthesis. Neither bioneutralizing antibody to activin A or activin B decreased basal oFSHbetaLuc expression or mouse FSH secretion significantly. Dose-dependent inhibition of FSH synthesis by anti-BMP7 was also observed in rat and sheep pituitary cultures. These results combined with the fact that the messenger RNAs for BMP-7 and BMP-6 were detected in mouse pituitaries and LbetaT2 cells indicate that BMP-7 and/or BMP-6 can function as FSH stimulators and may be significant physiological factors maintaining basal FSH expression in vivo.     

Retroviral transfer of a human tyrosine hydroxylase cDNA in various cell lines: regulated release of dopamine in mouse anterior pituitary AtT-20 cells.

Little is known about the molecular events mediating neurotransmitter release, a crucial step in synaptic transmission. In this paper, the biosynthesis and release of L-beta-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine were analyzed in three heterologous cell lines after retroviral-mediated gene transfer of tyrosine hydroxylase (EC 1.14.16.2), the rate-limiting enzyme in catecholamine synthesis. A recombinant retrovirus encoding human tyrosine hydroxylase type I as well as neomycin-resistance gene was used to infect a fibroblast (NIH 3T3), a neuroblastoma (NS20 Y), and a neuroendocrine (AtT-20) cell line. After selection in the presence of neomycin and in tyrosine-free medium, high levels of exogenous tyrosine hydroxylase activity were detected in extracts of the three cell lines. High-performance liquid chromatography of cell extracts and culture supernatants confirmed that the three cell lines hydroxylated tyrosine to form L-DOPA and released this metabolite into the culture medium. Interestingly, the neuroendocrine cell line AtT-20 synthesized not only L-DOPA but also dopamine. Evoked secretion studies established that AtT-20 cells released the transmitter upon depolarization in a regulated, calcium-dependent way. We discuss the implication of this approach for the analyses of neurotransmitter release as well as in the context of degenerative disorders such as Parkinson disease.     

Hypoprolactinemia decreases tyrosine hydroxylase activity in the tuberoinfundibular dopaminergic neurons acutely by protein dephosphorylation and chronically by changes in gene expression

This study evaluated the roles of protein dephosphorylation or suppressed gene expression in reducing tyrosine hydroxylase activity in tuberoinfundibular dopaminergic neurons after acute or chronic bromocriptine (BROMO) administration. Diestrous or ovariectomized rats were injected with BROMO (3 mg/kg, s.c.) at 1000 h and were sacrificed 4 h later or were injected with BROMO at 12 h intervals for 3 days.In vitro tyrosine hydroxylase activity was assessed by incubating hypothalamic explants with brocresine, an L-aromatic amino acid decarboxylase inhibitor, and measuring 3,4-dihydroxyphenylalanine (DOPA) accumulation in the stalk-median eminence (SME). The incubation medium also contained either 2 μM okadaic acid, a protein phosphatase 1 and 2A inhibitor, or its vehicle (0.25% dimethylsulfoxide). Acute (4 h) and chronic (3 days) BROMO treatment suppressed circulating PRL levels from 10–12 ng/ml to<1 ng/ml and reduced tyrosine hydroxylase activity in the SME by 60% or 40% in diestrous or ovariectomized rats, respectively. Okadaic acid increased tyrosine hydroxylase activity in the SME 2-fold in control diestrous or ovariectomized rats. The reduced tyrosine hydroxylase activity in the SME after acute BROMO treatment was increased by okadaic acid 5-or 3-fold in diestrous or ovariectomized rats respectively, to a value similar to the controls. In sharp contrast, after chronic BROMO treatment, the response to okadaic acid was blunted. As assessed byin situ hybridization, tyrosine hydroxylase mRNA signal levels in the arcuate nucleus of diestrous rats were not altered after acute BROMO treatment, but were reduced by 70% after chronic BROMO treatment. The acute BROMO-induced decrease in tyrosine hydroxylase activity was reversed by co-administration of oPRL or rPRL, indicating that the action of BROMO is via a reduction in PRL. The data suggest that protein dephosphorylation may be a primary mechanism for the rapid BROMO-dependent suppression of tyrosine hydroxylase activity, whereas suppression of tyrosine hydroxylase gene expression may contribute to the lower tyrosine hydroxylase activity after chronic BROMO treatment.     

Evidence for protein kinase-C mediation of the neurotensin-induced activation of tyrosine hydroxylase in tuberoinfundibular dopaminergic neurons.

The purpose of the present study was to determine whether neurotensin acts within the arcuate nucleus/median eminence to activate tyrosine hydroxylase (TH) within tuberoinfundibular dopamine neurons. The role of Ca2+/phospholipid-dependent protein kinase (protein kinase-C) in the regulation of TH and its involvement in the neurotensin-induced activation of TH within tuberoinfundibular dopamine (TIDA) neurons also was investigated. The activity of TH within TIDA neurons was assessed by quantification of the formation of 3,4-dihydroxyphenylalanine in the arcuate nucleus/median eminence after inhibition of 3,4-dihydroxyphenylalanine decarboxylase. Neurotensin (0.1-10 nM) increased the activity of TH within the arcuate nucleus/median eminence under in vitro conditions by approximately 80%. The activity of TH in the arcuate nucleus/median eminence also was increased approximately 55% by the phorbol ester 12-O-tetradecanoyl(phorbol-13-acetate) (1-100 nM), which activates protein kinase-C. Sphingosine (10 microM), an inhibitor of protein kinase-C, attenuated the activation of TH within TIDA neurons that was induced by both 12-O-tetradecanoyl(phorbol-13-acetate) and neurotensin. Sphingosine alone did not alter the activity of TH, nor did it alter the (Bu)2cAMP-induced activation of TH in the arcuate nucleus/median eminence. It is concluded that neurotensin acts directly within the arcuate nucleus/median eminence to activate TIDA neurons. Furthermore, it is suggested that the activity of TH within these neurons is enhanced after the activation of protein kinase-C and that protein kinase-C may mediate the neurotensin-induced activation of TH within these hypothalamic dopamine neurons.