SIGNALING AND SECRETION IN NEUROENDOCRINE CELLS
, Head, Section on Cellular Signaling
Melanija Tomić, PhD, Staff Scientist
Zonghe Yan, MD, PhD, Postdoctoral Fellow
Karla Kretschmannova, PhD, Visiting Fellow
Shuo Li, PhD, Visiting Fellow
Takayo Murano, MD, PhD, Visiting Fellow
Hana Weber-Zemkova, PhD, Guest Researcher1
We investigate cellular signaling cascades and secretion in pituitary cells, with special emphasis on the interactions between plasma membrane electrical events and receptor-controlled pathways. Pituitary cells exhibit spontaneous firing of action potentials and calcium transients, and hormonal stimulation leads to up- or downregulation of electrical activity and voltage-gated calcium influx–dependent secretion through a complex cascade of events. Our main objective is to elucidate the channels and receptors involved in calcium signaling and the role of calcium ions as messengers in controlling intracellular signaling and secretion. We are currently studying the biophysical basis of pituitary cell type–specific calcium signaling–secretion coupling, the metabolism and roles of cyclic nucleotides in the regulation of electrical activity and calcium signaling, the identification and role of endothelin receptor subtypes in the control of electrical activity, and the role and molecular properties of calcium-conducting purinergic receptor channels in pituitary functions.
Mechanism of spontaneous electrical activity in pituitary cells
This and other laboratories have characterized several plasma membrane channels in pituitary cells, but the mechanism underlying their pacemaking activity remains unknown. Our current efforts focus on the role of sodium-conducting channels in the control of pacemaking in pituitary cells. The removal of sodium from these cells reversibly hyperpolarized the membrane potential, suppressed calcium oscillations, and then led to a reduction in the level of intracellular calcium concentration to near steady-state levels. In contrast, the blockade by tetrodotoxin of voltage-dependent sodium channels, which are expressed in pituitary cells, was ineffective. One group of channels that could account for the effects of removed extracellular sodium is the hyperpolarization-activated (HCN) channels, which play a distinct role in the control of membrane excitability in spontaneously active cardiac and neuronal cells. RT-PCR analysis revealed the presence of mRNA transcripts for HCN2, HCN3, and HCN4 subunits in normal and immortalized cells. Furthermore, hyperpolarization of the membrane potential below −60 mV elicited a slowly activating voltage-dependent inward current in the majority of tested pituitary cells. Bath application of 1 mM cesium and 100 µM ZD7288 blocked the current. However, application of these blockers of HCN channels had no significant effect on resting membrane potential or electrical activity. Our results indicated that HCN channels in pituitary cells were under tonic activation by the basal level of cAMP. Our ongoing experiments focus on the expression and possible role of other sodium-conducting channels in spontaneous electrical activity and basal hormone release.
Using pituitary somatotrophs as a cell model, we combined experiments and theory to clarify the mechanisms underlying spontaneous and receptor-controlled electrical activity. Experiments support a role for (1) a sodium-conducting and tetrodotoxin-insensitive channel in controlling spontaneous and growth hormone-releasing hormone– (GHRH) stimulated pacemaking, the latter in a cAMP-dependent manner; (2) an opposing role of spontaneously active inwardly rectifying potassium (Kir) channels and G protein–regulated Kir channels in somatostatin-mediated inhibition of pacemaking; and (3) a role for voltage-gated calcium channels in spiking and large-conductance (BK-type) calcium-activated potassium channels in plateau bursting. The mathematical model is compatible with a wide variety of experimental data involving pharmacology and extracellular ion substitution and supports the importance of constitutively active tetrodotoxin-insensitive sodium and Kir channels in maintaining spontaneous pacemaking in pituitary somatotrophs. The model also suggests that these channels are involved in the up- and downregulation of electrical activity by GHRH and somatostatin. In the model, the plateau bursting is controlled by two functional populations of BK channels characterized by their distance from the voltage-gated calcium channels. The rapid activation of the proximal BK channels is critical for the establishment of the plateau, whereas slow recruitment of the distal BK channels terminates the plateau.
Gonzalez-Iglesias AE, Kretschmannova K, Tomic M, Stojilkovic SS. ZD7288 inhibits exocytosis in an HCN-independent manner and downstream of voltage-gated calcium influx in pituitary lactotrophs. Biochem Biophys Res Commun 2006;346:845-50.
Kretschmannova K, Gonzalez-Iglesias AE, Tomić M, Stojilkovic SS. Dependence of hyperpolarisation-activated cyclic nucleotide-gated channel activity on basal cyclic adenosine monophosphate production in spontaneously firing GH3 cells. J Neuroendocrinol 2006;16:484-93.
