Signaling and Secretion in Neuroendocrine Cells

Stanko S. Stojilkovic, PhD, Head, Section on Cellular Signaling
Melanija Tomić, PhD, Staff Scientist
Zonghe Yan, MD, PhD, Research Fellow
Karla Kretschmannova, PhD, Visiting Fellow
Marek Kucka, PhD, Visiting Fellow
Shuo Li, PhD, Visiting Fellow
Takayo Murano, MD, PhD, Visiting Fellow

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 spontaneous calcium transients while 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 role of G protein–coupled receptors in the control of electrical activity, and the molecular properties of calcium-conducting purinergic receptor channels in pituitary functions.

Cyclic nucleotides and electrical signaling pathways

Andric,¹ Kretschmannova, Kucka, Murano, Tomić, Stojilkovic; in collaboration with Sherman, Schechter

Numerous plasma membrane channels have been characterized in pituitary cells, but the mechanism underlying their pace-making activity remains unknown. We observed that, in all pituitary cell types, the removal of sodium reversibly hyperpolarized membrane potential and suppressed calcium oscillations, followed by a reduction in the intracellular calcium concentration to near steady-state levels. In contrast, blockade by tetrodotoxin of voltage-dependent sodium channels, which are expressed in all secretory cell types, was ineffective. We also found that inhibition of electrical activity by removal of bath sodium was accompanied by an instantaneous blockade of cyclic nucleotide efflux transporter, suggesting a coupling between electrical activity and cyclic nucleotide efflux. In addition, our experiments indicated that (1) a sodium-conducting and tetrodotoxin-insensitive channel plays a role in controlling pace-making stimulated by growth hormone–releasing hormone in a cAMP-dependent manner and (2) spontaneously active inwardly rectifying potassium (K_ir) channels and G protein–regulated K_ir channels play an opposing role in somatostatin-mediated inhibition of pace-making. We further described a role for voltage-gated calcium channels in spiking and for large-conductance (BK-type) calcium-activated potassium channels in plateau bursting.

In collaboration with Arthur Sherman and colleagues, we combined experiments and theory and used pituitary somatotrophs as a cell model to clarify the mechanisms underlying spontaneous and receptor-controlled electrical activity. Our mathematical model is compatible with a wide variety of experimental data involving pharmacology and extracellular ion substitution and points to the importance of constitutively active tetrodotoxin-insensitive sodium and K_ir channels in maintaining spontaneous pace-making in pituitary somatotrophs. The model also suggests that these channels are involved in the up- and downregulation of electrical activity by growth hormone–releasing hormone and somatostatin. In the model, two functional populations of BK channels, characterized by the distance from the voltage-gated calcium channels, control plateau bursting. 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. Our ongoing work focuses on identifying the channels responsible for the background sodium conductance and the relationship between spontaneous and receptor-controlled electrical activity and cyclic nucleotide efflux.

In collaboration with Alan Schechter and colleagues, we studied the effects of hydroxyurea on cyclic nucleotide signaling in human erythroid progenitor cells. Hydroxyurea, a drug widely used to treat myeloproliferative diseases, has been approved for the treatment of sickle cell disease because it raises fetal hemoglobin. Our earlier findings indicated that nitric oxide and the soluble guanylyl cyclase pathways are involved in hydroxyurea induction of fetal hemoglobin levels in erythroid progenitor cells. More recently, we showed that, during erythroid differentiation, endothelial nitric oxide synthase mRNA and protein levels decline steadily, as do the production of nitric oxide derivatives and cAMP levels, although cGMP levels remain stable. Hydroxyurea raised intracellular cGMP and cAMP levels in erythroid progenitor cells. The nitric oxide donor DEANONOate induced even higher cGMP levels but reduced cAMP levels. Hydroxyurea induced production of approximately 45 pM cGMP per minute per ng of purified soluble guanylyl cyclase, similar to induction by 1 µM DEANONOate. We found that hydroxyurea and ProliNONOate produced iron-nitrosyl derivatives of soluble guanylyl cyclase. Thus, we confirmed that hydroxyurea can directly interact with the deoxy-heme of soluble guanylyl cyclase, presumably by a free-radical nitroxide pathway, and activate cGMP production. These data add to an expanding appreciation of the role of hydroxyurea as an inducer of the nitric oxide/cGMP pathway in erythroid progenitor cells. Similar mechanisms may also be involved in the cytostatic effects of hydroxyurea as well as in the induction of fetal hemoglobin.

Cokic V, Andric SA, Stojilkovic SS, Noguchi CT, Schechter AN. Hydroxyurea nitrosylates and activates soluble guanylyl cyclase in human erythroid cells. Blood 2008;111:1117-1123.
Stojilkovic SS. Cyclic nucleotides and their regulation. In: Izzo JL, Sica DA, Black HR, eds. The Hypertension Primer. Lippincott Williams & Wilkins, 2008;8-11.
Stojilkovic SS. Ion channels, transporters and electrical signaling. In: Conn PM, ed. Neuroscience in Medicine. Humana Press, 2008;53-89.
Tsaneva-Atanasova 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.

