Biomedical subjects

J I Goldberg

Publications and source records attributed to J I Goldberg.

At least 19 recordsLinked to original sources

PubMed2001

Laser ablation reveals regulation of ciliary activity by serotonergic neurons in molluscan embryos.

Early in embryonic development, the pond snail Helisoma trivolvis exhibits a rotational behavior that is generated by beating of cilia in the dorsolateral and pedal bands. Although previous anatomical and pharmacological studies provided indirect evidence that a pair of serotonergic neurons, Embryonic Neurons C1 (ENC1s), is involved in regulating embryonic rotation, direct evidence linking ENC1 to ciliary function is still lacking. In the present study, we used laser microbeams to perturb ENC1 in vivo while monitoring ciliary activity in identified ciliary bands. A laser treatment protocol to specifically ablate ENC1 without damaging the surrounding cells was established. Unilateral laser treatment of ENC1 caused transient increases in the activity of the pedal and ipsidorsolateral cilia, lasting 30-50 min. In contrast, activity of cilia that were not anatomically associated with ENC1 was unaffected by laser treatment. Mianserin, an effective serotonin antagonist in Helisoma ciliated cells, decreased the overall CBF of pedal and dorsolateral cilia by reducing the occurrence of spontaneous CBF surges in these cilia. Finally, the cilioexcitatory action of ENC1 laser treatment was mimicked by serotonin and reduced in the presence of mianserin. These results suggest that laser treatment provokes a release of serotonin from ENC1, resulting in a prolonged elevation of activity in the target ciliary cells. We conclude that, in addition to their previously established role in regulating neurodevelopment, ENC1s also function as serotonergic motor neurons to regulate ciliary activity, and therefore the rotational behavior of early embryos.

Animals↗

PubMed2000

Agonist-specific Ca2+ signaling systems, composed of multiple intracellular Ca2+ stores, regulate gonadotropin secretion.

Ca2+ signals regulate many cellular functions, including hormone secretion. Agonist-specific Ca2+ signaling may arise from the differential mobilization of multiple Ca2+ stores. Although they act through the same receptor subtype, two gonadotropin-releasing hormones (sGnRH and cGnRH-II) generate quantifiably different Ca2+ signals in goldfish gonadotropes, suggesting that their Ca2+-dependent signaling cascades may differ. We combined electrophysiology, Ca2+ imaging, and radioimmunoassay detection of gonadotropin (GTH-II) secretion to determine the role of intracellular Ca2+ stores in GnRH-stimulated exocytosis. Our findings suggest that voltage-gated Ca2+ channels do not mediate acute GnRH-signaling. Instead, both sGnRH- and cGnRH-II-stimulated GTH-II releases are dependent on Ca2+ mobilized from TMB-8/CPA-sensitive compartments. However, sGnRH, but not cGnRH-II, utilizes intracellular stores sensitive to caffeine and xestospongin C. We also identified a homeostatic mechanism where reduced extracellular Ca2+ availability increase GTH-II release by mobilizing Ca2+ stores. Our results are the first to suggest that several classes of intracellular Ca2+ stores differentially participate in agonist signaling and homeostasis in gonadotropes.

Animals↗

PubMed2000

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.

Animals↗

PubMed1999

Involvement of protein kinase C in 5-HT-stimulated ciliary activity in Helisoma trivolvis embryos.

