Wavelength separation from extreme ultraviolet mirrors using phaseshift reflection.

A generic design and fabrication scheme of Mo/Si multilayer-grating phaseshift reflector systems is reported. Close to optimized extreme ultraviolet (EUV, λ=13.5 nm) reflectance values up to 64% are demonstrated, while the diffractive properties can be exploited in spectral filtering applications. The results can contribute to a wavelength-unspecific solution for the suppression of λ>100 nm out-of-band radiation in EUV lithography.

Generating ultrarelativistic attosecond electron bunches with laser wakefield accelerators.

Femtosecond electron bunches with ultrarelativistic energies were recently generated by laser wakefield accelerators. Here we predict that laser wakefield acceleration can generate even attosecond bunches, due to a strong chirp of the betatron frequency. We show how the bunch duration scales with the acceleration parameters and that, after acceleration, the bunches can propagate over many tens of centimeters without a significant increase in duration.

Interaction of free charged particles with a chirped electromagnetic pulse.

We study the effect of chirp on electromagnetic (EM) pulse interaction with a charged particle. Both the one-dimensional (1D) and 3D cases are considered. It is found that, in contrast to the case of a nonchirped pulse, the charged particle energy can be changed after the interaction with a 1D EM chirped pulse. Different types of chirp and pulse envelopes are considered. In the case of small chirp, an analytical expression is found for arbitrary temporal profiles of the chirp and the pulse envelope. In the 3D case, the interaction with a chirped pulse results in a polarization-dependent scattering of charged particles.

Paradoxical role of large-conductance calcium-activated K+ (BK) channels in controlling action potential-driven Ca2+ entry in anterior pituitary cells.

Activation of high-conductance Ca(2+)-activated K(+) (BK) channels normally limits action potential duration and the associated voltage-gated Ca(2+) entry by facilitating membrane repolarization. Here we report that BK channel activation in rat pituitary somatotrophs prolongs membrane depolarization, leading to the generation of plateau-bursting activity and facilitated Ca(2+) entry. Such a paradoxical role of BK channels is determined by their rapid activation by domain Ca(2+), which truncates the action potential amplitude and thereby limits the participation of delayed rectifying K(+) channels during membrane repolarization. Conversely, pituitary gonadotrophs express relatively few BK channels and fire single spikes with a low capacity to promote Ca(2+) entry, whereas an elevation in BK current expression in a gonadotroph model system leads to the generation of plateau-bursting activity and high-amplitude Ca(2+) transients.

Dependence of pituitary hormone secretion on the pattern of spontaneous voltage-gated calcium influx. Cell type-specific action potential secretion coupling.

In excitable cells, voltage-gated calcium influx provides an effective mechanism for the activation of exocytosis. In this study, we demonstrate 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 due to the proportion of spontaneously firing cells or spike frequency. Gonadotrophs exhibited calcium signals during prolonged depolarization comparable with signals 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 three 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 that generated high amplitude calcium signals. Furthermore, a shift in the pattern of firing from sharp spikes to plateau-like spikes in gonadotrophs triggered luteinizing hormone secretion. These 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.

Differential expression of ionic channels in rat anterior pituitary cells.

Secretory anterior pituitary cells are of the same origin, but exhibit cell type-specific patterns of spontaneous intracellular Ca2+ signaling and basal hormone secretion. To understand the underlying ionic mechanisms mediating these 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 a similar group of ionic channels are expressed in each cell type, including transient and sustained voltage-gated Ca2+ channels, tetrodotoxin-sensitive Na+ channels, transient and delayed rectifying K+ channels, and multiple Ca2+ -sensitive K+ channel subtypes. However, there were marked differences in the expression levels of some of the ionic channels. Specifically, lactotrophs and somatotrophs exhibited low expression levels of tetrodotoxin-sensitive Na+ channels and high expression levels of the large-conductance, Ca2+ -activated K+ channel compared with those observed in gonadotrophs. In addition, functional expression of the transient K+ channel was much higher in lactotrophs and gonadotrophs than in somatotrophs. Finally, the expression of the transient voltage-gated Ca2+ channels was higher in somatotrophs than in lactotrophs and gonadotrophs. These results indicate that there are cell type-specific patterns of ionic channel expression, which may be of physiological significance for the control of Ca2+ homeostasis and secretion in unstimulated and receptor-stimulated anterior pituitary cells.