Receptors and pathways controlling prolactin secretion

Gonzalez-Iglesias,² Hatae,³ Kretschmannova, Kucka, Murano, Tomić, Stojilkovic; in collaboration with Ortmann, Zorec

Our recent work has focused on the secretion of prolactin by pituitary lactotrophs and the role of dopamine D2 and endothelin-A receptors, testosterone, and hypotonicity in the secretion process. Dopamine secreted from hypophysial hypothalamic neurons is a principal inhibitory regulator of prolactin (PRL) release caused by pituitary lactotrophs’ activation of dopamine-2 receptors. Earlier work with dopamine D2 receptors revealed that the receptors’ activation in pituitary lactotrophs leads to inhibition of PRL release. Other researchers have suggested that such inhibition occurs through the G_i/o-alpha protein–mediated inhibition of cAMP production and/or G_i/o-beta/gamma dimer–mediated activation of K_ir channels and inhibition of voltage-gated calcium channels. We showed that the dopamine agonist–induced inhibition of spontaneous calcium influx and release of prestored PRL was preserved when we elevated cAMP levels by forskolin treatment. We further observed that dopamine agonists inhibited both spontaneous and depolarization-induced calcium influx in untreated, but not in pertussis toxin–treated, cells. We observed a similar inhibition in cells with blocked K_ir channels, suggesting that the dopamine effect on voltage-gated calcium channel gating is sufficient to inhibit spontaneous calcium influx. 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 the addition of wortmannin, an inhibitor of phosphatidylinositol 3-kinase, and lithium, an inhibitor of glycogen synthase kinase-3, but it was attenuated in the presence of phorbol 12-myristate 13-acetate, which inhibits Gz signaling pathway in a protein kinase C–dependent manner. Our results indicate for the first time that dopamine inhibits basal PRL release by (1) blocking voltage-gated calcium influx through the pertussis toxin–sensitive signaling pathway and (2) desensitizing calcium-secretion coupling through the pertussis toxin–insensitive and protein kinase C–sensitive signaling pathway.

In collaboration with Robert Zorec and colleagues, we studied the release by hypotonicity of the pituitary hormone PRL, which also contributes to osmoregulation. In perifused rat lactotrophs, hypotonicity resulted in a transient increase followed by a sustained depression of PRL release, as monitored by radioimmunoassay. In single cells imaged by confocal microscopy, hypotonicity elicited discharge of the fluorescently labeled atrial natriuretic peptide cargo from about 2 percent of vesicles per cell, which synchronously loaded the styryl dye FM 4-64 through secretory fusion pores. In contrast, high potassium–induced depolarization resulted in a response by about 10 percent of vesicles per cell, with a different unloading/loading time course for the two fluorescent probes. In cell-attached studies, we recorded discrete changes in membrane capacitance in both unstimulated and stimulated conditions, reflecting single vesicle fusions/fissions with the plasma membrane. In stimulated cells, the probability of full fusion events was low and unchanged, whereas over 95 percent of fusion events were transient, with increased open fusion–pore probability, average pore-dwell time, frequency of occurrence, and fusion-pore conductance. Hypotonicity only rarely elicited new fusion events in silent membrane patches. The results indicate that, in hypotonicity-stimulated lactotrophs, rapidly releasable vesicles appear prefused and release hormone in a “kiss-and-run” mode. Ongoing experiments focus on comparing single-cell exocytotic events in lactotrophs and somatotrophs.

Gonzalez-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 2008;149:1470-1479.
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.
Jorgacevski J, Stenovec M, Kreft M, Bajic A, Rituper B, Vardjan N, Stojilkovic SS, Zorec R. Hypotonicity and peptide discharge from a single vesicle. Am J Physiol Cell Physiol 2008;295:C624-31.
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;103:149-157.

Molecular, pharmacological, and functional properties of GABA-A receptors in anterior pituitary cells

Weber-Zemkova,⁴ Tomić, Stojilkovic; in collaboration with Zemkova

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. It acts through three structurally and pharmacologically distinct classes of receptors: GABA_A and GABA_C ligand–gated Cl⁻ channels and G protein–coupled GABA_B receptors. Earlier studies suggested that GABA also inhibits prolactin release in vitro, but the receptors involved in such release have not been characterized. During the past year, we studied the expression of GABA-A receptor channels in pituitary cells, their distribution within secretory anterior pituitary cell types, and the nature (stimulatory or inhibitory) of the channels’ actions. Our results show that mRNAs for all GABA_A receptor subunits are expressed in pituitary cells and that alpha1/beta1 subunit proteins are present in all secretory cells. In voltage-clamped gramicidin-perforated cells, GABA induced dose-dependent increases in current amplitude that were inhibited by bicuculline and picrotoxin and facilitated by diazepam and zolpidem in a concentration-dependent manner. In intact cells, GABA and the GABA-A receptor agonist muscimol caused a rapid and transient increase in intracellular calcium, whereas the GABA-B receptor agonist baclofen was ineffective, suggesting that chloride-mediated depolarization activates voltage-gated calcium channels. Consistent with this finding, RT-PCR analysis indicated a high level of NKCC1 expression in pituitary cells but not of KCC2 cation/chloride transporter mRNAs. Furthermore, the GABA-A channel reversal potential for chloride ions was positive to the baseline membrane potential in most cells, and the activation of ion channels by GABA resulted in depolarization of cells and modulation of spontaneous electrical activity. Thus, secretory pituitary cells express functional, depolarizing GABA-A receptor channels. These results raise the possibility that GABA might be a releasing factor in the pituitary, but the physiological relevance of its excitatory effects are yet to be fully understood (see Perspectives: J Physiol 2008;586:3023).

