We investigate cellular signaling cascades in pituitary cells and interactions between plasma membrane electrical events and receptor-controlled pathways. Our research focuses on calcium signaling and cellular regulation, and our main objective is to elucidate the channels and receptors involved in calcium signaling and the role of calcium ions as messengers in controlling signaling, secretion, and gene expression. Our approach calls for characterizing calcium signaling from biophysical, physiological, pharmacological, and molecular biology points of view. We are currently studying the biophysical basis of pituitary cell type–specific calcium signaling-secretion coupling, the molecular dissection of ATP-gated P2X receptor channels, and the roles of G protein–coupled and tyrosine kinase receptors in control of signaling, hormone secretion, and gene expression.
Voltage-gated channels and spontaneous electrical activity and calcium signaling
Kretschmannova, Gonzalez-Iglesias
The membrane potential of secretory anterior pituitary cells is not stable but rather oscillates around the baseline potential of about –60 mV. When membrane potential oscillations reach the threshold level, pituitary cells fire action potentials, a common activity of all secretory anterior pituitary cells that occurs independently of external stimuli. We previously found that pituitary cells exhibit different patterns of electrical activity and action potential–driven calcium signaling and secretion in vivo and in vitro. Cultured lactotrophs and somatotrophs and some immortalized pituitary cells frequently exhibit larger membrane potential oscillations, on top of which the depolarizing plateau and bursts of action potentials are generated, with spikes that usually do not reach the reverse potential. In contrast, membrane potential oscillations in cultured gonadotrophs and some immortalized pituitary cells generate single action potentials that are sharp and of short duration and that reverse polarization of membrane potential. We have progressed in understanding the role of cell type–specific spontaneous firing of action potential in calcium signaling and basal hormone secretion as well as the role of plasma membrane channels that participate in spiking. Our work included characterization of depolarizing sodium and calcium channels, cell type–specific expression of potassium channels, the paradoxical role of large-conductance calcium-activated channels (BK channels), in controlling action potential–driven calcium entry, and the role of small-conductance calcium-activated channels (SK channels) in plateau bursting in agonist-activated gonadotrophs.
Our current efforts focus on channels and pathways that participate in pacemaking. The finding that normal and immortalized pituitary cells express calcium-inhibitable adenylyl cyclases prompted us to examine the hypothesis that cyclic nucleotides play a role in spontaneous pacemaking and basal prolactin release in such pituitary cells by controlling hyperpolarization-activated cation channels and/or cyclic nucleotide–gated channels. Consistent with this hypothesis, stimulation of adenylyl cyclases by forskolin initiates firing of action potentials in quiescent lactotrophs and increases the spiking frequency in spontaneously active cells, which, in turn, facilitates voltage-gated calcium influx and prolactin secretion. Inhibition of phosphodiesterases by 3-isobutyl-1-methylxanthine also stimulates cyclic nucleotide accumulation and prolactin release. Conversely, MDL-12330A inhibits basal and forskolin-stimulated cyclic nucleotide production in a concentration-dependent manner, along with electrical activity, calcium transients, and prolactin secretion. Basal cyclic AMP production is augmented by removal of extracellular calcium and is attenuated by facilitation of voltage-gated calcium influx. The results suggest that the intrinsic activity of calcium-inhibitable adenylyl cyclases do indeed contribute to the control of spontaneous pacemaking and basal prolactin secretion.
Stojilkovic SS. Ca2+-regulated exocytosis and SNARE function. Trends Endocrinol Metab 2005;16:81-83.
Stojilkovic SS, Zemkova H, Van Goor F. Biophysical basis of pituitary cell type-specific Ca2+ signaling-secretion coupling. Trends Endocrinol Metab 2005;16:152-159.
