Ferenc A. Antoni

Vasopressinergic control of pituitary adrenocorticotropin secretion comes of age

This article summarizes the importance of arginine vasopressin (AVP) in the control of adrenocorticotropin (ACTH) secretion, with special reference to interactions with corticotropin releasing factor (CRF-41), glucocorticoids, and the purported corticotropin release inhibiting peptide atriopeptin. AVP that participates in the regulation of ACTH release at the pituitary level is produced in two main groups of neurons in the hypothalamus: parvicellular cells in the paraventricular nucleus, which also produce CRF-41, and magnocellular neurons in the supraoptic and paraventricular nuclei. The role of the latter in anterior pituitary hormone release has been debated for many years. Evidence generated in the last 5 years shows quite convincingly that AVP released by magnocellular neurons is, in fact, also involved in the control of ACTH. Nevertheless, it is clear that corticotrope cells require CRF-41 to maintain their capacity to secrete ACTH. This is at least due partly to the fact that AVP does not increase proopiomelanocortin mRNA transcription, while CRF-41 is a potent inducer of this gene. New developments in the area of corticotrope cell physiology are discussed, highlighting evidence for dual ACTH secreting pathways in anterior pituitary cells, which may be controlled separately by AVP and CRF-41. Evidence for interactions between ACTH secretagogues and peptidergic as well as glucocorticoid inhibitors of ACTH secretion is reviewed to demonstrate that an important aspect of AVP/CRF-41 dualism may be associated with the ability of the secretagogues to selectively modulate the efficacy of inhibitory factors. Finally, by citing examples from physiological studies on the regulation of ACTH secretion, it is shown how the multicomponent hypothalamic regulatory system operates, emphasizing the considerable signal integrating role of the adenohypophysial corticotrope cell.

Molecular diversity of cyclic AMP signalling.

Several neuroendocrine control systems are prominently controlled by G-protein coupled receptors that activate the cAMP signal transduction pathway. The discovery of multiple genes that encode the molecular machinery of cAMP metabolism has revolutionized our knowledge of cAMP mediated processes. This perhaps all too familiar second messenger can be generated by nine different membrane enzymes in the context of varied levels of activation of G proteins as well as Ca(2+)- and protein kinase C-dependent processes. The amplitude, length and subcellular distribution of the cAMP signal are further modulated by over twenty functionally distinct isotypes of cAMP-degrading phosphodiesterases in a cell- and stimulus-specific manner. The present review summarizes the key properties of the molecular machinery that generates the cAMP signal and highlights how it is deployed in neuroendocrine systems.

Alternative splicing determines sensitivity of murine calcium‐activated potassium channels to glucocorticoids

1 Large‐conductance Ca2+‐ and voltage‐activated potassium (BK) channels are important regulators of cellular excitability. Here, we present a patch‐clamp electrophysiological analysis of splice‐variant‐specific regulation by the synthetic glucocorticoid dexamethasone (DEX) of BK channels consisting of cloned STREX or ZERO α‐subunit variants expressed in human embryonic kidney (HEK 293) cells. 2 STREX channels in isolated membrane patches were inhibited by protein kinase A (PKA) and this was blocked on pre‐treatment of intact cells with DEX (100 nm) for 2 h. 3 The effect of DEX required the synthesis of new mRNA and protein. Furthermore, it required protein phosphatase 2A (PP2A)‐like activity intimately associated with the channels, as it was blocked by 10 nm okadaic acid but not by the specific protein phosphatase‐1 inhibitor peptide PPI−2. 4 ZERO variant channels that lack the STREX insert were activated by PKA but were not influenced by DEX. ZERO channels containing a mutant STREX domain (S4STREXA) were also activated by PKA. Importantly, DEX blocked PKA activation of S4STREXA channels in a PP2A‐dependent manner. 5 Taken together, the STREX domain is crucial for glucocorticoid regulation of BK channels through a PP2A‐type enzyme. Moreover, glucocorticoids appear to induce a generic set of proteins in different types of cells, the actions of which depend on the expression of cell‐specific targets.

Reciprocal regulation of calcium dependent and calcium independent cyclic AMP hydrolysis by protein phosphorylation.

