Stanko S. Stojilkovic, PhD, Head, Section on Cellular Signaling
Melanija Tomić, PhD, Staff Scientist
Arturo E. Gonzalez-Iglesias, PhD, Postdoctoral Fellow
Noriyuki Hatae, PhD, Postdoctoral Fellow
Yonghua Jiang, PhD, Postdoctoral Fellow
Karla Kretschmannová, PhD, Postdoctoral Fellow
Zonghe Yan, MD, PhD, Postdoctoral Fellow
Zhaodong Liang, MD, Contractor

We investigate cellular signaling cascades and secretion in neuroendocrine cells, with special emphasis on the interactions between plasma-membrane electrical events and receptor-controlled pathways. The majority of neuroendocrine 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, and the expression pattern and molecular properties of calcium-conducting P2X receptor channels.

Role of cyclic nucleotides in control of electrical activity, calcium signaling, and secretion

Kretschmannová, Gonzalez-Iglesias, Tomić

Normal and immortalized pituitary cells generate action potentials spontaneously. In pituitary lactotrophs, such electrical activity and the associated voltage-gated calcium influx (VGCI) are sufficient to maintain high and steady hormone release in vitro. We and others have characterized numerous plasma membrane channels, but the mechanism underlying their pacemaking activity is still not known. Our current efforts focus on the role of cyclic nucleotides in the control of pacemaking in pituitary lactotrophs. In anterior pituitary cells, both VGCI-inhibitable and -insensitive adenylyl cyclase (AC) subtypes contributed to basal cAMP production, and soluble guanylyl cyclase (sGC) was exclusively responsible for basal cGMP production. Inhibition of basal AC activity, but not of sGC activity, reduced prolactin release. In contrast, forskolin stimulated cAMP and cGMP production as well as pacemaking, VGCI, and prolactin secretion. Elevation of cAMP and cGMP levels by inhibition of phosphodiesterase activity was also accompanied by increased prolactin release. The AC inhibitors attenuated forskolin-stimulated cyclic nucleotide production, VGCI, and prolactin release. The cell-permeable 8-Br-cAMP stimulated firing of action potentials and prolactin release and rescued hormone secretion in cells with inhibited ACs in an extracellular calcium-dependent manner, whereas 8-Br-cGMP and 8-pCPT-2Me-cAMP were ineffective. Protein kinase A inhibitors did not change spontaneous and forskolin-stimulated pacemaking, VGCI, and prolactin release. These results indicate that cAMP facilitates pacemaking, VGCI, and prolactin release in lactotrophs, predominantly in a protein kinase A- and Epac cAMP receptor-independent manner.

Hyperpolarization-activated channels (HCNs) play a distinct role in the control of membrane excitability in spontaneously active cardiac and neuronal cells. HCNs are members of cyclic nucleotide-gated channels and may thus mediate the action of cAMP on pacemaking activity in pituitary cells. Consistent with this hypothesis, RT-PCR analysis revealed the presence of mRNA transcripts for HCN2, HCN3, and HCN4 subunits in these cells. Hyperpolarization of the membrane potential below -60 mV elicited a slowly activating voltage-dependent inward current (Ih) in the majority of tested cells. Bath application of 1 mM cesium, a commonly used inorganic blocker of Ih, and 100 µM ZD7288, a specific organic blocker of Ih, inhibited the current. Receptor- and non-receptor-mediated activation of AC and sGC and the addition of 8-Br-cAMP, a membrane-permeable cAMP analogue, did not affect Ih. Inhibition of basal AC activity, but not of basal sGC activity, caused reduction of the peak amplitude and a leftward shift in the activation curve of Ih. Such inhibition of current was reversed by stimulation of AC with forskolin and the addition of 8-Br-cAMP, but not by 8-Br-cGMP. Application of Cs+ had no significant effect on the resting membrane potential or electrical activity, whereas ZD7288 exhibited complex and Ih-independent effects on spontaneous electrical activity, calcium signaling, and prolactin release. These results indicate that HCN channels in pituitary cells are under tonic activation by the basal level of cAMP and are not critical for spontaneous firing of action potentials. The results further indicate that ZD7288 attenuates the exocytotic pathway downstream of calcium influx.