Receptor-controlled calcium signaling, secretion, and gene expression
Jiang, Hatae, Andric,1 Kostic1; in collaboration with Wong
Several G protein–coupled and tyrosine kinase receptors are expressed in pituitary cells and regulate various cellular functions. We studied the role of gonadotropin-releasing hormone, growth hormone–releasing hormone, and growth hormone receptors in signaling in pituitary cells. Specifically, we addressed the role of calcium influx in sustained and repetitive calcium signaling and the mechanism of calcium efflux and the kinetics of recovery of calcium signaling during repetitive stimulation with gonadotropin-releasing hormone in neonatal pituitary gonadotrophs. We found that growth hormone can serve as an intrapituitary autocrine/paracrine factor that maintains growth hormone gene expression in somatotrophs and that such maintenance is mediated by Janus kinase-2/MAPK and Janus kinase-2/phosphoinositide 3-kinase cascades coupled to growth hormone receptors. Finally, we studied the calcium-dependent and -independent roles of growth hormone–releasing hormone receptors in the control of soluble guanylyl cyclase activity in pituitary cells. Our current effort focuses on control of expression of this enzyme and its roles in signaling and secretion.
As discussed above, normal and immortalized pituitary cells, including lactotrophs, spontaneously generate action potentials. In lactotrophs in vitro, such spontaneous electrical activity and the associated voltage-gated calcium influx are sufficient to maintain high prolactin release. Thus, the control of spontaneous voltage-gated calcium influx in these cells is relevant not only for keeping serum prolactin levels within the physiological concentration range but also for preventing the effects of excessive calcium influx on other cellular functions. G protein–coupled dopamine receptors play an important role in the control of prolactin release by blocking spontaneous voltage-gated calcium influx. Our results indicate that endothelin receptors also inhibit voltage-gated calcium influx–dependent prolactin release. However, the receptors inhibit secretion downstream of voltage-gated calcium influx in a phospholipase C– and tyrosine kinase–independent manner. We also found that endothelin receptors are coupled to both pertussis toxin–sensitive and –insensitive Gi proteins. Finally, we discovered that the coupling of endothelin receptors to the Gz signaling pathway accounts for inhibition of prolactin secretion downstream of voltage-gated calcium influx. Sustained inhibition of secretion is achieved through downregulation of the adenylyl cyclase signaling cascade, whereas rapid inhibition also occurs at elevated cAMP levels regardless of the status of phospholipase C, tyrosine kinases, and protein kinase C. The results indicate that the coupling of seven-transmembrane–domain receptors to Gz proteins provides a pathway that effectively blocks hormone secretion for a prolonged time without interfering with pacemaking activity and calcium influx–dependent cellular functions. Our ongoing investigations focus on the identification of endothelin receptor subtypes responsible for coupling to the Gz signaling pathway.
Andric SA, Zivadinovic D, Gonzalez-Iglesias AE, Lachowicz A, Tomic M, Stojilkovic SS. Endothelin-induced, long lasting, and Ca2+ influx-independent blockade of intrinsic secretion in pituitary cells by Gz subunits. J Biol Chem 2005;280:26896-26903.
Kostic TS, Andric SA, Stojilkovic SS. Receptor-controlled phosphorylation of alpha-1 soluble guanylyl cyclase enhances nitric oxide-dependent cyclic guanosine 5´-monophosphate production in pituitary cells. Mol Endocrinol 2004;18:458-470.
Zemkova H, Balik A, Kretschmannova K, Mazna P, Stojilkovic SS. Recovery of Ins(1,4,5)-trisphosphate-dependent calcium signaling in neonatal gonadotrophs. Cell Calcium 2004;36:89-97.
Zhou H, Ko WKW, Ho WKK, Stojilkovic SS, Wong AOL. Novel aspects of growth hormone (GH) autoregulation: GH-induced GH gene expression in grass carp pituitary cells through autocrine/paracrine mechanisms. Endocrinology 2004;145:4615-4628.
ATP-gated receptor channels and calcium signaling
Yan, He,1 Liang, Tomic; in collaboration with Koshimizu, Obsil, Zemkova
We previously found that the purinergic signaling system is operative in normal and immortalized anterior pituitary cells. The cells release ATP under resting conditions and in response to activation of calcium-mobilizing receptors. However, there is no correlation between the rate of basal hormone and ATP release, suggesting that ATP is not co-secreted with hormones by regulated exocytosis. Experiments in progress are directed toward characterizing a pathway responsible for ATP release. The cells also express ecto-nucleotidases, which hydrolyze ATP, resulting in formation of the respective nucleoside and free phosphate. We found transcripts for the ecto-nucleotidase eNTPDase 1-3 in pituitary cells. ADP and adenosine, the products of this hydrolytic cascade, also act as extracellular messengers by activating distinct plasma membrane receptors, which are termed purinergic and belong to two groups: P1 and P2 receptors. Pituitary cells express both families of receptors. We identified the presence of G protein–coupled and ATP/ADP-gated P2Y1, P2Y4, P2Y6, and P2Y12 receptors and characterized the role of P2Y1 receptor subtype in the lactotroph fraction of cells. Calcium-conducting P2X receptor-channels (P2XRs), including P2X2aR and its spliced form P2X2bR, as well as P2X3R, P2X4R, and P2X7R are also expressed in these cells. In lactotrophs, the P2X4R subtype probably provides a major pathway for calcium influx–dependent signaling and hormone secretion.
