Electrical Activity and Calcium Channels in Neuroendocrine Cells (original) (raw)
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Journal of Neurophysiology, 2007
Cultured pituitary somatotrophs release growth hormone in response to spontaneous Ca 2+ entry through voltage-gated calcium channels (VGCCs), which is governed by plateau-bursting electrical activity and is regulated by several neurohormones, including GH-releasing hormone (GHRH) and somatostatin. Here we combine experiments and theory to clarify the mechanisms underlying spontaneous and receptor-controlled electrical activity. Experiments support a role of a Na + -conducting and tetrodotoxin-insensitive channel in controlling spontaneous and GHRHstimulated pacemaking, the latter in a cAMP-dependent manner; an opposing role of spontaneously active inwardly rectifying K + (K ir ) channels and G-protein-regulated K ir channels in somatostatin-mediated inhibition of pacemaking; as well as a role of VGCCs in spiking and BK-type Ca 2+ -activated K + channels in plateau bursting. The mathematical model is compatible with a wide variety of experimental data involving pharmacology and extracellular ion substitution and supports the importance of constitutively active tetrodotoxin-insensitive Na + and K ir channels in maintaining spontaneous pacemaking in pituitary somatotrophs. The model also suggests that these channels are involved in the up-and down-regulation of electrical activity by GHRH and somatostatin. In the model, the plateau bursting is controlled by two functional populations of BK channels, characterized by distance from the VGCCs. The rapid activation of the proximal BK channels is critical for the establishment of the plateau, whereas slow recruitment of the distal BK channels terminates the plateau. Page 2 of 47 Kato M, and Sakuma Y. Regulation by growth hormone-releasing hormone and somatostatin of a Na + current in the primary cultured rat somatotroph. Endocrinology 138: 5096-5100, 1997. Kaupp UB, and Seifert R. Cyclic nucleotide-gated ion channels. Physiol Rev 82: 769-824, 2002. Koshimizu T, Tomic M, Van Goor F, and Stojilkovic SS. Functional role of alternative splicing in pituitary P2X2 receptor-channel activation and desensitization. Mol Endocrinol 12: 901-913, 1998. Koshimizu TA, Tomic M, Wong AO, Zivadinovic D, and Stojilkovic SS. Characterization of purinergic receptors and receptor-channels expressed in anterior pituitary cells. Endocrinology 141: 4091-4099, 2000. Kostic TS, Andric SA, and Stojilkovic SS. Receptor-controlled phosphorylation of alpha 1 soluble guanylyl cyclase enhances nitric oxide-dependent cyclic guanosine 5'-monophosphate production in pituitary cells. Mol Endocrinol 18: 458-470, 2004. Kostic TS, Tomic M, Andric SA, and Stojilkovic SS. Calcium-independent and cAMPdependent modulation of soluble guanylyl cyclase activity by G protein-coupled receptors in pituitary cells. J Biol Chem 277: 16412-16418, 2002. Kretschmannova K, Gonzalez-Iglesias AE, Tomic M, and Stojilkovic SS. Dependence of hyperpolarisation-activated cyclic nucleotide-gated channel activity on basal cyclic adenosine monophosphate production in spontaneously firing GH3 cells. J Neuroendocrinol 18: 484-493, 2006. Kuryshev YA, Haak L, Childs GV, and Ritchie AK. Corticotropin releasing hormone inhibits an inwardly rectifying potassium current in rat corticotropes. J Physiol 502 ( Pt 2): 265-279, 1997. Kwiecien R, and Hammond C. Differential management of Ca2+ oscillations by anterior pituitary cells: a comparative overview. Neuroendocrinology 68: 135-151, 1998. Kwiecien R, Robert C, Cannon R, Vigues S, Arnoux A, Kordon C, and Hammond C. Endogenous pacemaker activity of rat tumour somatotrophs. J Physiol 508 ( Pt 3): 883-905, 1998. Kwiecien R, Tseeb V, Kurchikov A, Kordon C, and Hammond C. Growth hormone-releasing hormone triggers pacemaker activity and persistent Ca 2+ oscillations in rat somatotrophs. J Physiol 499 ( Pt 3): 613-623, 1997. Lee SH, Lee EH, Ryu SY, Rhim H, Baek HJ, Lim W, and Ho WK. Role of K(+) channels in frequency regulation of spontaneous action potentials in rat pituitary GH(3) cells. Neuroendocrinology 78: 260-269, 2003. Lewis DL, Goodman MB, St John PA, and Barker JL. Calcium currents and fura-2 signals in fluorescence-activated cell sorted lactotrophs and somatotrophs of rat anterior pituitary. Endocrinology 123: 611-621, 1988. Lewis DL, Weight FF, and Luini A. A guanine nucleotide-binding protein mediates the inhibition of voltage-dependent calcium current by somatostatin in a pituitary cell line. Proc Natl Acad Sci U S A 83: 9035-9039, 1986. Luini A, Lewis D, Guild S, Schofield G, and Weight F. Somatostatin, an inhibitor of ACTH secretion, decreases cytosolic free calcium and voltage-dependent calcium current in a pituitary cell line.