Characterization of calcium signaling by purinergic receptor-channels expressed in excitable cells.

ATP-gated purinergic receptors (P2XRs) are a family of cation-permeable channels that conduct Ca(2+) and facilitate voltage-sensitive Ca(2+) entry in excitable cells. To study Ca(2+) signaling by P2XRs and its dependence on voltage-sensitive Ca(2+) influx, we expressed eight cloned P2XR subtypes individually in gonadotropin-releasing hormone-secreting neurons. In all cases, ATP evoked an inward current and a rise in [Ca(2+)](i). P2XR subtypes differed in the peak amplitude of [Ca(2+)](i) response independently of the level of receptor expression, with the following order: P2X(1)R < P2X(3)R < P2X(4)R < P2X(2b)R < P2X(2a)R < P2X(7)R. During prolonged agonist stimulation, Ca(2+) signals desensitized with different rates: P2X(3)R > P2X(1)R > P2X(2b)R > P2X(4)R >> P2X(2a)R >> P2X(7)R. The pattern of [Ca(2+)](i) response for each P2XR subtype was highly comparable with that of the depolarizing current, but the activation and desensitization rates were faster for the current than for [Ca(2+)](i). The P2X(1)R, P2X(3)R, and P2X(4)R-derived [Ca(2+)](i) signals were predominantly dependent on activation of voltage-sensitive Ca(2+) influx, both voltage-sensitive and -insensitive Ca(2+) entry pathways equally contributed to [Ca(2+)](i) responses in P2X(2a)R- and P2X(2b)R-expressing cells, and P2X(7)R operated as a nonselective pore capable of conducting larger amounts of Ca(2+) independently on the status of voltage-gated Ca(2+) channels. Thus, Ca(2+) signaling by homomeric P2XRs expressed in an excitable cell is subtype-specific, which provides an effective mechanism for generating variable [Ca(2+)](i) patterns in response to a common agonist.

Amplitude-dependent spike-broadening and enhanced Ca(2+) signaling in GnRH-secreting neurons.

In GnRH-secreting (GT1) neurons, activation of Ca(2+)-mobilizing receptors induces a sustained membrane depolarization that shifts the profile of the action potential (AP) waveform from sharp, high-amplitude to broad, low-amplitude spikes. Here we characterize this shift in the firing pattern and its impact on Ca(2+) influx experimentally by using prerecorded sharp and broad APs as the voltage-clamp command pulse. As a quantitative test of the experimental data, a mathematical model based on the membrane and ionic current properties of GT1 neurons was also used. Both experimental and modeling results indicated that inactivation of the tetrodotoxin-sensitive Na(+) channels by sustained depolarization accounted for a reduction in the amplitude of the spike upstroke. The ensuing decrease in tetraethylammonium-sensitive K(+) current activation slowed membrane repolarization, leading to AP broadening. This change in firing pattern increased the total L-type Ca(2+) current and facilitated AP-driven Ca(2+) entry. The leftward shift in the current-voltage relation of the L-type Ca(2+) channels expressed in GT1 cells allowed the depolarization-induced AP broadening to facilitate Ca(2+) entry despite a decrease in spike amplitude. Thus the gating properties of the L-type Ca(2+) channels expressed in GT1 neurons are suitable for promoting AP-driven Ca(2+) influx in receptor- and non-receptor-depolarized cells.

Signal transduction mechanisms mediating secretion in goldfish gonadotropes and somatotropes.

The intracellular signal transduction mechanisms mediating maturational gonadotropin and somatotropin secretion in goldfish are reviewed. Several major signaling mechanisms, including changes in intracellular [Ca2+], arachidonic acid cascades, protein kinase C, cyclic AMP/protein kinase A, calmodulin, nitric oxide, and Na+/H+ antiport, are functional in both cell types. However, their relative importance in mediating basal secretion and neuroendocrine-factor-regulated hormone release differs according to cell type. Similarly, agonist- and cell-type-specificity are also present in the transduction pathways leading to neuroendocrine factor-modulated maturational gonadotropin and somatotropin release. Specificity is present not only in the actions of different regulators within the same cell type and with the same ligand in the two cell types, but this also exists between isoforms of the same neuroendocrine factor within a single cell type. Other evidence suggests that function-selectivity of signaling may also result from differential modulation of Ca2+ fluxes from different sources. The interaction of different second messenger systems provide the basis by which regulation of maturational gonadotropin and somatotropin release by multiple neuroendocrine factors can be integrated at the target cell level.