Stojilkovic S. Pituitary cell type-specific electrical activity, calcium signaling and secretion. Biol Res 2006;39:401-23.
Tsaneva-Antanasova K, Sherman A, Van Goor F, Stojilkovic SS. Mechanism of spontaneous and receptor-controlled electrical activity in pituitary somatotrophs: experiments and theory. J Neurophysiol 2007;98:13-44.
Receptor-controlled electrical activity in pituitary cells
Several hormones stimulate and inhibit spontaneous electrical activity of pituitary cells. Our ongoing work focuses on the role of endothelins, ATP, and androgens in control of voltage-gated calcium influx and hormone secretion. Pituitary cells produce endothelins, and all five major secretory cell types express functional endothelin-A receptors. In gonadotrophs, stimulation of the receptors leads to activation of the Gq/11 signaling pathway accompanied by oscillatory calcium release from intracellular pools, gonadotropin secretion, and facilitation of voltage-gated calcium influx. In somatotrophs and lactotrophs, however, endothelins inhibit spontaneous voltage-gated calcium influx through the Gi/o signaling pathway. These observations raised the possibility that several receptor subtypes are generated by alternative RNA splicing of the pituitary endothelin-A receptor and, though coupled to different G proteins and intracellular signaling, exhibit comparable binding characteristics.
Consistent with our hypothesis, we recently isolated from rat anterior pituitary cDNAs of endothelin-A transcripts, which are generated by alternative RNA splicing. Deletion of exon 2 and insertion of fragments from introns 1 and 2 accounted for the formation of three misplaced proteins, whereas insertion of a fragment from intron 6 resulted in the generation of a functional plasma membrane receptor, termed endothelin-A-C13 receptor. In this splice variant, a shorter 382A-to-399L sequence substituted for the C-terminal 382S-to-426N sequence of the wild-type receptor, resulting in alteration of the putative domains responsible for both coupling to Gq/11 and Gs proteins and endocytotic recycling as well as in deletion of the predicted protein kinase C/casein kinase 2 phosphorylation sites. We identified the mRNA transcripts for the splice receptor in normal and immortalized pituitary cells and several other tissues. The pharmacological profiles of recombinant wild-type and splice receptors were highly comparable, but the coupling of splice receptor to the calcium-mobilizing signaling pathway was attenuated, causing a rightward shift in the potency for agonist. Furthermore, the efficacy of splice receptor in stimulating the adenylyl cyclase–signaling pathway and internalizing it was significantly reduced. These results indicate, for the first time, the presence of a novel endothelin-A splice receptor, which could contribute to the functional heterogeneity among secretory pituitary cell types.
Dopamine secreted from hypophysial hypothalamic neurons is a principal inhibitory regulator of prolactin (PRL) release by pituitary lactotrophs through the activation of dopamine-2 receptors. The receptors signal through the pertussis toxin–sensitive Gi/o and pertussis toxin–insensitive Gz proteins as well as through a G protein–independent, b-arrestin/GSK3–dependent pathway. Activation of the receptors in pituitary lactotrophs leads to inhibition of PRL release. It has been suggested that such inhibition occurs through the Gi/o-a protein–mediated inhibition of cAMP production and/or Gi/o-bg dimer–mediated activation of Kir channels and inhibition of voltage-gated calcium channels. Recently, we found that the dopamine agonist–induced inhibition of spontaneous calcium influx and release of pre-stored PRL was preserved when cAMP levels were elevated by forskolin and Kir channels were inhibited by cesium. We further observed that dopamine agonists inhibited both spontaneous and depolarization-induced calcium influx in controls but not in pertussis toxin–treated cells, suggesting that control of voltage-gated calcium channel gating could represent the major pathway through which dopamine receptors block PRL release. However, agonist-induced inhibition of PRL release was only partially relieved in pertussis toxin–treated cells, indicating that dopamine receptors also inhibit exocytosis downstream of voltage-gated calcium influx. The pertussis toxin–insensitive step in agonist-induced inhibition of PRL release was not affected by inhibition of PI3-kinase with wortmannin or of GSK-3 with lithium but was attenuated by blockage of the Gz signaling pathway with phorbol esters. These results indicate that dopamine inhibits basal PRL release by blocking voltage-gated calcium influx through the Gi/o signaling pathway and by desensitizing calcium-secretion coupling, probably through the Gz signaling pathway.