P2X receptors are a family of ligand-gated cation channels composed of two transmembrane domains, N- and C-termini located intracellularly, and a large extracellular loop containing the ATP-binding domain. To identify regions important for binding and gating, our work with recombinant channels focuses on chimeras and point mutagenesis of conserved ectodomain residues. We expressed mutant channels in human embryonic kidney 293 cells and mouse gonadotropin-releasing hormone–secreting GT1 neurons and analyzed the results with calcium imaging and patch-clamp techniques. Experiments with chimeric P2XRs helped us characterize gating, ionic conduction, deactivation of receptors, structural determinants of receptor desensitization, and recovery from desensitization. To identify regions important for ATP binding, we used the known sequence and secondary structure similarities between the Lys180-Lys326 ectodomain region of P2X4 and the class II aminoacyl-tRNA synthetases as a guide to generate a three-dimensional model of the receptor binding site and to design mutants. The interplay between homology modeling and site-directed mutagenesis suggested that the Asp280 residue of P2X4R coordinates ATP binding via the magnesium ion, that Phe230 coordinates the binding of the adenine ring of ATP, and that Lys190, His286, and Arg278 coordinate the actions of negatively charged alpha, beta, and gamma phosphate groups, respectively. Until the crystal structure of the channel is solved, this model could provide a useful approach for future studies on the identification of the ATP-binding domain and gating of P2XRs. Using the P2X4R model and single-residue mutants, we are currently investigating the relevance of the two transmembrane regions and the Tyr315-Ile333 sequence in receptor gating.
He M-L, Gonzalez-Iglesias AE, Tomic M, Stojilkovic SS. Release and extracellular metabolism of ATP by ecto-nucleotidase eNTPDase 1-3 in hypothalamic and pituitary cells. Purinergic Signal 2005;1:135-144.
Stojilkovic SS, Tomic M, He M-L, Yan Z, Koshimizu T, Zemkova H. Molecular dissection of purinergic P2X receptor channels. Ann NY Acad Sci 2005;1048:116-130.
Yan Z, Liang Z, Tomic M, Obsil T, Stojilkovic SS. Molecular determinants of the agonist binding domain of a P2X receptor channel. Mol Pharmacol2005;67:1078-1088.
Zemkova H, He ML, Koshimizu T, Stojilkovic SS. Identification of ectodomain regions contributing to gating, deactivation, and resensitization of purinergic P2X receptors. J Neurosci 2004;24:6968-6978.
Publications Related to Other Work
Stojilkovic SS, Zivadinovic D, Hegedis A, Marjanovic M, Radoslav K. Andjus (1926-2003): a brief summary of his life and work. Ann NY Acad Sci 2005;1048:1-9.
Zemkova H, Balik A, Stojilkovic SS. Expression and signal transduction pathways of melatonin receptors in pituitary. In: Pandi-Perumal SR, Cardinali D, eds. Melatonin: Biological Basis of Its Function in Health and Disease. Georgetown, TX: Landis Bioscience, 2005;1-19.
1Ma-Lan He, PhD, former Postdoctoral Fellow
COLLABORATORS
Taka-aki Koshimizu, MD, PhD, Kyoto University, Kyoto, Japan
Tomas Obsil, PhD, Charles University, Prague, Czech Republic
Anderson Wong, PhD, University of Hong Kong, Hong Kong, China
Hana Zemkova, PhD, Czech Academy of Sciences, Prague, Czech Republic
For further information, contact stojilks@mail.nih.gov.