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Signaling and Secretion in Neuroendocrine Cells

Stanko S. Stojilkovic, PhD, Head, Section on Cellular Signaling
Silvana A. Andric, PhD, Special Volunteer
Claudio E. Coddou Alvarez, PhD, Visiting Fellow
Anne-Marie Heegaard, MD, PhD, Special Volunteer
Marek Kucka, PhD, Visiting Fellow
Shuo Li, PhD, Visiting Fellow
Melanija Tomic, PhD, Staff Scientist
Zonghe Yan, MD, PhD, Research Fellow

The Section investigates cellular signaling cascades and secretion in neuroendocrine cells, with special emphasis on the interactions between plasma membrane electrical events and receptor-controlled pathways. Using multidisciplinary and collaborative approaches, the Section investigates signaling pathways at the cellular and molecular levels, determines the manner in which hormones and neurotransmitters utilize calcium as an intracellular messenger, and characterizes channels involved in electrical activity and calcium signaling in hypothalamic, pituitary and other cell types. Currently, we are addressing the issues of how the structural features of pituitary channels relate to their functions and how plasma membrane receptors and the intracellular signaling milieu affect channel activity. For this purpose, we characterize both native and recombinant channels and receptors that were cloned from the pituitary gland. We also analyze the relevance of channels and pathways in calcium-regulated hormone secretion.

Expression and structural and functional characterization of pituitary P2X receptors

We have cloned five ATP-gated P2X receptor channels from the pituitary gland: P2X2R, P2X3R, P2X4, P2X6, and P2X7. Our ongoing work, done in collaboration with Hana Zemkova, has focused on their structural-functional characterization and identification of receptor subtypes expressed in individual cell types, with particular emphasis on two transmembrane domains of these receptors, using rat P2X4 as a receptor model. Experiments with cysteine- and alanine-scanning mutagenesis of transmembrane domains suggested a helical topology, with every third or fourth amino acid affected by substitution (Jelinkova et al., 2008; Jindrichova et al., 2009). The recently published crystal structure of the zebrafish P2X4 receptor confirmed the helical organization of transmembrane domains. We also studied the functional relevance of aromatic residues in the upper part of the transmembrane domain-1 of P2X receptors. Replacement of the conserved Tyr residue with Ala had a receptor-specific effect: the P2X1 receptor was nonfunctional, the P2X2, P2X4, and P2X3 receptors exhibited enhanced sensitivity to ATP and αβ-meATP accompanied by prolonged decay of current after washout of agonists, and the P2X7 receptor’s sensitivity for agonists was not affected, though decay of current was delayed. Replacement of the P2X4 receptor-Tyr42 with other amino acids revealed the relevance of an aromatic residue at this position. Mutation of the neighboring Phe and ipsilateral Tyr/Trp residues, but not the contralateral Phe residue, also affected the P2X2, P2X3, and P2X4 receptor function. Double mutation of ipsilateral Tyr-42 and Trp-46 P2X4 receptor residues restored receptor function, whereas the corresponding P2X2 receptor double mutant was not functional. In contrast, mutation of the contralateral Phe-48 residue in the P2X4-Y42A mutant had no effect (Jindrichova et al., 2009). These results indicate that aromatic residues in the TM1 play important roles in the three-dimensional structure of the P2X receptors in open state and that they are required not only for ion conductivity but also for specificity of agonist binding and/or channel gating.

Quantitative RT-PCR analysis revealed that mRNA transcripts for the P2X4 subunit were most abundant in rat anterior pituitary tissue, and western blot analysis confirmed its expression at the protein level. Single-cell patch clamp recordings showed that extracellular ATP induced an inward current in a majority of TRH-responsive pituitary cells that resembled the current generated by recombinant P2X4 receptors. Under current-clamped conditions, activation of these channels led to initiation of firing of action potentials in quiescent cells and increase in the firing frequency in spontaneously active cells. Channels activated and desensitized in a dose-dependent manner and deactivated rapidly. In the presence of ivermectin, a specific allosteric modulator of P2X4 receptors, we observed an approximate seven-fold increase in the maximum amplitude of ATP-induced inward current, accompanied by an increase in the sensitivity of receptors for ATP, delayed deactivation of receptors, and enhanced the ATP-induced prolactin release. These results indicate that TRH–responsive cells, including lactotrophs, express homomeric and/or heteromeric P2X4 receptors, which could operate as pacemaking channels.