Extracellular ATP is the other major intrapituitary factor that contributes to the control of calcium signaling and secretion in pituitary cells; it activates G protein–coupled P2Y receptors and calcium-conducting P2X channels. Initially, we characterized the expression and role of ATP-gated receptors and channels in lactotrophs. More recently, we characterized the expression and electrophysiological properties of purinergic receptors in pituitary gonadotrophs from embryonic, neonatal, and adult rats. In cells from all three age groups, ATP induced non-oscillatory, depolarizing, slowly desensitizing, and rapidly deactivating current, indicating that the cells express cation-conducting P2X channels but not calcium-mobilizing P2Y receptors. The amplitudes of P2X current response and rates of receptor desensitization were dependent on ATP concentration. The biophysical and pharmacological properties of P2X currents were consistent with the expression of the P2X2 subtype of channels in the rat cells. ATP-induced rapid depolarization of gonadotrophs led to initiation of firing in quiescent cells, increased frequency of action potentials in spontaneously active cells, and transient stimulation of luteinizing hormone release. ATP also influenced gonadotropin-releasing hormone–induced current and membrane potential oscillations and hormone release in an extracellular calcium–dependent manner. These inositol phosphate–dependent oscillations were facilitated, slowed, or stopped depending on ATP concentration, the time of its application, and the calcium content in intracellular stores. The results indicate that, in gonadotrophs, P2X receptors could operate as pacemaking channels and modulators of agonist-controlled electrical activity and secretion.
Gonzales-Iglesias AE, Murano T, Tomić M, Stojilkovic SS. Dopamine inhibits basal prolactin release in pituitary lactotrophs through pertussis toxin-sensitive and -insensitive signaling pathway. Endocrinology 2007, in press.
Hatae N, Aksentijevich N, Zemkova HW, Kretschmannova K, Tomić M, Stojilkovic SS. Cloning and functional identification of novel endothelin receptor type A isoforms in pituitary. Mol Endocrinol 2007;21:1192-1204.
Weiss JM, Stojilkovic SS, Diedrich K, Ortmann O. Effects of testosterone on hormonal content and calcium-dependent basal secretion in female rat pituitary cells. J Steroid Biochem Mol Biol 2007;103:149-57.
Zemkova H, Balik A, Jiang Y, Kretschmannova K, Stojilkovic SS. Roles of purinergic P2X receptors as pacemaking channels and modulators of calcium-mobilizing pathway in pituitary gonadotrophs. Mol Endocrinol 2006;20:1423-36.
Characterization of cyclic nucleotide signaling pathways in pituitary cells
We studied the relevance of cyclic nucleotide–signaling pathways to the control of electrical activity, voltage-gated calcium influx, and PRL release. Our results indicate that both voltage-gated calcium influx–inhibitable and –insensitive adenylyl cyclase subtypes contribute to basal cAMP production, with soluble guanylyl cyclases exclusively responsible for basal cGMP production. Inhibition of basal adenylyl cyclase activity, but not of soluble guanylyl cyclase activity, reduced PRL release. In contrast, activation of adenylyl cyclases by forskolin stimulated cAMP and cGMP production as well as electrical activity, calcium influx, and PRL secretion. Elevation of cAMP and cGMP levels by inhibition of phosphodiesterase activity was also accompanied by increased PRL release. The adenylyl cyclase inhibitors attenuated forskolin-stimulated cyclic nucleotide production, calcium influx, and PRL release. The cell-permeable 8-bromo-cAMP stimulated firing of action potentials and PRL release and rescued hormone secretion in cells with inhibited adenylyl cyclases in an extracellular calcium–dependent manner, whereas 8-bromo-cGMP and 8-(4-chlorophenylthio)-2¢-O-methyl-cAMP were ineffective. Protein kinase A inhibitors did not stop spontaneous and forskolin-stimulated pacemaking, voltage-gated calcium influx, and PRL release. The results indicate that cAMP facilitates voltage-gated calcium influx and secretion in a predominantly protein kinase A– and Epac cAMP receptor–independent manner. Our ongoing work focuses on characterization of cyclic nucleotide–gated channels in the action of cAMP on spontaneous electrical activity and basal hormone release.