The hydrolysis of cyclic nucleotide second messengers takes place through multiple cyclic nucleotide phosphodiesterases (PDEs). The significance of this diversification is not fully understood. Here we report the differential regulation of low Km Ca2+‐activated (PDE1C) and Ca2+‐independent, rolipram‐sensitive (PDE4) PDEs by protein phosphorylation in the neuroendocrine cell line AtT20. Incubation of cells with 8‐(4‐chlorophenylthio)‐cyclic AMP (CPT‐cAMP) enhanced PDE4 and reduced PDE1C activity. These effects were blocked by H89 indicating mediation by cAMP‐dependent protein kinase (PKA), furthermore in broken cell preparations PKA produced the same reciprocal changes of PDE activities. Calyculin A, an inhibitor of protein phosphatases 1 and 2 A, stimulated PDE4 and enhanced the inhibitory effect of CPT‐cAMP on PDE1C. The reduction of PDE1C activity was characterized by a marked attenuation of the activation by Ca2+/calmodulin. Stimulation of PDE4 activity by CPT‐cAMP or calyculin A was attributable to PDE4D3 and these effects could also be reproduced in human embryonic kidney cells expressing epitope‐tagged PDE4D3. Together, these data show reciprocal regulation of PDE1C and PDE4D by PKA, which represents a novel scheme for plasticity in intracellular signalling.

Early glucocorticoid induction of calmodulin and its suppression by corticotropin-releasing factor in pituitary corticotrope tumor (AtT20) cells

Glucocorticoids inhibit stimulus-evoked ACTH secretion by the rapid induction of new protein(s) that suppress intracellular free calcium signals. The present study examined whether the calcium receptor protein, calmodulin, is induced by glucocorticoids in the mouse pituitary corticotrope tumor (AtT20 D16:16) cell line. Treatment of AtT20 D16:16 cells with the synthetic glucocorticoid dexamethasone markedly (up to 10-fold) increased the level of a single (approximately 1.6kb) calmodulin mRNA 90 min after the application of steroid. Puromycin applied 15 min before and during dexamethasone treatment blocked the induction of this mRNA, suggesting that additional glucocorticoid induced transcription factor proteins may be required for enhanced calmodulin gene transcription. A two-fold increase in the intensity of an approximately 18K immunoreactive calmodulin protein band was detected by immunoblotting at 90 min after dexamethasone administration. Corticotropin releasing factor, added for 30 min at the start of steroid treatment, prevented the increase of calmodulin mRNA, as well as the suppression of corticotropin releasing factor-evoked ACTH release caused by dexamethasone. These data suggest that calmodulin may be involved in the early phase of glucocorticoid inhibition of pituitary ACTH release.

Selective Enhancement of an A Type Potassium Current by Dexamethasone in a Corticotroph Cell Line

Perforated patch recording was used to examine the effect of the synthetic steroid dexamethasone on the whole cell potassium (K+) current, in the mouse corticotroph tumour cell line AtT20/D16‐16. In 15 out of 52 control cells (29%) there was a rapidly‐activating, rapidly‐ inactivating K+ current of the A type, the amplitude of which was strongly dependent on the holding potential in use prior to its activation by depolarising voltage pulses, and which was blocked by 1 mM 4‐aminopyridine (4‐AP, n = 5). The effect of dexamethasone (100nM, 2h, 37°C) was that the A current increased in prevelance (24 out of 31 cells, 77%), lost its dependence on holding potential (over the range studied), and as a result became significantly larger than in controls, for certain voltage steps (peak A current density was 18.5 +2.4 pA/pF (n = 12) for control cells and 26.3 ± 3.9 pA/pF (n = 18) for dexamethasone treated cells, for a step to +30mV from ‐60mV, values are mean ± SEM). All cells exhibited a slowly‐activating, sustained K+ current, which was unaffected by changes in the holding potential, unaffected by 4‐AP and consisted of at least 3 components: one blocked by 30 mM tetraethylammonium(TEA) or 100 nM charybdotoxin (CTX); a second blocked by 100 nM apamin; and a third not blocked by TEA, CTX, apamin, clofilium (100 nM) or niflumic acid (0.1 mM). Dexamethasone produced no change in the slowly‐activating, sustained current nor in any of its individual components. The effect of dexamethasone on the A current was completely blocked by 0.1 mM puromycin, a protein synthesis blocker, while puromycin alone did not affect the size or frequency of the A current, nor alter the slowly‐activating, sustained current. Secretion studies using 4‐AP confirmed that the A current has a role in stimulated adrenocorticotrophic hormone (ACTH) secretion. In summary, AtT20 cells contain at least four types of K+ current: an A current and 3 currents contributing to the slowly‐activating current. Selective enhancement of the A current by dexamethasone, shown here to require synthesis of new protein, is one of the mechanisms whereby glucocorticoids exert inhibitory control on ACTH secretion.