Characterization of cyclic nucleotide signaling pathways in pituitary cells

Jiang, Andric, ¹ Kostic ²

Our earlier studies revealed that sGC represents the major pathway for synthesis of cGMP in pituitary cells. The pituitary enzyme is composed of alpha-1 and beta-1 subunits, and dimer activity is regulated directly at the protein level. Physiologically, the most relevant mode of such regulation is mediated by binding of nitric oxide (NO) to the heme group of sGC. The enzyme is also subjected to phosphorylation by protein kinase A, src-like kinases, and protein kinases C and G. However, our knowledge of the promoters of sGC genes and regulation of the enzyme activity at transcriptional and post-transcriptional levels is deficient. We recently analyzed the promoter region of the rat alpha1-sGC gene. Using pituitary tissue, we identified the transcriptional start site and cloned a 3.5 kb upstream promoter of the alpha-1sGC subunit. Sequence analysis of this TATA-less promoter revealed the presence of several putative binding sites for transcriptional factors, including a CCAAT site at -41 to -32 and a 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 -49 to +156 region was critical for basal transcriptional activity. Using nuclear proteins extracted from normal and immortalized pituitary cells, electrophoretic mobility shift assay indicated that the CCAAT/Sp1 site within the -49 to +156 region was able to interact specifically with the CCAAT binding factor and Sp1. The two sites partly overlapped, and both conferred stimulatory effects. We confirmed in vivo recruitment of CCAAT-binding factor and Sp1 by chromatin immunoprecipitation. Our results indicate that the composite CCAAT/Sp1 cis-element contributes to expression of alpha1-sGC subunit in resting pituitary cells.

Shortly after Sutherland first described the biosynthesis of cAMP in response to hormonal stimulation, he observed not only increased intracellular cAMP levels but also a concurrent rise in extracellular cAMP. Lipid membranes are virtually impermeable to cyclic nucleotides, and extrusion of the nucleotides against a concentration gradient is consistent with operation of ATP-dependent and probenecid-sensitive transport mediated by plasma membrane proteins, which are termed cyclic nucleotide efflux pumps. Such energy-dependent cyclic nucleotide cellular efflux is operative in several eukaryotic cells and could be mediated by the multidrug-resistance proteins MRP4, MRP5, and MRP8. In pituitary cells, however, the operation of export pumps and their contribution to the control of intracellular cyclic nucleotide levels had not been studied. Our recent experiments indicated that cellular efflux of cyclic nucleotides was detectable in normal and immortalized pituitary cells under resting conditions and was enhanced after concurrent stimulation of cAMP and cGMP production. In resting and stimulated cells, the efflux pumps transported most of the of cGMP produced de novo, limiting its intracellular accumulation to a concentration range of 1-2 µM. In contrast, only a small fraction of cAMP was released, and we observed time- and concentration-dependent accumulation of this messenger in the cytosol, ranging from 1-100 µM. Stimulation and inhibition of cGMP production alone did not affect cAMP efflux, suggesting the operation of two different transport pathways in pituitary cells. Rates of cAMP and cGMP effluxes were comparable, and both pathways were blocked by probenecid and progesterone. Normal pituitary cells expressed mRNA transcripts for MRP4, MRP5, and MRP8, whereas GH3 immortalized pituitary cells expressed only transcripts for MRP5. Downregulation of MRP5 expression in GH3 cells lowered cGMP release without affecting cAMP efflux. These results indicate that, in pituitary cells, cyclic nucleotide cellular efflux plays a critical role in eliminating intracellular cGMP but not cAMP and that such selectivity is achieved by expression of MRP5.