Our earlier studies revealed that soluble guanylyl cyclase represents the major pathway for synthesis of cGMP in pituitary cells. The pituitary enzyme is composed of alpha-1 and beta-1 subunits, with dimer activity regulated directly at the protein level. Physiologically, the most relevant mode of such regulation is mediated by the binding of nitric oxide to the heme group of sGC. Recently, we focused on characterizing the alpha1-soluble guanylyl cyclase gene promoter in rat pituitary cells. We identified the transcription start site of the alpha1 soluble guanylyl cyclase gene in the rat cells and cloned the 3.5 kb 5¢ promoter. Sequence analysis of this TATA-less promoter revealed several putative binding sites for transcriptional factors, including the CCAAT site at −41 to −32 and the Sp1 site at −34 to −24. Transfection of pituitary cells with constructs of variable lengths confirmed the relevance of different promoter regions in the control of transcriptional activity. Among them, the region from −49 to + 156 was critical for basal transcriptional activity. Electrophoretic mobility shift assay using nuclear proteins extracted from normal and immortalized pituitary cells indicated that the CCAAT/Sp1 site within the region from −49 to +156 interacted specifically with CCAAT-binding factor and Sp1. The latter two sites partly overlapped, and both conferred stimulatory effects. Chromatin immunoprecipitation confirmed the in vivo recruitment of CCAT-binding factor and Sp1. The results indicate that the composite CCAAT/Sp1 cis-element contributes to the expression of the alpha1-sGC subunit in resting pituitary cells.
Our recent experiments indicated that the cellular efflux of cyclic nucleotides was detectable in normal and immortalized pituitary cells under resting conditions and was enlarged after concurrent stimulation of cAMP and cGMP production. We also observed in resting and stimulated cells that the efflux pumps transported the majority of de novo–produced cGMP, but not cAMP, limiting cGMP’s intracellular accumulation to a concentration range of 1 to 2 µM. We further observed that stimulation and inhibition of cGMP production alone did not affect cAMP efflux, suggesting the operation of two distinct transport pathways in pituitary cells. The rates of cAMP and cGMP effluxes were comparable, and probenecid and progesterone blocked both pathways. In a search for a pathway responsible for cGMP-specific efflux, we analyzed the expression of mRNA transcripts for multidrug resistance proteins in normal and immortalized pituitary cells. Our results indicated the expression of transcripts for MRP4, MRP5, and MRP8 in normal pituitary cells, whereas GH3 immortalized pituitary cells expressed only transcripts for MRP5. Downregulation of MRP5 expression in GH3 cells diminished cGMP release without affecting cAMP efflux. The results suggest that, in pituitary cells, cyclic nucleotide cellular efflux plays a critical role in eliminating intracellular cGMP but not cAMP, with such selectivity achieved by the expression of MRP5.
Andric SA, Kostic TS, Stojilkovic SS. Contribution of multidrug resistance protein MRP5 in control of cyclic guanosine 5¢-monophosphate intracellular signaling in anterior pituitary cells. Endocrinology 2006;147:3435-45.
Gonzales-Iglesias AE, Jiang Y, Tomić M, Kretschmannova K, Andric SA, Zemkova H, Stojilkovic SS. Dependence of electrical activity and calcium influx-controlled prolactin release on adenylyl cyclase signaling pathway in pituitary lactotrophs. Mol Endocrinol 2006;20:2231-46.
Jiang Y, Stojilkovic SS. Molecular clonic and characterization of alpha1-soluble guanylyl cyclase gene promoter in rat pituitary cells. J Mol Endocrinol 2006;37:503-15.
Stojilkovic SS. Cyclic nucleotides and their regulation. In: Izzo JL, ed. The Hypertension Primer. 2007, in press.
Pituitary purinergic receptor channels
P2X receptors are a family of ligand-gated cation channels composed of two transmembrane domains, with N- and C-termini located intracellularly and a large extracellular loop containing the ATP-binding domain. We mutated the conserved Lys313, Tyr315, Gly316, Ile317, Arg318, Asp320, Val323, Lys329, Phe330, and Ile333 residues in order to study the role of the Lys313-to-Ile333 ectodomain sequence of the rat P2X4 receptor in both ATP binding and transduction of signals to the channel gate. We made current recordings on lifted cells and applied ATP by using an ultrafast solution-switching system. The rates of wild-type channel opening and closing in the presence of ATP, but not the rate of wash-out–induced closing, depended on agonist concentration. All mutants other than I317A were expressed in the plasma membrane at comparable levels. The majority of mutants showed significant changes in the peak amplitude of responses and the EC50 values for ATP. When stimulated with the supramaximal ATP concentration, mutants also differed in the kinetics of their activation, deactivation, and/or desensitization. The results suggest a critical role of the Lys313 residue in receptor function beyond coordinating the phosphate group of ATP and even a possible contribution of the Tyr315 residue to the agonist-binding module. The pattern of changes of receptor function by mutation of other residues was consistent with the operation of the Gly316-to-Ile333 sequence as a signal transduction module between the ligand-binding domain and the channel gate in the second transmembrane domain.