Autocrine regulation of calcium influx and gonadotropin-releasing hormone secretion in hypothalamic neurons.

Gonadotropin-releasing hormone (GnRH) receptors are expressed in hypothalamic tissues from adult rats, cultured fetal hypothalamic cells, and immortalized GnRH-secreting neurons (GT1 cells). Their activation by GnRH agonists leads to an overall increase in the extracellular Ca2+-dependent pulsatile release of GnRH. Electrophysiological studies showed that GT1 cells exhibit spontaneous, extracellular Ca2+-dependent action potentials, and that their inward currents include Na+, T-type and L-type Ca2+ components. Several types of potassium channels, including apamin-sensitive Ca2+-controlled potassium (SK) channels, are also expressed in GT1 cells. Activation of GnRH receptors leads to biphasic changes in intracellular Ca2+ concentration ([Ca2+]i), with an early and extracellular Ca2+-independent peak and a sustained and extracellular Ca2+-dependent plateau phase. During the peak [Ca2+]i response, electrical activity is abolished due to transient hyperpolarization that is mediated by SK channels. This is followed by sustained depolarization and resumption of firing with increased spike frequency and duration. The agonist-induced depolarization and increased firing are independent of [Ca2+]i and are not mediated by inhibition of K+ currents, but by facilitation of a voltage-insensitive and store depletion-activated Ca2+-conducting inward current. The dual control of pacemaker activity by SK and store depletion-activated Ca2+ channels facilitates voltage-gated Ca2+ influx at elevated [Ca2+]i levels, but also protects cells from Ca2+ overload. This process accounts for the autoregulatory action of GnRH on its release from hypothalamic neurons.

Expression of purinergic P2X2 receptor-channels and their role in calcium signaling in pituitary cells.

Pituitary cells express purinergic receptor-channels (P2XR), the activation of which by ATP is associated with the facilitation of Ca2+ influx. Pharmacological, RT-PCR, and nucleotide sequence analyses confirm the presence of a wild type P2X2aR and a spliced isoform P2X2bR, which lacks a portion of carboxyl terminal amino acids. Wild type and spliced isoform receptors have a similar EC50 for ATP and time-course for activation, but the spliced isoform exhibits rapid and complete desensitization, whereas the wild type channel desensitizes slowly and incompletely. Deletion and insertion studies have revealed that a 6 residue sequence located in carboxyl tail (Arg371-Pro376) is required for sustained Ca2+ influx through wild type receptors. When co-expressed, the wild type and spliced channels form functional heteropolymeric channels. The patterns of Ca2+ signaling in the majority of pituitary cells expressing ATP-gated receptor-channels are highly comparable to those observed in cells co-transfected with P2X2aR and P2X2bR. ATP-induced [Ca2+]i response in pituitary cells is partially inhibited by nifedipine, a blocker of voltage-gated L-type Ca2+ channels, suggesting that P2X2R not only drive Ca2+ into the cell, but also activate voltage-gated Ca2+ entry. Our results indicate that ATP represents a paracrine and (or) autocrine factor in the regulation of Ca2+ signaling, and that its actions are mediated in part by heteropolymeric P2X2R.

Modeling of membrane excitability in gonadotropin-releasing hormone-secreting hypothalamic neurons regulated by Ca2+-mobilizing and adenylyl cyclase-coupled receptors.

