Multiple cholinergic signaling pathways in pituitary gonadotrophs

Abstract

Acetylcholine (ACh) has been established as a paracrine factor in the anterior pituitary gland, but the receptors mediating ACh action and the cell types bearing these receptors have not been identified. Our results showed that the expression of the nicotinic subunits mRNAs followed the order β2 > β1 = α9 > α4 in cultured rat pituitary cells. The expression of the subunits in immortalized LβT2 mouse gonadotrophs followed the order β2 > α4 = α1. M4 > M3 muscarinic receptor mRNA were also identified in pituitary and LβT2 cells. The treatment of cultured pituitary cells with GnRH down-regulated the expression of α9 and α4 mRNAs, without affecting the expression of M3 and M4 receptor mRNAs, and ACh did not alter the expression of GnRH receptor mRNA. We also performed double immunostaining to show the expression of β2-subunit and M4 receptor proteins in gonadotrophs. Functional nicotinic channels capable of generating an inward current, facilitation of electrical activity, and Ca(2+) influx were identified in single gonadotrophs and LβT2 cells. In both cell types, the M3 receptor-mediated, phospholipase C-dependent Ca(2+) mobilization activated an outward apamin-sensitive K(+) current and caused hyperpolarization. The activation of M4 receptors by ACh inhibited cAMP production and GnRH-induced LH release in a pertussis toxin-sensitive manner. We concluded that multiple cholinergic receptors are expressed in gonadotrophs and that the main secretory action of ACh is inhibitory through M4 receptor-mediated down-regulation of cAMP production. The expression of nicotinic receptors in vitro compensates for the lack of regular GnRH stimulation of gonadotrophs.

Fig. 1.

Expression of ACh receptor subunits in pituitary cells. A and B, qRT-PCR analysis of expression of mRNAs for nAChR subunits (A) and mAChRs (B) in pituitary cells (closed bars) and LβT2 cells (open bars). Data shown are means ± sem values from four (A) and five (B) experiments. C and D, Time course of down-regulation of α4 (C) and α9 (D) mRNAs expression by GnRH in pituitary cells. E, The lack of effect of ACh on GnRH receptor mRNA expression in pituitary cells.

Fig. 2.

Triple immunofluorescence labeling of β2-subunit of nAChR, M4 subtype of mAChR, and LH in anterior pituitary sections. A, β2-subunit (green fluorescence). B, M4 subunit (red fluorescence). C, LH-containing cells (blue fluorescence). D, Overlay, both β2-subunit and M4 subunit were observed in LH-containing cells. Nuclei were devoid of staining in all cases. Scale bar, 20 μm.

Fig. 3.

Agonist-induced oscillatory calcium and current responses in identified gonadotrophs. A and B, Examples of Ca²⁺ responses to application of ACh and GnRH in gonadotrophs bathed in Ca²⁺-containing (A) and Ca²⁺-deficient (B) medium. C and D, Oxotremorine (OX) stimulated Ca²⁺ oscillations (C) and atropine inhibited effects of ACh on [Ca²⁺]_i response (D) in GnRH-responsive cells bathed in Ca²⁺-deficient medium. In this and following figures, arrows indicate the start of agonist application. E and F, Voltage clamp amphotericin-perforated whole-cell recording of Ca²⁺ oscillations monitored as an outward Ca²⁺-activated current in gonadotrophs stimulated with GnRH (E) and ACh (F). G, In a fraction of gonadotrophs, ACh stimulated inward and outward currents. All cells were voltage clamped at −60 mV. Horizontal bars indicate duration of agonist application.

Fig. 4.

ACh-induced Ca²⁺ mobilization and activation of SK-type potassium channels in LβT2 gonadotrophs. A, Representative records of oscillatory and nonoscillatory [Ca²⁺]_i responses induced by variable ACh concentrations (top) and extracellular calcium dependence of sustained but not early spiking (bottom). Gray area indicates period during which cells were exposed to Ca²⁺-deficient medium. B, Pharmacological identification of receptors responsible for ACh-induced Ca²⁺ mobilization. Top, Inhibition of Ca²⁺-mobilizing action of 20 μm ACh in cells bathed in Ca²⁺-deficient medium and treated with 100 nm DAU 5884, a M3-mAChR-specific inhibitor. Bottom, Insensitivity of ACh-induced Ca²⁺ response to DpGlu, a specific antagonist of GnRH receptor (left), and inhibition by atropine, a general mAChR blocker (right), in cells bathed in Ca²⁺-deficient medium. C and D, Current-clamp traces of ACh (C)- and GnRH (D)-induced transient hyperpolarization. E and F, Activation of K_Ca channels by ACh in LβT2 gonadotrophs. E, Effect of the holding potential (HP) on the amplitude of ACh-induced outward current: representative traces (top) and mean ± sem values (bottom). The reversal potential (E_rev = −97 ± 5 mV, n = 3) was near to the calculated reversal potential for K⁺ ions. F, Sensitivity of ACh-induced current to K_Ca channel blockers. Top, Inhibition of ACh-induced current by apamin (200 nm), a small conductance K_Ca channel blocker, but not IBTX (200 nm), a highly specific blocker for K_Ca1.1 channels. Bottom, Summary histogram showing effects of removal of bath Ca²⁺ and addition of K⁺ channel blockers: apamin (200 nm), IBTX (100 nm), BMI (30 μm), TRAM 34 (1 μm), and Xe991 (1 μm), in cell bathed in Ca²⁺-containing medium. *, P < 0.01. LβT2 cells were voltage clamped at −40 mV using amphotericin-perforated whole-cell recording.

