We investigate the cellular signalling cascades in endocrine and neuroendocrine cells and the interactions between plasma membrane electrical events and receptor-controlled pathways. Our objective is to elucidate the mechanisms controlling calcium signalling and the role of calcium ions as messengers in control of signalling, secretion, and gene expression. Our approach has been to characterize calcium signalling from a biophysical, physiological, and pharmacological point of view. More recently, we have begun to use molecular biological approaches to identify pathways participating in the generation and control of calcium signals and to address how the calcium-signalling function in neuroendocrine cells is related to the structures of individual receptors and channels. Our current investigations focus on the calcium-signalling function of purinergic receptors, the mechanism of endothelin and thyrotropin-releasing hormone actions in pituitary lactotrophs, and the role of calcium as an extracellular and intracellular messenger.

Calcium Signalling by Purinergic Receptors
He, Tomic, Stojilkovic; in collaboration with Koshimizu
Pituitary cells secrete ATP, which acts as an autocrine and/or paracrine extracellular messenger on two families of purinergic receptors termed P2YRs and P2XRs. P2YRs are G protein–coupled receptors, and P2XRs are cation-permeable channels that conduct calcium and facilitate voltage-sensitive calcium entry if expressed in excitable cells. Even though it is known that these channels vary with respect to their ligand preferences and channel kinetics during activation, desensitization, and recovery, the contributions of distinct receptor subdomains to the subtype-specific behavior have been incompletely characterized. We recently showed that the wild-type P2X2aR and its splice form lacking the intracellular Val370-Gln438 C-terminal sequence (P2X2bR) respond to ATP stimulation with comparable EC50s and peak current/calcium responses but desensitize in a receptor-specific manner: P2X2aR desensitizes slowly and P2X2bR desensitizes rapidly.

We studied the effects of different agonists and of substituting the ectodomain on the pattern of calcium signalling by P2X2aR and P2X2bR. Both receptors showed similar EC50s (estimated from the peak current/calcium response) and IC50s (estimated from the rate of current/calcium signal desensitization) for agonists in the order 2-MeS-ATP < ATP < ATP-g-S < BzATP << ab-methylene ATP; the IC50s for agonists shifted to the right compared with their EC50s. Furthermore, the ATP-induced receptor-subtype–specific pattern of desensitization was mimicked by high- but not low-efficacy agonists.

Figure 11

Influence of the substitution of ectodomain at P2X2Rs on agonistic potency of ATP (left panels), BzATP (central panels), and ab-methylene ATP (right panels). A, Change in the EC50s for agonists in cells expressing P2X2a+X3Rs. B, Comparison of the peak amplitude of [Ca2+]i signals in P2X2a+X3R-expressing cells. P2X2b+X3Rs showed comparable leftward shifts in EC50s for three agonists and a decrease in peak amplitude of calcium responses.

These results suggested a ligand-specific desensitization pattern, which we sought to confirm by generating chimeric P2X2aR and P2X2bR containing the Val60-Phe301 ectodomain sequence of P2X3R and the Val61-Phe313 ectodomain sequence of P2X7R instead of the native Ile66-Tyr310 sequence. The homomeric receptors bearing the extracellular domain of the P2X3 subunit in the P2X2a-based backbone mimicked two intrinsic functions of P2X3R: sensitivity to ab-methylene ATP and ecto-ATPasedependent recovery from endogenous desensitization; the two functions were localized to the N- and C-terminal halves of the P2X3R extracellular loop, respectively. Furthermore, the mutated P2X2a+X3R and P2X2b+X3R exhibited comparable EC50s for ATP, BzATP, and ab-methylene ATP in the submicromolar concentration range and desensitized in a receptor-specific and ligand-nonspecific manner. On the other hand, the chimeric P2X2+X7R exhibited decreased sensitivity for ATP and desensitized in a receptor-nonspecific manner. The results suggest that the efficacy of agonists for the ligand-binding domain of P2X2Rs reflects the strength of desensitization controlled by their C-terminal structures.

We have also analyzed the agonist selectivity and desensitization rates of P2XRs in heteromeric configuration. First, we demonstrated the physical and functional heteromerization of native and mutant P2X2a and P2X2b subunits. In heteromeric receptors, the ectodomain of P2X3 was a structural determinant for ligand selectivity and recovery from desensitization, and the C-terminus of P2X2R was an important factor for the desensitization rate. Furthermore, [g-32P]8-azido ATP, a photoreactive agonist, was effectively cross-linked to the P2X3 subunit in homomeric receptors but not in heteromeric P2X2+P2X3Rs. The results indicate that heteromeric receptors formed by distinct P2XR subunits develop new functions resulting from integrative effects of the participating extracellular and C-terminal subdomains.