Cellular localisation of adenylyl cyclase : A post-genome perspective

The intracellular messenger cAMP is essential for vital processes ranging from ovulation to cognition. There are 10 genes for adenylyl cyclase (AC), the biosynthetic enzyme of cAMP. Nine of these encode membrane-bound proteins and one gives rise to soluble AC. The understanding of the biological significance of this molecular diversity is incomplete. Membrane-bound ACs conform to the same structural blueprint but have markedly different regulatory characteristics. AC mRNAs are differentially distributed in the body suggesting non-redundant physiological functions. The subcellular localisation of AC isoforms has not been examined in detail. Here we discuss the current knowledge on the intracellular targeting of AC isoforms, and highlight the technical problems of AC detection, some of which appear to be caused by the poor quality-control of commercially supplied antibodies. The principal message is that intracellular targeting of ACs may be isoform-specific and also dependent on the cellular context of expression.

Circadian changes of type II adenylyl cyclase mRNA in the rat suprachiasmatic nuclei

Circadian functions of the suprachiasmatic nuclei (SCN) are influenced by cyclic AMP (cAMP). Adenylyl cyclase type II (AC-II) is a cAMP-generating enzyme which, in the context of activation by Gsalpha, is further stimulated by protein kinase C or G protein betagamma subunits. Using in situ hybridization we have found a biphasic variation in AC-II mRNA within the rat SCN during the light-dark cycle (peaks at Zeitgeber time 6 and 18) and also in constant darkness (peaks at circadian time 2 and 14). The cingulate cortex showed no such variation. These findings suggest that circadian changes in AC-II expression may be pertinent to the rhythmic functions of the SCN.

Early Glucocorticoid Inhibition of Hormone Release in Pituitary Corticotrope Cells Is Voltage Dependent

The suppression of adrenocorticotropic hormone (ACTH) secretion by adrenal corticosteroids is a functionally significant element of homeostatic regulation in the hypothalamic-pituitary-adrenocortical axis.’ Previous work suggests that a pivotal early effect of corticosteroids in anterior pituitary tumor (AtT20) cells is the reduction of corticotropin releasing factor (CRF)-induced intracellular free calcium transients.* Electrophysiological studies in hippocampal neuron^^.^ and pituitary tumor cell line^^.^ have indicated that glucocorticoids may “stabilize,” i.e., hyperpolarize, the membrane potential primarily through the enhancement of potassium currents, which would reduce the activation of voltage-operated calcium currents. As further tests of this hypothesis the present study examined whether 1) the ACI’H-release inhibiting action of glucocorticoids could be influenced by manipulations that alter the activity of ion channels in the plasma membrane and bring about depolarization; 2) glucocorticoid inhibition persists in electrically permeabilized AtT20 cells.

Vasupressin and the Endocrine Response To Stress

This paper highlights the role of vasopressin in the endocrine response to stress at various levels of regulation in the hypothalamic-pituitary-adrenocortical system. Novel data arc presented regarding the interaction of vasopressin and corticotropin-releasing factor (CRF) in rat anterior pituitary cells. In this system CRF-induced cyclic AMP synthesis is under feedback inhibition by intracellular free calcium. Vasopressin enhances the cyclic AMP response to CRF apparently by reducing the efficiency of calcium inhibition. As corticosteroid mediated inhibition of ACTH release is also mediated by a calcium-dependent process, these findings show a mechanism whereby vasopressin may change the set-point of glucocorticoid negative feedback at the pituitary level.