Calcium Signaling by Purinergic Receptors
He, Koshimizu, Tomic, Stojilkovic
Purinergic receptors (P2XR) are a family of cation-permeable channels that conduct calcium and facilitate voltage-sensitive calcium entry in excitable cells. To study calcium signaling by P2XR and its dependence on voltage-sensitive calcium influx, eight cloned P2XR subtypes were expressed individually in GT1 neurons. In all cases, ATP evoked an inward current and a rise in intracellular calcium concentration. P2XR subtypes differed in the peak amplitude of calcium response and the rate of signal desensitization. The P2X1R-, P2X3R-, and P2X4R-derived calcium signals predominantly depended on activation of voltage-sensitive calcium influx. Both voltage-sensitive and -insensitive calcium entry pathways contributed equally to intracellular calcium responses in P2X2aR- and P2X2bR-expressing cells while P2X7R operated as a nonselective pore capable of conducting larger amounts of calcium independently of the status of voltage-gated calcium channels. Thus, calcium signaling by homomeric P2XR expressed in an excitable cell is subtype-specific, which provides an effective mechanism for generating variable calcium signaling patterns in response to a common agonist.

In further experiments, we studied the desensitization of recombinant wild-type and mutant channels. The investigations focused on the relevance of the P2XR C-terminus and ligand-binding domains in controlling receptor desensitization. By substituting six amino acid (6-aa) sequences of the C-termini of P2X4R and P2X3R with the corresponding arginine371-proline376 sequence of P2X2aR, the slow desensitizing pattern of P2X2aR was mimicked fully by P2X4R but only partially by P2X3R. Changing the total net charge in 6-aa of P2X4R so that it became more positive also slowed the receptor desensitization. On the other hand, substitution of the arginine371-proline376 sequence of P2X2aR with the corresponding sequences of P2X1R, P2X3R, and P2X4R increased the rate of receptor desensitization. Furthermore, heterologous polymerization of wild-type P2X2aR and mutant P2X3R with the C-terminal 6-aa of P2X2aR at its analogous position resulted in a functional channel with significantly delayed desensitization.

We also constructed P2X2a+X3 and P2X2b+X3 chimeric receptors containing the extracellular domain sequence of P2X3R instead of the native sequence. Chimeric channels responded to ATP and alpha, beta-methylene ATP with EC50 values comparable to that of P2X3R. The rates of calcium signal desensitization by P2X2+X3R mutants increased, although not to the level observed in P2X3R-expressing cells. In parallel to the wild-type P2X2aR and P2X2bR, calcium signals generated by P2X2b+X3R desensitized faster than those generated by P2X2a+X3R. Chimeric channels induced calcium signals of the peak amplitudes between those observed in native P2X2Rs and P2X3R. The results indicate that the ligand-binding domain contributes to the control of P2X2R desensitization through a mechanism independent of C-terminal domain-controlled desensitization. Further studies should clarify the generality of this mechanism within the family members.

Differential Expression of Ionic Channels in Pituitary Cells
Van Goor, Zivadinovic, Stojilkovic
Secretory anterior pituitary cells originate from same stem cell-type, but during differentiation they gain the cell type–specific function patterns of spontaneous intracellular calcium signaling and basal hormone secretion. To understand the underlying ionic mechanisms mediating such differences, we compared the ionic channels expressed in somatotrophs, lactotrophs, and gonadotrophs from randomly cycling female rats under identical cell culture and recording conditions. Our results indicate that each cell type expresses a similar series of ionic channels, including transient and sustained voltage-gated calcium channels, tetrodotoxin-sensitive sodium channels, transient and delayed rectifying potassium channels, and multiple calcium-sensitive potassium channel subtypes. However, we observed marked differences in the expression levels of some of the ionic channels. Specifically, lactotrophs and somatotrophs exhibited low expression levels of tetrodotoxin-sensitive sodium channels and high expression levels of the large-conductance, calcium-activated potassium channel compared with those observed in gonadotrophs. In addition, functional expression of the transient potassium channel was much higher in lactotrophs and gonadotrophs than in somatotrophs. Finally, the expression of the transient voltage-gated calcium channels was higher in somatotrophs than in lactotrophs and gonadotrophs. The results indicate that cell type–specific patterns of ionic channel expression may be of physiological significance for the control of calcium homeostasis and secretion in unstimulated and receptor-stimulated anterior pituitary cells.

Dependence of the Pattern of Electrical Activity on Intracellular Calcium
Van Goor, Zivadinovic, Stojilkovic in collaboration with Andrew P. LaBeau and Arthur Sherman, Mathematical Research Branch, NIDDK, and Y-X Li, Department of Mathematics and Zoology, University of British Columbia, Vancouver, Canada
Activation of high conductance, calcium-activated potassium (BK) channels normally limits action potential duration and the associated voltage-gated calcium entry by facilitating membrane repolarization. However, we recently found that BK channel activation in rat pituitary somatotrophs prolonged membrane depolarization, leading to the generation of plateau-bursting activity and facilitated calcium entry. Rapid activation by domain calcium pointed to a paradoxical role for BK channels, which limited the participation of delayed-rectifying amplitude potassium channels during membrane repolarization by truncating the action potential. Conversely, pituitary gonadotrophs expressed relatively few BK channels and fired single spikes with a low capacity to promote calcium entry, whereas elevation in BK current expression in a gonadotroph model-system led to the generation of plateau-bursting activity and high amplitude Ca2+ transients.