Gonadotropin-releasing hormone (GnRH) secretion from native and immortalized hypothalamic neurons is regulated by endogenous Ca(2+)-mobilizing and adenylyl cyclase (AC)-coupled receptors. Activation of both receptor types leads to an increase in action potential firing frequency and a rise in the intracellular Ca(2+) concentration ([Ca(2+)](i)) and neuropeptide secretion. The stimulatory action of Ca(2+)-mobilizing agonists on voltage-gated Ca(2+) influx is determined by depletion of the intracellular Ca(2+) pool, whereas AC agonist-stimulated Ca(2+) influx occurs independently of stored Ca(2+) and is controlled by cAMP, possibly through cyclic nucleotide-gated channels. Here, experimental records from immortalized GnRH-secreting neurons are simulated with a mathematical model to determine the requirements for generating complex membrane potential (V(m)) and [Ca(2+)](i) responses to Ca(2+)-mobilizing and AC agonists. Included in the model are three pacemaker currents: a store-operated Ca(2+) current (I(SOC)), an SK-type Ca(2+)-activated K(+) current (I(SK)), and an inward current that is modulated by cAMP and [Ca(2+)](i) (I(d)). Spontaneous electrical activity and Ca(2+) signaling in the model are predominantly controlled by I(d), which is activated by cAMP and inhibited by high [Ca(2+)](i). Depletion of the intracellular Ca(2+) pool mimics the receptor-induced activation of I(SOC) and I(SK), leading to an increase in the firing frequency and Ca(2+) influx after a transient cessation of electrical activity. However, increasing the activity of I(d) simulates the experimental response to forskolin-induced activation of AC. Analysis of the behaviors of I(SOC), I(d), and I(SK) in the model reveals the complexity in the interplay of these currents that is necessary to fully account for the experimental results.

Two endogenous gonadotropin-releasing hormones generate dissimilar Ca(2+) signals in identified goldfish gonadotropes.

Ca(2+) signals are involved in the signal transduction of neuroendocrine regulators. In goldfish, two endogenous gonadotropin-releasing hormones, salmon (s)GnRH and chicken (c)GnRH-II, control maturational gonadotropin secretion. Although considerable evidence suggests that sGnRH and cGnRH-II exert their activity on goldfish gonadotropes through a single population of receptors, differences in signal transduction mechanisms between these peptides have been demonstrated. We used ratiometric Fura-2 Ca(2+) imaging of single morphologically identified gonadotropes to quantitatively compare the Ca(2+) signals evoked by sGnRH and cGnRH-II. The amplitude and the rate of rise of sGnRH- and cGnRH-II-evoked Ca(2+) signals increased with concentration. At maximal concentrations, Ca(2+) signals generated by cGnRH-II rose significantly faster than those elicited by sGnRH, while other parameters such as the maximum amplitude, average Ca(2+) increase, and latency did not differ between the two peptides. Ca(2+) signals evoked by sGnRH or cGnRH-II were often spatially restricted to one part of the cell over the duration of the response. We provide a comprehensive account of the spatial and temporal aspects, including calculated kinetics, of GnRH-evoked Ca(2+) signals in single identified gonadotropes. This is the first report of quantified differences in Ca(2+) signals generated by two endogenous GnRH neuropeptides, which may act through the same receptor population in this cell type.

Expression of Ca(2+)-mobilizing endothelin(A) receptors and their role in the control of Ca(2+) influx and growth hormone secretion in pituitary somatotrophs.

The expression and coupling of endothelin (ET) receptors were studied in rat pituitary somatotrophs. These cells exhibited periods of spontaneous action potential firing that generated high-amplitude fluctuations in cytosolic calcium concentration ([Ca(2+)](i)). The message and the specific binding sites for ET(A), but not ET(B), receptors were found in mixed pituitary cells and in highly purified somatotrophs. The activation of these receptors by ET-1 led to an increase in inositol 1,4,5-trisphosphate production and the associated rise in [Ca(2+)](i) and growth hormone (GH) secretion. The Ca(2+)-mobilizing action of ET-1 lasted for 2-3 min and was followed by an inhibition of action potential-driven Ca(2+) influx and GH secretion to below the basal levels. As in somatostatin-treated cells, the ET-1-induced inhibition of spontaneous electrical activity and Ca(2+) influx was accompanied by the inhibition of adenylyl cyclase and by the stimulation of inward rectifier potassium current. In contrast to somatostatin, ET-1 did not inhibit voltage-gated Ca(2+) channels. During prolonged agonist stimulation a gradual recovery of Ca(2+) influx and GH secretion occurred. In somatotrophs treated with pertussis toxin overnight, the ET-1-induced Ca(2+)-mobilizing phase was preserved, but it was followed immediately by facilitated Ca(2+) influx and GH secretion. Both somatostatin- and ET-1-induced inhibitions of adenylyl cyclase activity were abolished in pertussis toxin-treated cells. These results indicate that the transient cross-coupling of Ca(2+)-mobilizing ET(A) receptors to the G(i)/G(o) pathway in somatotrophs provides an effective mechanism to change the rhythm of [Ca(2+)](i) signaling and GH secretion during continuous agonist stimulation.