Gating properties of P2X7 receptor channels

We and others have observed that activation of the P2X7 receptor induced not only the rapid opening of an integral ion channel that was permeable to small cations, but also a gradual increase in permeability to fluorescent dyes. Such bi-functional permeation properties of this receptor could reflect a dilation of integral pore of the channels or integration of another permeation pathway by activated channels. Recently, we provided several lines of evidence indicating that the P2X7 receptor pore dilates. First, during prolonged agonist application, a rapid current that peaked within 200 ms was accompanied by a slower current that required tens of seconds to reach its peak. Second, the secondary rise in current was observed under different ionic conditions and temporally coincided with the development of conductivity to larger organic cations. Third, the biphasic response was also observed in cells with blocked pannexin channels and in cells not expressing these channels endogenously. Fourth, the biphasic current was preserved in N-terminal T15A, T15S, and T15V mutants that have low or no permeability to organic cations, reflecting enhanced permeability to inorganic cations. In contrast, the T15E, T15K, and T15W mutants, and a mutant with deleted P2X7 receptor–specific 18-amino acid C-terminal segment, were instantaneously permeable to organic cations and generated high amplitude monophasic currents (Yan et al., 2008). Together, these results indicate that the P2X7 receptor channel dilates under physiological ion conditions, leading to generation of biphasic current, and that this process is controlled by residues near the intracellular side of the channel pore. Our ongoing work on this topic concerns the relevance of sensitization of P2X7 receptors induced by repetitive and prolonged agonist application to the kinetics of pore dilation.

Our experiments also revealed that the kinetics of P2X7 receptor pore dilation differs from the kinetics of fluorescent dye uptake and release, suggesting that pore dilation could not account for the transport of fluorescent dyes across the plasma membrane. We are currently working on a hypothesis that association of pannexin hemichannels with P2X receptors is responsible for the fluorescent dye uptake. Initial studies showed that the full-size pannexin1 channel and several novel splice forms, as well as the full-size pannexin2 channel, are expressed in hypothalamic and pituitary cells. We are also investigated the interactions of the full-size pannexin 1 with its two splice forms, their interactions with pannexin2 channels and P2X receptors that are expressed in rat pituitary, as well as the relevance of phosphorylation and glycosylation modification to the trafficking and oligomerization of pannexin1 channels.

Allosteric regulation of P2X receptor channels

The heavy metal mercury is associated with cell damage; this metal has high affinity for sulfhydryl groups, thereby inactivating enzymes, cysteine residues, and sulfur-containing antioxidants. Excess mercury suppresses the antioxidant defense leading to a subsequent increase in oxidative stress; thus mercury may act as an oxidative stress inducer. In the brain, mercury also causes behavioral and/or cognitive disturbances. This could reflect its action as a stress-inducer, but also as a direct modulator of ion channels, as indicated by its ability to interact with voltage- and ligand-gated ionic channels. Mercury also affects the function of P2X receptors. It potentiates the ATP-evoked currents in cells expressing P2X2 receptor and attenuates agonist-induced current in P2X4 and P2X7 receptor–expressing cells. Because modulation of P2X receptor activity induced by mercury shares the same pattern as copper, it was reasonable to hypothesize that both metals bind to a common allosteric site. However, the mercury modulation is not related to the extracellular residues critical for copper modulation.

Our collaborative work with Pablo Huidobro-Toro focused on identification of the ectodomain site(s) for mercury action at P2X receptors. We generated two chimeras using the full-size P2X2 subunit termed P2X2a and a splice variant lacking a 69-residue segment in the C-terminal, termed P2X2b, as donors for intracellular and transmembrane segments and the P2X4 subunit as the donor for ectodomain segment of chimeras. The potentiating effect of mercury on ATP-induced current was preserved in Xenopus oocytes expressing P2X4/2a chimera, but was absent from oocytes expressing P2X4/2b chimera. Site-directed mutagenesis experiments revealed that the Cys-430 residue mediates the effects of mercury on the P2X2a receptor activity. Because mercury could act as an oxidative stress inducer, we also tested whether hydrogen peroxide and the mitochondrial stress inducers myxothiazol and rotenone mimicked mercury effects. These agents potentiated the ATP-evoked P2X2a receptor and P2X4/2a receptor currents, but not P2X2b receptor and P2X2a-C430A and P2X2a-C430S mutant currents, whereas the antioxidants dithiothreitrol and N-acetylcysteine prevented the hydrogen peroxide potentiation. Alkylation of Cys-430 residue with methylmethane-thiosulfonate also abolished the mercury and hydrogen peroxide potentiation. These results, which recently appeared (Coddou et al., J Neurosci 2009;29:12284), are consistent with the hypothesis that the Cys-430 residue is an intracellular P2X2a receptor redox sensor. The present findings will allow the identification of other members of the P2X receptor family that may also have a redox sensor, unraveling the implications of intracellular cysteine residues on receptor physiology. Taken together, these findings highlight the fact that P2X receptors are not only sensors of extracellular ATP and allosteric modulators but may also respond to intracellular stimuli that vary depending on the metabolic state of the cell.