The EMBO journal, 1995
Hormonal stimulation of voltage-dependent Ca2+ channels in pituitary cells is thought to contribute to the sustained phase of Ca2+ entry and secretion induced by secretion stimulating hormones and has been suggested as a mechanism for refilling the Ca2+ stores. Using the cell-attached patch-clamp technique, we studied the stimulation of single Ca2+ channels by thyrotropin-releasing hormone (TRH) in rat GH3 cells. We show that TRH applied from the bath switched the activity of single L-type Ca2+ channels from a gating mode with very low open probability (po) to a gating mode with slightly smaller conductance but 10 times higher po. Interconversions between these two gating modes were also observed under basal conditions, where the equilibrium was shifted towards the low po mode. TRH applied from the pipette had no effect, indicating the involvement of a cytosolic compound in the stimulatory pathway. We show that TRH does not potentiate all the L-type Ca2+ channels in a given membrane...
Proceedings of the National Academy of Sciences, 1990
The relationships between the activation status of voltage-sensitive Ca2+ channels and secretory responses were analyzed in perfused rat gonadotrophs during stimulation by high extracellular K+ concentration ([K+]e) or the physiological agonist, gonadotropin-releasing hormone (GnRH). Increase of [K+]e to 50 mM evokes an on-off secretory response, with a rapid rise in luteinizing hormone (LH) secretion to a peak at 35 sec (on response) followed by an exponential decrease to the steady-state level. Cessation of K+ stimulation elicits a transient (off) response followed by an exponential decrease to the basal level. The LH response to high [K+]e is nifedipine-sensitive and its amplitude depends on membrane potential. There is a close relationship between the LH secretory response to high [K+]e and the amplitude of the inward Ca2+ current measured at 100 msec in whole-cell patch clamp experiments. In addition, the profile of the LH secretory response is similar to that of the response o...
Cell Calcium, 2002
Simultaneous measurements of electrical activity and intracellular Ca 2; levels were performed in perforatedpatch current-clamped individual GH 3 cells. Both in cells showing brief (:100 ms) and long action potentials (APs), we found a good correlation between the averaged intracellular Ca 2; concentration ([Ca 2; ] i) and AP frequency, but not between the mean [Ca 2; ] i and AP duration. Nevertheless, the magnitude of spontaneous Ca 2; oscillations was highly dependent on the size and duration of the APs. The decay of the Ca 2; transients was not slowed when the size of the oscillations was varied either spontaneously or after elongation of the AP with the K ; channel blocker tetraethyl ammonium. Furthermore, the recovery from Ca 2; loads similar to those induced by the APs was slightly retarded after treatment of the cells with intracellular store Ca 2;-ATPase inhibitors. Among previous results showing that caffeine-induced [Ca 2; ] i increases are secondary to electrical activity enhancements in GH 3 cells, these data indicate that the Ca 2; entry triggered via APs is the primary determinant of the [Ca 2; ] i variations, and that Ca 2;-induced Ca 2; release has a minor contribution to Ca 2; oscillations recorded during spontaneous activity. They also point to modulation of electrical activity patterns as a crucial factor regulating spontaneous [Ca 2; ] i signalling, and hence pituitary cell functions in response to physiological secretagogues.
Immunology & Cell Biology, 2000
Influx of Ca 2+ via Ca 2+ channels is the major step triggering exocytosis of pituitary somatotropes to release growth hormone (GH). Voltage-gated Ca 2+ and K + channels, the primary determinants of the influx of Ca 2+ , are regulated by GH-releasing hormone (GHRH) through G-protein-coupled intracellular signalling systems. Using whole-cell patch-clamp techniques, the changes of the Ca 2+ and K + currents in primary cultured ovine and human somatotropes were recorded. Growth hormone-releasing hormone (10 nmol/L) increased both Land T-type voltage-gated Ca 2+ currents. Inhibition of the cAMP/protein kinase A (PKA) pathway by either Rp-cAMP or H 89 blocked this increase in both Land T-type Ca 2+ currents. Growth hormone-releasing hormone also decreased voltage-gated transient (I A) and delayed rectified (I K) K + currents. Protein kinase C (PKC) inhibitors, such as calphostin C, chelerythrine or downregulation of PKC, blocked the effect of GHRH on K + currents, whereas an acute activation of PKC by phorbol 12,13-dibutyrate (1 µmol/L) mimicked the effect of GHRH. Intracellular dialysis of a specific PKC inhibitor (PKC 19-36) also prevented the reduction in K + currents by GHRH. It is therefore concluded that GHRH increases voltage-gated Ca 2+ currents via cAMP/PKA, but decreases voltage-gated K + currents via the PKC signalling system. The GHRH-induced alteration of Ca 2+ and K + currents augments the influx of Ca 2+ , leading to an increase in [Ca 2+ ]i and the GH secretion.