Control of action potential-driven calcium influx in GT1 neurons by the activation status of sodium and calcium channels.

An analysis of the relationship between electrical membrane activity and Ca2+ influx in differentiated GnRH-secreting (GT1) neurons revealed that most cells exhibited spontaneous, extracellular Ca(2+)-dependent action potentials (APs). Spiking was initiated by a slow pacemaker depolarization from a baseline potential between -75 and -50 mV, and AP frequency increased with membrane depolarization. More hyperpolarized cells fired sharp APs with limited capacity to promote Ca2+ influx, whereas more depolarized cells fired broad APs with enhanced capacity for Ca2+ influx. Characterization of the inward currents in GT1 cells revealed the presence of tetrodotoxin-sensitive Na+, Ni(2+)-sensitive T-type Ca2+, and dihydropyridine-sensitive L-type Ca2+ components. The availability of Na+ and T-type Ca2+ channels was dependent on the baseline potential, which determined the activation/inactivation status of these channels. Whereas all three channels were involved in the generation of sharp APs, L-type channels were solely responsible for the spike depolarization in cells exhibiting broad APs. Activation of GnRH receptors led to biphasic changes in cytosolic Ca2+ concentration ([Ca2+]i), with an early, extracellular Ca(2+)-independent peak and a sustained, extracellular Ca(2+)-dependent phase. During the peak [Ca2+]i response, electrical activity was abolished due to transient hyperpolarization. This was followed by sustained depolarization of cells and resumption of firing of increased frequency with a shift from sharp to broad APs. The GnRH-induced change in firing pattern accounted for about 50% of the elevated Ca2+ influx, the remainder being independent of spiking. Basal [Ca2+]i was also dependent on Ca2+ influx through AP-driven and voltage-insensitive pathways. Thus, in both resting and agonist-stimulated GT1 cells, membrane depolarization limits the participation of Na+ and T-type channels in firing, but facilitates AP-driven Ca2+ influx.

Coordinate regulation of gonadotropin-releasing hormone neuronal firing patterns by cytosolic calcium and store depletion.

Elevation of cytosolic free Ca2+ concentration ([Ca2+]i) in excitable cells often acts as a negative feedback signal on firing of action potentials and the associated voltage-gated Ca2+ influx. Increased [Ca2+]i stimulates Ca2+-sensitive K+ channels (IK-Ca), and this, in turn, hyperpolarizes the cell and inhibits Ca2+ influx. However, in some cells expressing IK-Ca the elevation in [Ca2+]i by depletion of intracellular stores facilitates voltage-gated Ca2+ influx. This phenomenon was studied in hypothalamic GT1 neuronal cells during store depletion caused by activation of gonadotropin-releasing hormone (GnRH) receptors and inhibition of endoplasmic reticulum (Ca2+)ATPase with thapsigargin. GnRH induced a rapid spike increase in [Ca2+]i accompanied by transient hyperpolarization, followed by a sustained [Ca2+]i plateau during which the depolarized cells fired with higher frequency. The transient hyperpolarization was caused by the initial spike in [Ca2+]i and was mediated by apamin-sensitive IK-Ca channels, which also were operative during the subsequent depolarization phase. Agonist-induced depolarization and increased firing were independent of [Ca2+]i and were not mediated by inhibition of K+ current, but by facilitation of a voltage-insensitive, Ca2+-conducting inward current. Store depletion by thapsigargin also activated this inward depolarizing current and increased the firing frequency. Thus, the pattern of firing in GT1 neurons is regulated coordinately by apamin-sensitive SK current and store depletion-activated Ca2+ current. This dual control of pacemaker activity facilitates voltage-gated Ca2+ influx at elevated [Ca2+]i levels, but also protects cells from Ca2+ overload. This process may also provide a general mechanism for the integration of voltage-gated Ca2+ influx into receptor-controlled Ca2+ mobilization.

Functional role of alternative splicing in pituitary P2X2 receptor-channel activation and desensitization.