The P2X4 receptor is sensitive to ivermectin (IVM), a high molecular weight lipophilic compound used as an antiparasitic agent in human and veterinary medicine. Extracellularly applied IVM increases current amplitude in response to supramaximal agonist concentration as well as sensitivity to agonists. IVM also reduces the desensitization rate and greatly prolongs the deactivation of current after ATP removal. Thus, a leftward shift in the sensitivity of wild-type receptor for ATP in the presence of IVM could provide a valuable pharmacological tool for evaluating the response of P2X4 receptor mutants to increased concentrations of ATP. We therefore studied the current responses of previously generated K190A, K190R, F230A, F230W, R278A, R278K, D280A, D280E, K313A, and K313R-P2X4R mutants in the presence and absence of IVM. We also generated novel K67A, K67R, F185A, F185W, F294A, F294W, R295A, and R295K mutants of the P2X4 receptor because of the mutants’ potential importance for ATP binding. We performed all experiments with enhanced green fluorescent protein–tagged receptors in order to identify cells expressing receptors for electrophysiological recordings and to visualize the subcellular distribution of receptors by confocal microscopy. In the presence of IVM, all low or non-responsive mutants responded to ATP in a dose-dependent manner, with the EC50 values for ATP of about 1, 2, 4, 20, 60, 125, 270, 420, 1,000 and 2,300 µM at D280A, R278A, F185A, K190A, R295K, K313R, R295A, K313A, K67A, and K67R mutants, respectively. The results indicate that lysines 67 and 313 and arginine 295 play a critical role in forming the proper three-dimensional structure of the P2X4 receptor for agonist binding and/or channel gating.
To characterize the IVM binding site of the P2X4 receptor, we generated several chimeric and single-point mutants. Experiments with chimeric receptors revealed that the Val49-to-Val61 but not the Val64-to-Tyr315 ectodomain sequence is important for the effects of IVM on channel deactivation. Receptor-specific mutations placed in the Gly29-to-Val61 and Asp338-to-Leu358 regions showed the importance of Trp50, Val60, and Val357 residues in IVM regulation of the rate of channel deactivation, but not in maximum current amplitude. The results suggest that the transmembrane domains and nearby ectodomain region contribute to the effects of IVM on channel deactivation. Our ongoing experiments focus on identifying residues in the transmembrane domain responsible for IVM binding.
Jelinkova I, Yan Z, Liang Z, Moonat S, Teisinger J, Stojilkovic SS, Zemkova H. Identification of P2X4 receptor-specific residues contributing to the ivermectin effects on channel deactivation. Biochem Biophys Res Commun 2006;349:619-25.
Koshimizu T, Kretschmannova K, He M-L, Ueno S, Tanoue A, Yanagihara N, Stojilkovic SS, Tsujimoto G. Carboxyl-terminal splicing enhances physical interactions between the cytoplasmic tails of purinergic P2X receptors. Mol Pharmacol 2006;69:1588-98.
Yan Z, Liang Z, Obsil T, Stojilkovic SS. Participation of the Lys313-Ile333 sequence of the purinergic P2X4 receptor in agonist binding and transduction of signals to the channel gate. J Biol Chem 2006;282:32649-59.
Zemkova H, Yan Z, Liang Z, Jelinkova I, Tomic M, Stojilkovic SS. Role of aromatic and charged ectodomain residues in the P2X4 receptor function. J Neurochem 2007;102:1139-50.
1 Czech Academy of Sciences, Prague, Czech Republic
2 Arturo E. Gonzalez-Iglesias, PhD, former Postdoctoral Fellow
3 Noriyuki Hatae, PhD, former Postdoctoral Fellow
4 Yonghua Jiang, PhD, former Postdoctoral Fellow
5 Silvana A. Andric, PhD, former Postdoctoral Fellow
6 Tatjana S. Kostic, PhD, former Postdoctoral Fellow
COLLABORATOR
Taka-aki Koshimizu, MD, PhD, Kyoto University, Kyoto, Japan
Tomas Obsil, PhD, Charles University, Prague, Czech Republic
Olaf Ortmann, MD, PhD, Universität Regensburg, Regensburg, Germany
Arthur Sherman, PhD, Laboratory of Biological Modeling, NIDDK, Bethesda, MD
For further information, contactstojilks@mail.nih.gov.