1. During development, embryos of the pulmonate gastropod, Helisoma trivolvis, undergo a rotation behaviour due to the co-ordinated beating of three bands of ciliated epithelial cells. This behaviour is in part mediated by the neurotransmitter serotonin (5-HT) released from a pair of identified embryonic neurons. Using time-lapse videomicroscopy to measure ciliary beat frequency (CBF) in response to pharmacological manipulations, we determined whether protein kinase C (PKC) is involved in mediating 5-HT-stimulated ciliary beating. 2. Diacylglycerol (DAG) analogues sn-1,2-dioctanoyl glycerol (DiC8; 100 microM) and 1-oleoyl-2-acetyl-sn-glycerol (OAG; 100 microM), partially mimicked the 5-HT-induced increase in CBF. In contrast, application of OAG in the absence of extracellular Ca2+ did not result in an increase in CBF. 3. 5-HT-stimulated CBF was effectively blocked by PKC inhibitors bisindolylmaleimide (10 and 100 nM) and calphostin C (10 nM). In addition, bisindolylmaleimide (100 nM) inhibited DiC8-induced increases in CBF. At a higher concentration (200 nM), bisindolylmaleimide did not significantly reduce 5-HT-stimulated cilio-excitation. 4. Two different phorbol esters, phorbol 12-myristate 13-acetate (TPA; 0.1, 10 or 1000 nM) and phorbol 12beta, 13alpha-dibenzoate (PDBn; 10 microM) did not alter basal CBF. TPA (1 microM) did not alter 5-HT-stimulated CBF. Likewise, the synthetic form of phosphatidylserine, N-(6-phenylhexyl)-5-chloro-1-naphthalenesulphonamide (SC-9; 10 microM), did not increase CBF, whereas a strong increase in CBF was observed upon exposure to 5-HT. 5. The results suggest that a DAG-dependent, phorbol ester-insensitive isoform of PKC mediates 5-HT-stimulated CBF in ciliated epithelial cells from embryos of Helisoma trivolvis.

Animals↗

PubMed1999

Gonadotropin-releasing hormone neuronal system of the freshwater snails Helisoma trivolvis and Lymnaea stagnalis: possible involvement in reproduction.

Peptides of the gonadotropin-releasing hormone (GnRH) family are present in neural and nonneural tissues throughout the chordate phylum. Although GnRH peptides have been implicated in nonreproductive functions, their primary function is to control reproduction by regulating sexual behaviors and inducing gonadotropin hormone release from the pituitary. Evidence suggesting the presence of a similar peptide in the central nervous system (CNS) of the gastropod mollusc Helisoma trivolvis has recently been provided. In the present study, we examined the tissue distribution of the peptide and found that it is likely restricted to the nervous system. The neuronal system containing the endogenous GnRH-like peptide is described further and is shown, in part, to innervate the male reproductive tract. Immunostaining in the closely related snail, Lymnaea stagnalis, showed a conservation in the locations of some immunoreactive neurons. Notably, staining occurred in and adjacent to the lateral lobes of both snails. Because these lobes contain neurons involved in the stimulation of egg laying and GnRH staining occurred in additional areas in the Helisoma CNS that are involved in reproduction, we suggest that the endogenous GnRH-like peptide plays a role in regulating reproduction in freshwater snails.

Animals↗

PubMed1999

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.

Animals↗

PubMed1999

Treatment of the macrophage-like P388D.1 cells with bacterial lipopolysaccharide and interferon-gamma causes long-term alterations in calcium metabolism.

The intracellular calcium concentrations ([Ca2+]i) of P338D.1 macrophage-like cells, activated with interferon-gamma (IFN-gamma) and/or bacterial lipopolysaccharide (LPS) were determined using fura-2/AM and ratiometric imaging techniques. Treatment of macrophages with IFN-gamma and LPS resulted in significant downward shift in [Ca2+]i, 8, 16 and 24 h but not at 1 and 4 h after treatment. The decrease in [Ca2+]i also occurred when macrophages were treated with LPS only, but not after exposure of the cells to recombinant IFN-gamma, indicating that LPS was an essential signal in the observed changes in [Ca2+]i of activated macrophages. The IFN-gamma and/or LPS alteration in the [Ca2+]i, paralleled the in vitro nitric oxide production of the activated macrophages, 8, 16 and 24 h after treatment. The decrease in the [Ca2+]i may be caused by vigorous buffering and storing of Ca2+ by macrophages to below the normal resting quantities, following the reported transient increase in Ca2+ during the priming stage of macrophage activation. Thus, the downward shift in [Ca2+]I may play a physiological role in the activation processes of macrophages for antimicrobial responses.

Animals↗

PubMed1998

Early development of an identified serotonergic neuron in Helisoma trivolvis embryos: serotonin expression, de-expression, and uptake.