The relationship between intracellular calcium, BK channels, and delayed rectifier potassium channels is also tissue-specific. In GnRH-secreting neurons, activation of calcium-mobilizing receptors induced a sustained membrane depolarization that shifted the profile of the action potential waveform from a sharp, high amplitude to broad, low amplitude spikes. We experimentally characterized the shift in the firing pattern and its impact on voltage-gated calcium influx by using the prerecorded sharp and broad action potentials, the latter fired from depolarizing levels as the voltage-clamp command pulse. As a quantitative test of the experimental data, we also used a mathematical model based on the membrane and ionic current properties of GnRH neurons. Both the experimental and modeling results indicated that inactivation of the tetrodotoxin-sensitive sodium channels by sustained depolarization accounted for a reduction in the amplitude of the spike upstroke. The ensuing decrease in tetraethylammonium-sensitive potassium current activation slowed membrane repolarization, leading to the broadening of action potentials. The change in firing pattern increased the total L-type current and facilitated action potential-driven calcium entry. The voltage sensitivity of L-type channels expressed in the neuroendocrine cells enabled the depolarization-induced action potential broadening to facilitate calcium entry despite a decrease in spike amplitude. The findings indicate that the gating properties of L-channels expressed in GT1 neurons are suitable to promote action potential-driven calcium influx in receptor- and nonreceptor-depolarized cells.

Cell Type–Specific Calcium Signaling and Section in Pituitary Cells
Van Goor, Zivadinovic, Stojilkovic
In excitable cells, voltage-gated calcium influx provides an effective mechanism for the activation of exocytosis. In our recent study, we demonstrated that, although rat anterior pituitary lactotrophs, somatotrophs, and gonadotrophs exhibited spontaneous and extracellular calcium-dependent electrical activity, voltage-gated calcium influx triggered secretion only in lactotrophs and somatotrophs. The lack of action potential–driven secretion in gonadotrophs was not attributable to the proportion of spontaneously firing cells or spike frequency. Gonadotrophs exhibited calcium signals during prolonged depolarization that were comparable to those observed in somatotrophs and lactotrophs. The secretory vesicles in all three cell types also had a similar sensitivity to voltage-gated calcium influx. However, the pattern of action potential calcium influx differed among the cell types. Spontaneous activity in gonadotrophs was characterized by high amplitude, sharp spikes that had a limited capacity to promote calcium influx, whereas lactotrophs and somatotrophs fired plateau-bursting action potentials, which generated high amplitude calcium signals. Furthermore, a shift in the pattern of firing from sharp spikes to plateau-like spikes in gonadotrophs triggered LH secretion. The results indicate that the cell type–specific action potential-secretion coupling in pituitary cells is determined by the capacity of their plasma membrane oscillator to generate threshold calcium signals.

Calcium Signaling and Cyclic Nucleotide Production
Andric, Kostic, Tomic, Stojilkovic
We have also addressed the relevance of spontaneous firing of action potentials and calcium signaling on cyclic nucleotide production in pituitary cells, focusing on investigations of soluble guanylyl cyclase (sGC)-mediated cGMP production. This enzyme was expressed in pituitary tissue and dispersed cells, in enriched lactotrophs and somatotrophs, and in immortalized GH pituitary cells and was exclusively responsible for cGMP production in unstimulated cells. Basal sGC activity was partially dependent on voltage-gated calcium influx, and both the neuronal and endothelial calcium-sensitive nitric oxide (NO) synthases (NOS) were expressed in pituitary tissue and mixed cells, in enriched lactotrophs and somatotrophs, and in GH cells. Calcium-independent inducible NOS was transiently expressed in cultured lactotrophs and somatotrophs following the dispersion of cells, but not in GH cells and pituitary tissue. The enzyme participated in the control of basal sGC activity in cultured pituitary cells. The overexpression of inducible NOS by lipopolysaccharide plus interferon-gamma further increased NO and cGMP levels, and most of the cGMP produced de novo was rapidly released. Addition of an NO donor to perifused pituitary cells also led to a rapid cGMP release. Calcium-mobilizing agonists TRH and GnRH slightly increased basal cGMP production, but only when added in high concentrations. In contrast, adenylyl cyclase agonists GHRH and CRF induced a robust increase in cGMP production, with EC50 values in the physiological concentration range. As in cells overexpressing inducible NOS, the stimulatory action of GHRH and CRF was preserved in cells bathed in calcium-deficient medium but was not associated with a measurable increase in NO production. Furthermore, depolarization of cells by high potassium and Bay K 8644, an L-type calcium channel agonist, inhibited sGC activity. Depolarization-induced down-regulation of sGC activity was also observed in cells with inhibited cGMP-dependent phosphodiesterases, but not in cells bathed in Ca2+-deficient medium. This inhibition was independent of the pattern of calcium signaling (oscillatory versus nonoscillatory) and NO levels and was determined by the averaged concentration of intracellular calcium. The results indicate that sGC is present in secretory anterior pituitary cells and is regulated by spontaneous voltage-gated calcium influx through constitutively expressed neuronal and endothelial NOS and transiently expressed inducible NOS as well as independently of NO by adenylyl cyclase coupled-receptors. Calcium stimulates sGC through NOS but also serves as a negative feedback to break the stimulatory action of NO on enzyme activity.