Although ATP-gated ion channel (P2XR) expression is high among anterior pituitary cells, identification of the receptor subtypes and their selective expression within subpopulations of cell types, as well as their physiological role(s), are incompletely characterized. In this study, we focused on the expression and activity of the P2X2R subtype in anterior pituitary cells. Our results indicate that the primary P2X2R gene transcript in pituitary cells undergoes extensive alternative splicing, with generation of six isoforms. Two of these isoforms encode functional channels when expressed in GT1 or HEK293 cells: the wild-type P2X2R and the spliced isoform P2X2-2R, which lacks a stretch of carboxyl-terminal amino acids (Val370-Gln438). Four other clones showed different alterations, including an interfered reading frame starting in the first transmembrane domain and a 27-amino acid deletion in the large extracellular loop. When expressed separately or in combination with wild-type channels, these clones were nonfunctional. In single cell Ca2+ current and cytosolic Ca2+ concentration ([Ca2+)i) measurements, the P2X2R and P2X2-2R had similar EC50 values for ATP and time courses for activation and recovery from desensitization but differed significantly in their desensitization rates. The spliced isoform exhibited rapid and complete desensitization, whereas the wild-type channel desensitized slowly and incompletely. The mRNAs for wild-type and spliced channels were identified in enriched somatotroph, but not gonadotroph or lactotroph fractions. Expression of a functional ATP-gated channel in somatotrophs was confirmed by the ability of ATP to increase the frequency of [Ca2+]i spikes in spontaneously active cells or initiate spiking in quiescent cells. When voltage-gated Ca2+ influx was blocked, ATP increased [Ca2+]i, with a similar profile and EC50 to those observed in GT1 cells heterologously expressing wild-type or spliced P2X2R. The ligand-selectivity profile of native channels was consistent with the presence of P2X2R in somatotrophs. Finally, the desensitization rate of P2X2R in a majority of somatotrophs was comparable to that observed in neurons coexpressing wild-type and spliced channels. These data indicate that alternative splicing of P2X2R and coexpression of P2X2R and P2X2-2R subunits provide effective mechanisms for controlled cationic influx in somatotrophs.

Dopamine-D2 actions on voltage-dependent calcium current and gonadotropin-II secretion in cultured goldfish gonadotrophs.

Dopamine D2-receptor activation directly inhibits GnRH-induced gonadotropin-II (maturational gonadotropin, GTH-II) secretion from goldfish pituitary cells. In this study, we show that dopamine and its D2 agonist, quinpirole, reduced GTH-II secretion induced by either high extracellular K+ concentration or the voltage-gated Ca2+ channel agonist, Bay K 8644. These actions of dopamine were blocked by addition of the dopamine D2-receptor antagonist, spiperone. The actions of dopamine on Ca2+ current in single identified goldfish gonadotrophs were assessed in voltage-clamp experiments using Ba2+ as the charge carrier through voltage-gated Ca2+ channels. Dopamine caused a concentration-dependent reduction in Ba2+ current amplitude with an EC50 of 1.0+/-0.3 nM, but did not shift the current-voltage relationship. The D2 agonist quinpirole also caused a dose-dependent reduction in the Ba2+ current amplitude with an EC50 of 2.7+/-1.4 nM. Quinpirole slowed the activation and inactivation kinetics, as well as removing the steady-state inactivation properties of the Ba2+ current. In contrast to the actions of quinpirole, the dopamine D1-receptor agonist, SKF 38393, did not affect the Ba2+ current. The inhibitory action of dopamine on voltage-dependent Ca2+ currents was reversed by spiperone, but not by the D1 antagonist SKF 83566. Voltage-dependent Na+ and K+ currents were not affected by dopamine or dopamine agonists. These data indicate that dopamine D2-receptor activation reduces Ca2+ influx through voltage-dependent Ca2+ channels to inhibit GTH-II secretion.

Uncoupling of calcium mobilization and entry pathways in endothelin-stimulated pituitary lactotrophs.