In early-stage embryos of Helisoma trivolvis, a bilateral pair of identified neurons (ENC1) express serotonin and project primary descending neurites that ramify in the pedal region of the embryo prior to the formation of central ganglia. Pharmacological studies suggest that serotonin released from ENC1 acts in an autoregulatory pathway to regulate its own neurite branching and in a paracrine or synaptic pathway to regulate the activity of pedal ciliary cells. In the present study, several key features of early ENC1 development were characterized as a necessary foundation for further experimental studies on the mechanisms underlying ENC1 development and its physiological role during embryogenesis. ENC1 morphology was determined by confocal microscopy of serotonin-immunostained embryos and by differential-interference contrast (DIC) microscopy of live embryos. The soma was located at an anteriolateral superficial position and contained several distinguishing features, including a large spherical nucleus with prominent central nucleolus, large granules in the apical cytoplasm, a broad apical dendrite ending in a sensory-like structure at the embryonic surface, and a ventral neurite. ENC1 first expressed serotonin immunoreactivity around stage E13, followed immediately by the appearance of an immunoreactive neurite (stage E14). Both the intensity of immunoreactivity and primary neurite length were consistently greater in the right ENC1 at early stages. Serotonin uptake, as indicated by 5,7-dihydroxytryptamine-induced fluorescence, first occurred between stages E18 and E25. At later stages of embryogenesis (after stage E65), serotonin immunoreactivity disappeared, whereas serotonin uptake and normal cell morphology were retained.

Animals↗

PubMed1998

Induction of nitric oxide (NO) synthesis in murine macrophages requires potassium channel activity.

The activation of macrophages for antimicrobial responses is a multistage event involving numerous intracellular signalling cascades that makes possible target cell destruction by these effector cells. This study examined the effects of different potassium channel inhibitors and activators on the NO production of murine macrophage-like cell lines P388D.1 and B10-4(S). We found that the potassium channel inhibitors tetraethylammonium, 4-aminopyridine, and quinine caused dose-dependent reductions in the NO production of macrophages, and that the potassium channel activator, minoxidol, caused a dose-dependent enhancement of NO production. The inhibition of NO production was due to involvement of potassium channels in the priming stage of macrophage activation, since pretreatment with the priming agent interferon-gamma partially restored the NO response of the macrophages. The results of this study demonstrate a link between potassium channel activity and the activation of anitimicrobial functions of murine macrophages.

4-Aminopyridine↗

PubMed1998

Serotonin regulation of neurite outgrowth in identified neurons from mature and embryonic Helisoma trivolvis.

Neurite outgrowth and growth cone motility are among the many aspects of neuronal development that can be affected by specific neurotransmitters. This was first demonstrated in experiments on identified molluscan neurons that were isolated from mature ganglia and cultured under conditions that promote the regeneration of new neurites. The application of serotonin to a regenerating Helisoma neuron B19 produced an abrupt, reversible cessation of neurite outgrowth and growth cone motility. While this type of response would subsequently be demonstrated for other neurons and neurotransmitters in many different invertebrate and vertebrate species, experiments on Helisoma neurons have continued to play a pivotal role in advancing this field. In this paper, the mechanisms and sites of serotonin action and how these responses are manifested in vivo during embryonic development are discussed. Experiments primarily on neuron B19 have shown that serotonin acts on a novel serotonin receptor that is coupled to the elevation of cyclic AMP. This intracellular messenger directly activates a class of cyclic-nucleotide-gated sodium channels, leading to sodium influx, membrane depolarization, and activation of voltage-gated calcium channels. The resulting elevation of intracellular calcium acts through a calcium/calmodulin-dependent pathway to inhibit neurite outgrowth and growth cone motility. Although the final steps have yet to be completely resolved, they undoubtedly involve calcium-dependent regulation of cytoskeletal components. Regarding the sites of serotonin action, serotonin responses have been localized to growth cones and even filopodia in specific neurons. However, some studies suggest that neurite development may actually be regulated by serotonin in a paracrine, non-localized manner in a surprisingly large percentage of Helisoma neurons. Finally, experiments on Helisoma embryos have investigated how serotonin actually regulates the in vivo development of specific neurons. Pharmacological treatments that reduce the serotonin concentration in embryos affected the neurite morphology and synaptic efficacy of neuron B19 and the amount of neurite branching in embryonic neuron C1. All of these responses were consistent with the primary action of serotonin being the inhibition of neurite outgrowth, as predicted by the original cell culture studies.

Animals↗

PubMed1998

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.

Animals↗

PubMed1997

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.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1996

Involvement of extracellular sodium in agonist-induced gonadotropin release from goldfish (Carassius auratus) gonadotrophs.