Pituitary purinergic receptor channels

Yan, Liang, Kretschmannová, Tomić; in collaboration with Koshimizu, Obsil, Zemková

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. In anterior pituitary cells from adult rats, we identified the mRNA transcripts for several P2X receptors, including P2X2, P2X3, P2X4, and P2X7. We also documented functional expression of P2X receptors by using single-cell calcium measurements. Recently, we characterized the expression and electrophysiological properties of P2X receptors in pituitary gonadotrophs from embryonic, neonatal, and adult rats. In cells from all three age groups, the calcium-mobilizing agonist GnRH induced oscillatory, hyperpolarizing, non-desensitizing, and slowly deactivating currents. In contrast, ATP induced non-oscillatory, depolarizing, slowly desensitizing, and rapidly deactivating currents, indicating that the rat 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 P2X2-subtype channels in these cells. ATP-induced rapid depolarization of gonadotrophs led to initiation of firing in quiescent cells, an increase in the frequency of action potentials in spontaneously active cells, and a transient stimulation of LH release. ATP also influenced GnRH-induced current and membrane-potential oscillations and LH release in an extracellular Ca2+-dependent manner. These inositol phosphate-dependent oscillations were facilitated, slowed, or stopped, depending on ATP concentration, the time of its application, and the level of calcium content in intracellular stores. The results indicate that, in gonadotrophs, P2X receptors could operate as pacemaking channels and modulators of GnRH-controlled electrical activity and secretion.

Earlier, we cloned and characterized several P2X splice isoforms in rat anterior pituitary cells. Although alternative splicing extensively modifies the C-terminal sequences of P2X subunits, the direct influence of such post-transcriptional modifications on receptor architecture and function remains poorly understood. In a recent study, we focused on mouse pituitary P2X2 receptors. In this tissue, progressive splicing of the P2X2 C-terminus generated P2X2b and P2X2e, two functional subunit variants that exhibited accelerated desensitization rates and attenuated calcium signals when the receptors were in homomeric states. To measure the inter-subunit interaction in living cells, we used the efficient transfer of bioluminescent resonance energy between luciferase and fluorescent proteins attached to the N- or C-subunit termini of these subunits. The constitutive interactions between the full-length C-termini of P2X2 receptor were detected by a significantly higher fluorescence/luminescence intensity ratio than in negative controls. Moreover, interactions between C-termini and between C- and N-termini of adjacent subunits were significantly enhanced in homomeric and heteromeric receptors containing P2X2b or P2X2e subunits. Finally, deletion of two amino acids at the splicing junction, but not at the C-terminal end of the P2X2b receptor, resulted in the enhancement of channel desensitization and luminescence resonance energy transfer. These results indicate that C-terminal structure plays a critical role in cytoplasmic inter-subunit interactions and suggest that the extent of subunit interactions before ATP application could contribute to the subsequent channel activity and conformation changes associated with agonist-dependent desensitization.

The P2X4 receptor is sensitive to ivermectin (IVM), a high-molecular weight lipophilic compound used as an antiparasitic agent in human and veterinary medicine. IVM applied extracellularly increases both current amplitude in response to supramaximal agonist concentration and sensitivity to ATP and alpha,beta-meATP in rat receptor expressed in Xenopus oocytes and human receptor expressed in HEK293 cells. In both cell types, IVM also reduces the desensitization rate and greatly prolongs the deactivation of current after ATP removal. At present, there is no information about localization of the IVM binding site(s) at P2X4R. Initial experiments indicated that IVM is effective only when applied extracellularly. Using the wild-type rat homomeric P2X4R, several chimeric P2X2/P2X4 receptors, and single-point P2X4 receptor-specific mutants in the ectodomain and two transmembrane domains, our ongoing work focuses on the identification of receptor-specific residues responsible for IVM effects on channel gating. Experiments with chimeric receptors revealed that the Val49-Val61 but not the Val64-Tyr315 ectodomain sequence is important for the effects of IVM on channel deactivation. Receptor-specific mutations placed in the Gly29-Val61 and Asp338-Leu358 regions showed the importance of Trp50, Val60, and Val357 residues in IVM regulation of the rate of channel deactivation, but not on the maximum current amplitude. These results suggest that the transmembrane domains and nearby ectodomain region contribute to the effects of IVM on channel deactivation.

Publications Related to Other Work

¹ Silvana A. Andric, PhD, former Postdoctoral Fellow
² Tatjana S. Kostic, PhD, former Postdoctoral Fellow

COLLABORATORS

Taka-aki Koshimizu, MD, PhD, Kyoto University, Kyoto, Japan
Tomas Obsil, PhD, Charles University, Prague, Czech Republic
Hana Zemková, PhD, Czech Academy of Sciences, Prague, Czech Republic

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