In cells expressing Ca2+-mobilizing receptors, InsP3-induced Ca2+ release from intracellular stores is commonly associated with extracellular Ca2+ influx. Operation of these two Ca2+ signaling pathways mediates thyrotropin-releasing hormone (TRH) and angiotensin II (AII)-induced prolactin secretion from rat pituitary lactotrophs. After an initial hyperpolarization induced by Ca2+ mobilization from the endoplasmic reticulum (ER), these agonists generated an increase in the steady-state firing of action potentials, further facilitating extracellular Ca2+ influx and prolactin release. Like TRH and AII, endothelin-1 (ET-1) also induced a rapid release of Ca2+ from the ER and a concomitant spike prolactin secretion during the first 3-5 min of stimulation. However, unlike TRH and AII actions, Ca2+ mobilization was not coupled to Ca2+ influx during sustained ET-1 stimulation, as ET-1 induced a long-lasting abolition of action potential firing. This lead to a depletion of the ER Ca2+ pool, a prolonged decrease in [Ca2+]i, and sustained inhibition of prolactin release. ET-1-induced inhibition and TRH/AII-induced stimulation of Ca2+ influx and hormone secretion were reduced in the presence of the L-type Ca2+ channel blocker, nifedipine. Basal [Ca2+]i and prolactin release were also reduced in the presence of nifedipine. Furthermore, TRH-induced Ca2+ influx and secretion were abolished by ET-1, as TRH was unable to reactivate Ca2+ influx and prolactin release in ET-1-stimulated cells. Depolarization of the cells during sustained inhibitory action of ET-1, however, increased [Ca2+]i and prolactin release. These results indicate that L-type Ca2+ channel represents a common Ca2+ influx pathway that controls basal [Ca2+]i and secretion and is regulated by TRH/AII and ET-1 in an opposite manner. Thus, the receptor-mediated uncoupling of Ca2+ entry from Ca2+ mobilization provides an effective control mechanism in terminating the stimulatory action of ET-1. Moreover, it makes electrically active lactotrophs quiescent and unresponsive to other calcium-mobilizing agonists.

Extracellular sodium dependence of GnRH-stimulated growth hormone release in goldfish pituitary cells.

In goldfish, gonadotropin-releasing hormone (GnRH) stimulation of growth hormone (GH) release has been shown to involve extracellular Ca2+ entry through voltage-sensitive Ca2+ channels and the activation of protein kinase C (PKC). In this study, the possible involvement of extracellular Na+ in mediating the GH response to GnRH was examined using dispersed pituitary cells. Perifusion with Na(+)-depleted medium reversibly reduced the acute GH response to 5-min pulses of either 10 nM salmon (s)GnRH or 10 nM chicken (c)GnRH-II. Similarly, replacement of normal medium with Na(+)-depleted medium attenuated the long-term GH release response to sGnRH and cGnRH-II under static incubation conditions. These results suggest that GnRH-induced GH release requires the presence of extracellular Na+. Treatment with 5-min pulses of the Na(+)-channel agonist veratridine (10 microM) increased GH release in an extracellular Ca(2+)-dependent manner, presumably due to activation of voltage-sensitive Ca2+ channels resulting from the depolarizing effect of increased Na+ influx. On the other hand, Na+ entry through tetrodotoxin (TTX)-sensitive, voltage-dependent Na+ channels is not involved in GnRH-induced GH release. Application of 250 nM TTX, which abolished the voltage-sensitive Na+ currents in identified goldfish somatotropes, did not affect the acute GH responses to 5-min pulses of sGnRH and cGnRH-II. The possible participation of Na+/H+ antiport in mediating the extracellular Na(+)-dependent GnRH action on GH release was then examined. In static incubation experiments, sGnRH- and cGnRH-II-induced GH secretion were reduced by inhibitors of the Na+/H+ antiport, amiloride and dimethylamiloride (DMA). Likewise, the GH response to the PKC activator, tetradecanoyl phorbol acetate, was attenuated by treatment with Na(+)-depleted medium, amiloride, and DMA. The inhibitory actions of amiloride and DMA were selective as these drugs did not affect the GH response elicited by the Ca2+ ionophore ionomycin and the voltage-sensitive Ca2+ channel agonist, Bay K 8644. Taken together, these results indicate that extracellular Na+ and the Na+/H+ exchanger are involved in the mediation of GnRH-stimulated GH release in goldfish. Furthermore, this dependence on Na+ and Na+/H+ antiport probably occurs distal to the activation of PKC by GnRH.