In goldfish, gonadotropin (GTH-II) responses to the two endogenous GnRHs, salmon-GnRH and chicken-GnRH-II, are mediated by activation of protein kinase C (PKC) and voltage-sensitive Ca2+ channels. In this study, we investigated the role of extracellular Na+, voltage-dependent Na+ channels, and the plasma membrane Na+/H+ exchanger in mediating GnRH-stimulated GTH-II release from dispersed goldfish pituitary cells. Perifusion with Na+-depleted medium reduced the GTH-II response to both GnRHs and the response to the protein kinase C activator, phorbol 12-myristate 13-acetate. Conversely, increasing Na+ influx with veratridine (100 microM) stimulated GTH-II release in the presence and in the absence of extracellular Ca2+. However, the voltage-sensitive Na+ channel blocker, tetrodotoxin (1 microM), did not affect GnRH- stimulated GTH-II release, and the GnRHs did not affect voltage-sensitive Na+ currents. In contrast, the Na+/H+ antiport inhibitors, amiloride or its analog, DMA, reduced GTH-II responses to the GnRHs and phorbol 12-myristate 13-acetate. The Na+/H+ antiport inhibitors did not affect voltage-sensitive Ca2+ or Na+ currents or the GTH-II release response to the Ca2+ ionophore, ionomycin. These findings indicate that extracellular Na+ and the Na+/H+ exchanger are involved in the mediation of GnRH-stimulated GTH-II release. In addition, Na+ entry may modulate GTH-II release independent of extracellular Ca2+.

Animals↗

PubMed1996

Electrical membrane properties and ionic currents in cultured goldfish gonadotrophs.

The electrical membrane properties and ionic currents of cultured identified goldfish gonadotrophs were characterized using whole-cell patch-clamp recordings. Under current-clamp recording conditions, goldfish gonadotrophs had a resting membrane potential of approximately -60 mV and an input resistance of 8.2 +/- 1.6 G omega. In response to depolarizing current injections, one or more action potentials could be induced from resting membrane potential or more hyperpolarized holding potentials. In addition, some cells exhibited spontaneous action potential activity. Under voltage-clamp recording conditions, several outward and inward currents were isolated and characterized using pharmacological and ionic substitution studies. The outward currents included a fast-activating, transient current and an inactivation-resistant current similar to IA-type and delayed rectifier currents, respectively. Unlike mammalian gonadotrophs, apamin-sensitive K+ currents were not detected. Inward currents included a fast-activating and inactivating Na+ current and a high-voltage activated, dihydropyridine-sensitive Ca2+ current. The value for half-maximal steady-state inactivation of the Na+ and Ca2+ currents was -50.8 and -16.7 mV, respectively, indicating that a significant proportion of both Na+ and Ca2+ channels are available for activation at the resting membrane potential of these cells.

Animals↗

PubMed1995

Neurite branch development of an identified serotonergic neuron from embryonic Helisoma: evidence for autoregulation by serotonin.

Previous studies have shown that in select neurons, neurite outgrowth can be regulated by the same neurotransmitter that is synthesized and released by those neurons. However, it is not known whether such an autoregulatory mechanism is utilized during the normal course of nervous system development in either invertebrates or vertebrates. In the present study, we tested this hypothesis on the first pair of identified serotonergic neurons to be expressed in embryos of the pulmonate gastropod, Helisoma trivolvis. Embryonic neurons C1 (ENC1) elaborate a stereotyped pattern of neurite outgrowth prior to the differentiation of subsequent serotonergic neurons. Embryos were treated with either p-chlorophenylalanine (pCPA) or 5-hydroxytryptophan (5-HTP) to lower or raise embryonic serotonin content, respectively. High-performance liquid chromatography with electrochemical detection was used to measure the effects of these treatments on serotonin content, and serotonin immunohistochemistry was carried out to quantify the extent of neurite outgrowth of ENC1. Embryonic serotonin content was significantly reduced at both 24 and 48 hr after treatment with 0.02% pCPA, whereas dopamine levels were unchanged. Although the proximal neurite outgrowth of ENC1 appeared unaffected by the pCPA treatment at both of these time points, the distal outgrowth in the target cell region appeared more profuse. This effect on outgrowth was quantified by counting the number of neurite branch points, which was significantly increased both 24 and 48 hr after pCPA treatment. In contrast, 5-HTP treatment resulted in an increase in embryonic serotonin content and a significant decrease in the number of ENC1 branch points. Treatment with dopamine had no effect on the pattern of ENC1 neurite outgrowth. Together, these results support the hypothesis that a neuron may utilize its own transmitter in an autoregulatory fashion to regulate neurite formation during embryonic development.