Fig. 5.

Characterization of mAChR expressed in LβT2 gonadotrophs. A, Dependence of ACh-induced Ca²⁺ mobilization on phospholipase C signaling pathway. ACh-induced outward current from LβT2 cells persisted in Ca²⁺-deficient medium (top), was completely inhibited by 1 min of pretreatment with U73122, a specific phospholipase C inhibitor (middle), and was not affected by pretreatment with inactive analog U73343 (bottom). B, ACh-induced outward current was insensitive to dTC (top) but was abolished by atropine (middle) and DAU 5884 (bottom). C, Summary histogram showing the effect of atropine, pirenzepine, DAU 5884, and AF-DX 116 on the amplitude of ACh-induced currents. Voltage clamp amphotericin-perforated whole-cell recording from LβT2 cell voltage clamped at −40 mV are shown. Cells were stimulated with 10 μm ACh. *, P < 0.01.

Fig. 6.

Agonist-induced Ca²⁺ influx in single pituitary cells and LβT2 gonadotrophs. Top, Characterization of Ca²⁺ influx pathway in pituitary gonadotrophs. Effects of ACh (A) and nicotine (B) on [Ca²⁺]_i in gonadotrophs bathed in Ca²⁺-containing medium. Nicotine was ineffective in cells bathed in medium containing cytisine, a specific blocker of β2-subunit-containing nAChR (B). In most of the cells, ACh induced additional rise in [Ca²⁺]_i in the presence of cytisine (C) or nicotine (D). GnRH was added at the end of recording to identify gonadotrophs. Bottom, Characterization of Ca²⁺ influx pathway in LβT2 cells. Nicotine induced a rise in [Ca²⁺]_i in cells with blocked M3-mAChR by DAU 5884 (E). Stimulatory effects of nicotine on Ca²⁺ influx was abolished in cells bathed with 4 μm cytisine (F). In a fraction of cells, ACh induced additional rise in [Ca²⁺]_i in the presence of cytisine (G) or nicotine (H).

Fig. 7.

Electrophysiological characterization of nAChR in pituitary gonadotrophs. A and B, Current-clamp traces of GnRH (A) and ACh (B) induced electrical activity. Notice that GnRH-induced increase in the frequency of action potentials was periodically interrupted with transient hyperpolarization, whereas ACh application caused a sustained depolarization in this particular gonadotroph. C, Concentration-dependent effect of ACh on the amplitude of inward current in identified gonadotrophs. Top, Representative traces. Bottom, Mean ± sem values, with the estimated EC₅₀ of 8.6 μm (n = 5). D, Stimulation of inward current by 100 μm ACh, 30 μm nicotine, but not by 1 μm PNU 282987. E, Inhibition of ACh-induced inward current by dTC. F–H, The lack of effect of αBTX, atropine (ATR), and ivermectin (IVM) on ACh-induced inward current in identified gonadotrophs. All traces were obtained using standard whole-cell patch-clamp recording. Gray areas and horizontal bars indicate duration of drug application.

Fig. 8.

ACh-induced inhibition of adenylyl cyclase and LH release in perifused pituitary cells. A–C, Effects of ACh on cAMP release in cultured pituitary cells. A, Inhibition of basal cAMP release by ACh. B, PTX sensitivity of 10 μm ACh-induced inhibition of cAMP release. C, ACh (10 μm)-induced inhibition of forskolin (1 μm) stimulated cAMP release. D, Effect of ACh on cAMP release in LβT2 cells. Horizontal bars indicate duration of ACh treatment. E, Effects of ACh on GnRH induced LH release. Inhibitory effect on GnRH-stimulated LH release was observed after pretreatment with ACh (10 μm) for 15 min. Inset shows a small increase in LH release triggered by ACh alone.