Zemkova HW, Bjelobaba I, Tomić M, Zemkova H, Stojilkovic SS. Molecular, pharmacological, and functional properties of GABA-A receptors in anterior pituitary cells. J Physiol 2008;586:3097-3111.

Structural and functional characterization of purinergic receptor channels

Yan, Li, Tomić, Stojilkovic; in collaboration with Zemkova

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. The P2X4 receptor, but not other members of this family of receptors, is sensitive to ivermectin (IVM), a high–molecular weight lipophilic compound used as an antiparasitic agent in human and veterinary medicine. Our initial whole-cell current recordings showed that IVM raises the sensitivity of the P2X4 receptor to agonists, 2-fold enhances the current amplitude in response to supramaximal agonist concentrations, and greatly prolongs the deactivation of current. An increase in the sensitivity of the 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-P2X₄ mutants in the presence and absence of IVM. We also generated novel K67A, K67R, F185A, F185W, F294A, F294W, R295A, and R295K mutations of the P2X4 receptor because of the potential importance of these residues for ATP binding. To identify cells expressing receptors for electrophysiological recordings and to visualize the subcellular distribution of receptors by confocal microscopy, we used receptors tagged with enhanced green fluorescent protein at their C-termini. In the presence of IVM, all low or non-responsive mutants responded to ATP in a dose-dependent manner, with 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.

In further work on the structural-functional characterization of recombinant P2X4 receptor, we focused on the identification of residues contributing to allosteric regulation of the channels by IVM. Initial experiments demonstrated that IVM affects P2X4 receptor function when applied extracellularly but not intracellularly, suggesting that IVM could bind at the ectodomain. However, we further showed that transfer of the P2X4 ectodomain sequence to the backbone of the P2X2 receptor did not transfer the sensitivity for IVM. These initial observations led us to examine the potential relevance of transmembrane helices for IVM binding. To that end, we used cysteine-scanning mutagenesis of rat P2X₄ transmembrane regions. Mutations in 29 residues did not change the receptor function; among the residues, IVM effects were altered in Gln36, Leu40, Val43, Val47, Trp50, Asn338, Gly342, Leu346, Ala349, and Ile356 mutants. In addition, the substitution-sensitive Arg33 and Cys353 mutants could be considered IVM-sensitive hits. The pattern of these 12 residues was consistent with the helical topology of both transmembrane regions, with every third or fourth amino acid affected by substitution. These predominantly hydrophobic, non-polar residues are also present in the IVM-sensitive Schistosoma mansoni P2X subunit. The residues lie on the same side of their helices and could conceivably face lipids in the open conformation state and provide the binding pocket for IVM. In contrast, the IVM-independent hits Met31, Tyr42, Gly45, Val49, Gly340, Leu343, Ala344, Gly347, Thr350, Asp354, and Val357 map on the opposite side of their helices, probably facing the pore of the receptor or protein and playing important roles in gating. Once the receptor architecture becomes available from crystal studies, we will know the precise topology of these functionally important residues.

Jelínkova I, Vávra V, Jindrichova M, Obsil T, Zemkova HW, Zemkova H, Stojilkovic SS. Identification of P2X4 receptor transmembrane residues contributing to channel gating and interaction with ivermectin. Pflügers Arch Eur J Physiol 2008;456:939-950.
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;102:1139-1150.

¹ Silvana A. Andric, PhD, former Visiting Fellow
² Arturo E. Gonzalez-Iglesias, PhD, former Visiting Fellow
³ Noriyuki Hatae, PhD, former Visiting Fellow
⁴ Hana Weber-Zemkova, PhD, former Visiting Fellow

Collaborators

Olaf Ortmann, MD, PhD, University of Regensburg, Regensburg, Germany
Alan N. Schechter, MD, PhD, Molecular Medicine Branch, NIDDK, Bethesda, MD
Arthur Sherman, PhD, Laboratory of Biological Modeling, NIDDK, Bethesda, MD
Hana Zemkova, PhD, Institute of Physiology of the Academy of Science of the Czech Republic, Prague, Czech Republic
Robert Zorec, MD, PhD, Faculty of Medicine, Ljubljana, Slovenia

For further information, contact stojilks@mail.nih.gov.