Action potentials and membrane ion channels in clonal anterior pituitary cells
Proceedings of the National Academy of Sciences, 1983
The electrophysiological properties of the mouse anterior pituitary cell line AtT-20/D16-16 were investigated with intracellular and patch-clamp techniques. Clonal AtT-20/D16-16 cells were found to be electrically excitable, with most cells exhibiting spontaneous bursting action potentials. The mean burst rates varied from 1.4 Hz at -55 mV to 8.2 Hz at -25 mV, showing an approximately linear frequency-current relationship in the low current range. The bursts consisted of one to several fast Na4 spikes superimposed on a slow pacemaker potential, followed by a Ca2+ spike and a Ca2+-sensitive afterhyperpolarization. Removal of either Na+ or Ca2+ from the bathing medium led to cessation of spontaneous activity and the appearance of arrhythmic firing patterns. Single channel recordings revealed the presence of Ca2+dependent K+ channels with unitary conductances of =130 pS in physiological medium. These channels were activated by both intracellular Ca2+ and membrane depolarization. Addition of norepinephrine (10 ,LM) led to increases in burst frequency and f8endorphin secretion mediated by activation of f3-adrenergic receptors. Our results, in conjunction with previous work, suggest that the Ca2+ that enters the cell during the burst may be involved in hormone secretion.
Electrophysiology of Neurosecretory Cells from the Pituitary Intermediate Lobe
Journal of Experimental Biology, 1988
One of the goals in studying the electrical properties of neurosecretory cells is to relate their electrical activity to the process of secretion. A central question in these studies concerns the role of transmembrane calcium ion flux in the initiation of the secretory event. With regard to the secretory process in pituitary cells, several research groups have addressed this question in vitro using mixed primary anterior pituitary cell cultures or clonal cell lines derived from pituitary tumours. Other workers, including ourselves, have used homogeneous cell cultures derived from the pituitary intermediate lobes of rats to examine the characteristics of voltagedependent conductances, the contribution of these conductances to action potentials and their role in stimulus-secretion coupling. Pars intermedia (PI) cells often fire spontaneous action potentials whose frequency can be modified by the injection of sustained currents through the recording electrode. In quiescent cells action potentials can also be evoked by the injection of depolarizing current stimuli. At around 20°C these action potentials have a duration of about 5 ms. Although most of the inward current during action potentials is carried by sodium ions, a calcium ion component can be demonstrated under abnormal conditions. Voltage-clamp experiments have revealed that the membrane of these cells contains high-threshold, L-type, Ca 2+ channels and low-threshold Ca 2+ channels. Since hormone release from PI cells appears not to be dependent on action potential activity but does depend on external calcium ions, it is not clear what role these Ca 2+ channels play in stimulus-secretion coupling in cells of the pituitary pars intermedia. One possibility is that the low-threshold Ca 2+ channels are more important to the secretory process than the high-threshold channels.
Dual modulation of K channels by thyrotropin-releasing hormone in clonal pituitary cells
Proceedings of the National Academy of Sciences, 1985
Transmembrane electrical activity in pituitary tumor cells can be altered by substances that either stimulate or inhibit their secretory activity. Using patch recording techniques, we have measured the resting membrane potentials, action potentials, transmembrane macroscopic ionic currents, and single Ca2+-activated K channel currents of GH3 and GH4/C1 rat pituitary tumor cells in response to thyrotropin-releasing hormone (TRH). TRH, which stimulates prolactin secretion, causes a transient hyperpolarization of the membrane potential followed by a period of elevated action potential frequency. In single cells voltage clamped and internally dialyzed with solutions containing K+, TRH application results in a transient increase in Ca2+-activated K currents and a more protracted decrease in voltage-dependent K currents. However, in cells internally dialyzed with K+-free solutions, TRH produces no changes in inward Ca2+ or Ba2+ currents through voltage-dependent Ca channels. The time cour...
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
Somatostatin is a neurohormone peptide that inhibits a variety of secretory responses in different cell types. We have investigated the effects of somatostatin on calcium current and intracellular free calcium in AtT-20 cells, a pituitary tumor line in which the inhibitory actions of this peptide have been well characterized. At concentrations similar to those that inhibit adrenocorticotropic hormone (ACTH) release, somatostatin and its analogs reduced the levels of intracellular free calcium (as measured by the Quin-2 technique). Nifedipine and other blockers of voltage-dependent calcium channels also reduced cytosolic calcium levels. The effects of somatostatin and nifedipine were not additive, suggesting that somatostatin might inhibit calcium channels. Experiments using the whole-cell patch-clamp technique showed that somatostatin reduces voltage-dependent calcium current. The effects of somatostatin on cytosolic calcium and calcium current appear to be independent of its abilit...