Fredrick Van Goor

Morphological identification of live gonadotropin, growth-hormone, and prolactin cells in goldfish (Carassius auratus) pituitary-cell cultures

To better understand neuroendocrine regulation and the intracellular mechanisms mediating pituitary-hormone release, it is necessary to study the physiology of identified single cells. We have developed a system to identify gonadotropin, growth-hormone, and prolactin cells in primary cultures of goldfish pituitary cells. Using Nomarski differential interference-contrast microscopy, the unique morphologies of discrete subpopulations of cells were characterized. To aid in the initial characterization of different pituitary-cell types, a discontinuous Percoll density-gradient cell-separation technique was developed. This method provided fractions enriched with functional gonadotropin, growth-hormone, and prolactin cells. The morphology of each cell type was initially characterized in enriched fractions of immunofluorescently labelled cells using differential interference-contrast microscopy. The cell type-specific morphologies were then confirmed in live pituitary-cell cultures. Gonadotropin, growth-hormone, and prolactin cells were correctly identified in live pituitary-cell cultures. Gonadotropin, growth-hormone, and prolactin cells were correctly identified in live mixed cultures in 92, 94, and 100% of the trials, respectively. The ability to directly identify cells in primary cultures allows the physiological study of identified single cells with minimal pretreatment.

Influences of Norepinephrine, and Adrenergic Agonists and Antagonists on Gonadotropin Secretion from Dispersed Pituitary Cells of Goldfish, Carassius auratus

Static incubations of dispersed goldfish pituitary cells with 1-100 nM norepinephrine (NE) stimulated gonadotropin (GTH) release. Additions of the alpha-agonist phenylephrine, and the alpha 1-agonist 6-fluoronorepinephrine, but not the alpha 2-agonist clonidine, nor the beta-agonist isoproterenol, also enhanced GTH secretion. The GTH responses to 1 nM NE was significantly inhibited by coincubations with 1 microM of the alpha-antagonist phentolamine, the alpha 1-antagonists prazosine and benoxathian, but not the alpha 2-antagonist yohimbine nor the beta-antagonist propranolol. The GTH responses to NE and phenylephrine were also additive to salmon GTH-releasing hormone (sGnRH)-induced GTH release. These results suggests that NE directly stimulates GTH secretion independent of sGnRH receptors via alpha 1-like adrenergic receptors.

Amplitude-Dependent Spike-Broadening and Enhanced Ca2+ 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.

Relationship between cyclic AMP-stimulated and native gonadotropin-releasing hormone-stimulated gonadotropin release in the goldfish.

The relationship between drugs elevating intracellular cAMP levels and gonadotropin (GTH)-releasing hormone (GnRH) in the stimulation of GTH secretion in the goldfish was investigated using dispersed goldfish pituitary cells in primary culture. In static incubation experiments, activation of adenylyl cyclase by forskolin and the inhibition of cAMP phosphodiesterase by 3 isobutyl-1-methylxanthine (IBMX) increased cAMP release and stimulated GTH secretion. The addition of membrane permeant cAMP analogs, 8-bromoadenosine 3:5-cyclic monophosphate (8Br-cAMP), and dibutyryl cAMP also increased GTH release, suggesting that elevation of cAMP levels can induce GTH secretion. In the goldfish, dopamine is a physiological inhibitor of GTH release. Application of the dopamine agonist apomorphine decreased the GTH responses to forskolin, 8Br-cAMP, and salmon GTH-releasing hormone (sGnRH). The ability of agents that elevate cAMP levels to mimic GnRH action on GTH release suggests that cAMP may mediate GnRH-stimulated GTH secretion in the goldfish; however, this possibility was not substantiated by results from further experiments. In 2-hr static incubation studies, the GTH responses to sGnRH and chicken GnRH-II (cGnRH-II) were enhanced by coincubations with forskolin, IBMX, and 8Br-cAMP. The magnitudes of these enhancements were at least additive, if not synergistic. The levels of cAMP released into the media were unaffected by treatment with sGnRH and cGnRH-II, either in the absence or in the presence of IBMX. Replacement of normal testing media with Ca(2+)-deficient media (without Ca2+ salts and in the presence of 0.1 mM EGTA) decreased sGnRH and cGnRH-II stimulation of GTH release but did not affect forskolin and 8Br-cAMP actions. These results indicate that sGnRH and cGnRH-II stimulation of short term (less than or equal to 2-h) GTH release in the goldfish is not mediated by cAMP. The kinetics of the interactions between sGnRH, forskolin, and IBMX were also investigated in cell column perifusion studies. Applications of 5-min pulses of forskolin and IBMX stimulated rapid increases in GTH release; the latencies of these responses were similar to that observed with sGnRH. The simultaneous applications of sGnRH with either forskolin or IBMX resulted in GTH responses that were of greater magnitude and longer duration than those in response to sGnRH alone. These results together indicate that elevation of cAMP levels can potentiate the GTH response to the native GnRHs by increasing the magnitude of the acute GTH release and by prolonging the duration of GnRH action; however, cAMP does not appear to be involved directly in mediating GnRH stimulation of GTH release.(ABSTRACT TRUNCATED AT 400 WORDS)

GnRH signaling in goldfish pituitary cells.

In goldfish, maturational gonadotropin (GTH) and growth hormone (GH) release are stimulated by two native GTH-releasing hormones (sGnRH and cGnRH-II). Both GnRHs stimulate GTH and GH release via activation of phospholipase C, protein kinase C, Ca2+ entry through voltage-sensitive channels and calmodulin. However, sGnRH-induced GTH release also involves arachidonic acid and intracellular Ca2+ components absent from its action on GH, as well as from cGnRH-II action on GTH and GH secretion. The relative roles and interactions of these signaling pathways in mediating sGnRH and cGnRH-II action on acute and prolonged GTH and GH release are compared. How two GnRHs bind to similar receptors but induce similar and dissimilar transduction mechanisms in two cell types and within one cell type is unknown.

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±0.3u2003nM, 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±1.4u2003nM. 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.

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 10u2003nM salmon (s)GnRH or 10u2003nM 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 (10u2003μM) increased GH release in an extracellular Ca2+‐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 250u2003nM 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.

Calcium Ions as Intracellular Messengers

Calcium acts as both an extracellular (first) and an intracellular (second) messenger to regulate a diverse array of cellular functions, from cell division and differentiation to cell death. Accordingly, Ca2+ concentration in the plasma, as well as in the intercellular and intracellular environment, is under tight neuronal and hormonal control. The availability of Ca2+ for controlling Ca2+ concentration in the plasma and extracellular environment is secured from both diet and the bone matrix, thereby preventing any risk of Ca2+ deprivation. Changes in the extracellular free Ca2+ concentration ([Ca2+]e) signal the activation of Ca2+-sensing receptors, which act to restore basal [Ca2+]e. Although these receptors are found in several tissues, those expressed in parathyroid cells are intimately involved in the control of [Ca2+]e. This chapter focuses on the role of Ca2+ as an intracellular messenger and the pathways that regulate its intracellular concentration. All selected references are review articles that can direct the readers to more specific aspects of Ca2+ signaling. Also, several chapters in this textbook are critical for understanding some of the aspects of Ca2+ signaling. This includes, but is not limited to, chapters on Ca2+ and Ca2+-binding proteinmediated pathways, G protein-coupled receptors, phospholipases, adenylyl cyclases, protein kinase, ion channels, and apoptosis.