Nitric oxide induces [Ca²⁺]_i oscillations in pituitary GH₃ cells: involvement of I_DR and ERG K⁺ currents

Abstract

The role of nitric oxide (NO) in the occurrence of intracellular Ca2+ concentration ([Ca2+]i) oscillations in pituitary GH3 cells was evaluated by studying the effect of increasing or decreasing endogenous NO synthesis with L-arginine and nitro-L-arginine methyl ester (L-NAME), respectively. When NO synthesis was blocked with L-NAME (1 mM) [Ca2+]i, oscillations disappeared in 68% of spontaneously active cells, whereas 41% of the quiescent cells showed [Ca2+]i oscillations in response to the NO synthase (NOS) substrate L-arginine (10 mM). This effect was reproduced by the NO donors NOC-18 and S-nitroso-N-acetylpenicillamine (SNAP). NOC-18 was ineffective in the presence of the L-type voltage-dependent Ca2+ channels (VDCC) blocker nimodipine (1 microM) or in Ca2+-free medium. Conversely, its effect was preserved when Ca2+ release from intracellular Ca2+ stores was inhibited either with the ryanodine-receptor blocker ryanodine (500 microM) or with the inositol 1,4,5-trisphosphate receptor blocker xestospongin C (3 microM). These results suggest that NO induces the appearance of [Ca2+]i oscillations by determining Ca2+ influx. Patch-clamp experiments excluded that NO acted directly on VDCC but suggested that NO determined membrane depolarization because of the inhibition of voltage-gated K+ channels. NOC-18 and SNAP caused a decrease in the amplitude of slow-inactivating (IDR) and ether-à-go-go-related gene (ERG) hyperpolarization-evoked, deactivating K+ currents. Similar results were obtained when GH3 cells were treated with L-arginine. The present study suggests that in GH3 cells, endogenous NO plays a permissive role for the occurrence of spontaneous [Ca2+]i oscillations through an inhibitory effect on IDR and on IERG.

Intracellular and plasma membrane-initiated pathways involved in the [Ca2+]ielevations induced by iodothyronines (T3 and T2) in pituitary GH3cells

“…Cells were loaded with 10 M 4,5-diaminofluorescein-2-diacetate in normal Krebs solution (5.5 mM KCl, 160 mM NaCl, 1.2 mM MgCl 2, 1.5 mM CaCl2, 10 mM glucose, and 10 mM HEPES-NaOH, pH 7.4) in the presence [Ca 2ϩ ] i . In line with our laboratory's previous results (43,45), the majority of pituitary GH 3 cells showed significant [Ca 2ϩ ] i oscillations over time, while ϳ30% were quiescent (Fig. 1).…”

“…Fluorescence images were acquired at excitation wavelengths of 340 nm and 360 nm every 3 s. Sodium levels are expressed as fluorescence ratios (F 340/F360). [Ca 2ϩ ]i oscillations were identified, and their amplitude and frequency determined, using a computer program written in Java language, as previously described in detail (45). Briefly, for each single cell, the software ]i oscillations, these values were used to define a cutoff, which was set at the mean [Ca 2ϩ ]i Ϯ 2 SD.…”

“…Briefly, for each single cell, the software ]i oscillations, these values were used to define a cutoff, which was set at the mean [Ca 2ϩ ]i Ϯ 2 SD. Then the oscillation frequency was calculated as previously reported (45).…”

“…The first comprises the ryanodine-and inositol trisphosphate (IP 3 )-sensitive intracellular Ca 2ϩ stores and their refilling channels, whereas the second relies on the concerted activity of different classes of plasma membrane ion channels, such as Na ϩ , Ca 2ϩ , and K ϩ channels (43,50). Intriguingly, the ion channels involved in the complex genesis of [Ca 2ϩ ] i oscillations are regulated by nitric oxide (NO), which is itself able to trigger this electrical phenomenon in GH 3 cells (3,45). Since mitochondria are crucial in the regulation of cytosolic Ca 2ϩ signaling, in this study we also investigated the role of these organelles in the modulation of cytosolic Ca 2ϩ homeostasis by thyroid hormones.…”

Nitric Oxide Stimulates NCX1 and NCX2 but Inhibits NCX3 Isoform by Three Distinct Molecular Determinants

“…On the other hand, it has already been demonstrated that NO can regulate numerous classes of ion channels and iontransporting mechanisms involved in the maintenance of intracellular Na ϩ and Ca 2ϩ homeostasis in the central nervous system. In particular, NO can interact with the sodium-potassium pump (Na ϩ /K ϩ ATPase); with plasma membrane channels, including voltage-dependent calcium channels, voltage-dependent tetrodotoxin-sensitive Na ϩ , ether-a-go-go-related K ϩ (Taglialatela et al, 1999;Secondo et al, 2006) and voltage-dependent at big-and small-conductance Ca 2ϩ -dependent K ϩ channels (Brakemeier et al, 2003); and, finally, with intracellular ligand-gated calcium channels, such as inositol trisphosphate-and ryanodinesensitive channels (Annunziato et al, 2002). In all of these cases, NO acts by either activating or inhibiting these ioniccontrolling mechanisms (Taglialatela et al, 2001;Annunziato et al, 2002;Francis et al, 2010).…”