Receptor-Controlled Action Potential Secretion Coupling
Tomic, He, Van Goor,a Stojilkovic
Two Ca2+-mobilizing receptors expressed in lactotrophs, endothelin-A (ET-A) and thyrotropin-releasing hormone (TRH), induce both rapid calcium release from intracellular stores and prolactin secretion but differ in their actions during sustained stimulation. TRH facilitates and ET-1 inhibits voltage-gated calcium influx and prolactin secretion. ET-1-induced the depolarization of cells, and the enhancement of calcium influx upon calcium mobilization was established in both quiescent and firing lactotrophs treated overnight with pertussis toxin. Activation of adenylyl cyclase by forskolin and the addition of cell permeable 8Br-cAMP did not affect the ET-1–induced sustained inhibition of voltage-gated calcium influx, suggesting that the cAMP-protein kinase A signalling pathway does not mediate the inhibitory action of ET-1 on calcium influx.

Consistent with the role of pertussis toxin–sensitive potassium channels in ET-1–induced hyperpolarization of controls but not pertussis toxin-treated cells, ET-1 decreased the cell input resistance and activated a cesium-sensitive potassium current. The addition of cesium also mimicked the action of pertussis toxin on the pattern of sustained calcium signalling, but only in about half of ET-1–stimulated cells, and did not change ET-1–induced inhibition of prolactin secretion. We observed similar effects of pertussis toxin and cesium in immortalized GH3 cells transiently expressing ET-A receptors. Moreover, the following blockers did not affect the agonist-specific patterns of calcium signalling and prolactin secretion: apamin and paxilline, specific blockers of calcium-activated SK- and BK-type potassium channels, respectively; E-4031, a blocker of the ether a-go-go potassium channel; and linopirdine, a blocker of the M-type potassium channel. The results suggest that ET-1 inhibits voltage-gated calcium influx through activation of cesium-sensitive channels, presumably the Gi/o-controlled inward rectifier potassium channels, and that the agonist also inhibits prolactin release, but downstream of calcium influx. Further studies are required to identify the mechanism of sustained TRH-induced facilitation of voltage-gated calcium influx and prolactin secretion.

Extra- and Intracellular Messenger Functions of Calcium in Pituitary Cells
Andric, Kostic, Tomic, Zivadinovic,b Stojilkovic
It is well established that G protein–coupled receptors stimulate nitric oxide–sensitive soluble guanylyl cyclase by increasing intracellular calcium and activating calcium-dependent nitric oxide synthases. In pituitary cells, receptors that stimulated adenylyl cyclase, growth hormone–releasing hormone, corticotropin-releasing factor, and thyrotropin-releasing hormone also stimulated calcium signalling and increased cGMP levels, whereas receptors that inhibited adenylyl cyclase, endothelin-A and dopamine2, also inhibited spontaneous calcium transients and decreased cGMP levels. However, abolition of calcium signalling did not block receptor-controlled up- and down-regulation of cyclic nucleotide accumulation, suggesting that cAMP production affects cGMP accumulation. We observed agonist-induced cGMP accumulation in cells incubated in the presence of various phosphodiesterase and soluble guanylyl cyclase inhibitors, confirming that Gs-coupled receptors stimulated de novo cGMP production. In addition, cholera toxin (an activator of Gs), forskolin (an activator of adenylyl cyclase), and 8-Br-cAMP (a permeable cAMP analog) mimicked the stimulatory action of Gs-coupled receptors on cGMP production. Basal, agonist-, cholera toxin–, and forskolin-stimulated cGMP production, but not cAMP production, was significantly reduced in cells treated with H89, a protein kinase A inhibitor. The results indicate that coupling of seven plasma membrane–domain receptors to an adenylyl cyclase signalling pathway provides an additional calcium-independent and cAMP-dependent mechanism for modulating soluble guanylyl cyclase activity in pituitary cells.

Calcium can serve not only as an intracellular messenger but also as an extracellular messenger controlling the gating properties of plasma membrane channels; it also acts as an agonist for G protein-coupled calcium-sensing receptors. We studied the potential extracellular messenger functions of this ion in anterior pituitary cells. Depletion and repletion of extracellular calcium concentration–induced transient elevations in the intracellular calcium concentration and elevations in extracellular calcium above physiological levels decreased intracellular calcium in somatotrophs and lactotrophs, but not in gonadotrophs. The amplitudes and duration of intracellular calcium signals depended on the extracellular calcium concentration and its rate of change, which resulted exclusively from modulation of spontaneous voltage-gated calcium influx. Changes in extracellular calcium concentration also affected growth hormone and prolactin secretion. The prolactin secretory profiles paralleled the intracellular calcium profiles in lactotrophs, whereas growth hormone secretion was also stimulated by extracellular calcium independently of the status of voltage-gated calcium influx. Extracellular calcium modulated growth hormone secretion in a dose-dependent manner, with EC50s of 0.75 and 2.25 mM, and with minimum secretion at about 1.5 mM. In a parallel experiment, cAMP accumulation progressively increased with elevation in extracellular calcium, whereas inositol phosphate levels remained unaffected. The results indicate the cell type–specific role of extracellular calcium in the control of calcium signalling and secretion.