Collaborative work on other topics

In collaboration with the Section on Molecular Signal Transduction of NICHD headed by Tamás Balla, our group has also contributed to the characterization of calcium entry mediated by Orai1 plasma membrane channels. This collaborative project focused on the dependence of current generated by activated Orai1 channels, termed I_CRAC, on phosphoinositides in STIM1/Orai1-expressing cells (Korzeniowski et al, 2009). We showed that cells expressing STIM1 and Orai1 responded with the generation of I_CRAC current when depletion of the endoplasmic reticulum was achieved by injecting InsP3 through the recording pipette. Treatment of cells with Wortmannin, a PI4 kinase inhibitor, induced significant reduction in the peak amplitude of current. We also showed that activation of phospholipase C signaling pathway by angiotensin II receptors caused rapid but incomplete inhibition of I_CRAC current. These results indicate that PtdIns4P rather than PtdIns(4,5)P2 is a likely determinant of Orai channel activity.

Our laboratory has also contributed to the work on control of calcium signaling in GnRH-secreting hypothalamic neurons, work that is led by Susan Wray, head of Cellular and Developmental Neurobiology Section, NINDS. This collaboration focused on the role of kisspeptins, the natural ligands of the G protein–coupled receptor GPR-54 in calcium signaling. GnRH neurons maintained in mouse nasal explants exhibited spontaneous baseline oscillations in intracellular calcium concentration, which were critically dependent on the operation of voltage-gated, tetrodotoxin- (TTX) sensitive sodium channels and were not coupled to calcium release from intracellular pools. Activation of native GPR54 by kisspeptin-10 initiated calcium oscillations in quiescent GnRH cells, increased the frequency of calcium spiking in oscillating cells that led to summation of individual spikes into plateau-bursting type of calcium signals in a subset of active cells. These changes predominantly reflected the stimulatory effect of GPR54 activation on the plasma membrane oscillator activity by coupling this receptor to phospholipase C signaling pathways. Both components of this pathway, InsP3 and protein kinase C, contributed to the receptor-mediated modulation of baseline calcium oscillations. TTX and 2-aminoethyl diphenylborinate together abolished agonist-induced elevation in calcium in almost all cells, whereas flufenamic acid was less effective (Constantin et al., 2009). Together these results indicate that a plasma membrane calcium oscillator is spontaneously operative in the majority of prenatal GnRH neurons and is facilitated by kisspeptin-10 through phosphatidyl inositol diphosphate hydrolysis and depolarization of neurons by activating TTX-sensitive sodium channels and nonselective cationic channels.

Additional Funding

International fellowship award to Dr. Silvana Andric by the Serbian Ministry for Science, Belgrade (451-03-00275) – ongoing.
Sabbatical fellowship to Dr. Anne-Marie Heegaard by the Danish Council for Independent Research - Medical Sciences and the Faculty of Pharmaceutical Sciences, University of Copenhagen (2009) – ongoing.

Publications

Yan Z, LI S, Liang Z, Tomic M, Stojilkovic SS. The P2X7 receptor channel pore dilates under physiological ion conditions. J Gen Physiol 2008 132:563-573.
Jindrichova M, Varva V, Obsil T, Stojilkovic SS, Zemkova H. Functional relevance of aromatic residues in the first transmembrane domain of P2X receptors. J Neurochem 2009 109:923-934.
Korzeniowski MK, Popovic MA, Szentpetery Z, Varnai P, Stojilkovic SS, Balla T. Dependence of STIM1/Orai-mediated calcium entry on plasma membrane phosphoinositides. J Biol Chem 2009 284:21027-21035.
Constantin S, Caligioni CS, Stojilkovic S, Wray S. Kisspeptin-10 facilitates a plasma membrane-driven calcium oscillator in gonadotropin-releasing hormone-1 neurons. Endocrinology 2009 150:1400-1412.
Jelinkova I, Varva 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. Eur J Physiol 2008 456:939-950.

Collaborators

Tamás Balla, MD, PhD, Program in Developmental Neuroscience, NICHD, Bethesda, MD
Susan Wray, PhD, Cellular and Developmental Neurobiology Section, NINDS, NIH, Bethesda, MD
Norman J. Haughey, MD, PhD, The Johns Hopkins University School of Medicine, Baltimore, MD
J. Pablo Huidobro-Toro, PhD, Catholic University, Santiago, Chile
Hana Zemkova, PhD, Institute of Physiology of the Academy of Science of the Czech Republic, Prague, Czech Republic

Contact

For more information, email stankos@helix.nih.gov or visit http://neuroscience.nih.gov/Lab.asp?Org_ID=362.

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