5-Hydroxytryptophan↗

PubMed1994

Pharmacological characterization of a serotonin receptor involved in an early embryonic behavior of Helisoma trivolvis.

In contrast to the abundance of information on the many physiological and developmental actions of serotonin in molluscan nervous systems, comparatively little is known about the serotonin receptors involved in these responses. Embryos of the pulmonate gastropod, Helisoma trivolvis, display a cilia-driven rotational behavior that is regulated by endogenous serotonin. In the present study, two functional assays were used to determine some of the pharmacological properties of the receptors that mediate the cilio-excitatory action of serotonin. Time-lapse video microscopy was used to measure whole embryo rotation rate and cilia beat frequency in isolated cells. In dose-response experiments, serotonin was approximately 10 times more potent in stimulating cilia beat frequency over embryo rotation. In rotation experiments, 5-carboxyamidotryptamine and methysergide had effective agonist activity in dose ranges similar to that of serotonin (1 to 100 microM). In contrast, 8-hydroxydipropylaminotetralin HBr (8-OH-DPAT) displayed agonist activity of lower potency and effectiveness. Several compounds displayed antagonist activity in the 1 to 100 microM dose range, including mianserin, spiperone, ritanserin, 1-(1-naphthyl)piperazine, and propranolol. alpha-Methylserotonin had mixed agonist-antagonist activity, and metoclopramide, MDL-72222, and ketanserin were inactive. Experiments on isolated cells suggested that the extremely effective antagonism displayed by mianserin in the embryo rotation assay was due to its specific activity at ciliary serotonin receptors. These results implicate the presence of a novel serotonin receptor on embryonic ciliated cells that is pharmacologically distinct from those previously characterized in vertebrate or invertebrate systems.

Animals↗

PubMed1993

Presence and biological activity of a GnRH-like factor in the nervous system of Helisoma trivolvis.

Gonadotropin-releasing hormone (GnRH) constitutes a family of neuropeptides found throughout the vertebrates. Although a GnRH-like peptide has also been isolated from yeast (alpha-mating factor), the presence of GnRH has not been clearly demonstrated in invertebrate phyla. In this study, we tested the hypothesis that GnRH-like peptides are present and functional in the central nervous system (CNS) of the gastropod mollusc, Helisoma trivolvis. The presence of a GnRH-like peptide was examined by three methods: (1) in immunofluorescence studies with four different antibodies generated against several GnRH peptides, select neurons and putative neurosecretory cells were specifically and consistently labelled throughout the CNS; (2) reverse-phase high performance liquid chromatography (HPLC) and radioimmunoassay (RIA) analysis revealed a GnRH-like factor which co-migrates with mammalian (m)GnRH; and (3) in bioactivity experiments, extracts of Helisoma trivolvis CNS mimicked GnRH in stimulating gonadotropin release from dispersed goldfish pituitary cells in static culture. Two functional assays were carried out to examine the potential biological roles of GnRH-like peptides in Helisoma. (1) Intracellular recordings of left-parietal and visceral ganglion neurons revealed diverse electrophysiological responses to mGnRH. These effects were attenuated by a mGnRH antagonist. (2) Addition of mGnRH arrested neurite outgrowth in a subpopulation of dissociated embryonic Helisoma neurons in culture. Taken together, these results strongly suggest that a mGnRH-like peptide is an important neuropeptide in Helisoma. A hypothesis is presented that GnRH-like peptides may be ancient factors that are conserved both structurally and functionally in the evolution of animals.

Animals↗

PubMed1993

Voltage-activated ionic currents in goldfish pituitary cells.

The release of gonadotropin and growth hormone from goldfish pituitary cells has been shown to be dependent on the entry of extracellular Ca2+ through voltage-sensitive Ca2+ channels by pharmacological studies. As a first step to further investigate the involvement of voltage-dependent ion channels in the regulation of anterior pituitary hormone release in the goldfish, cell excitability and voltage-dependent ion currents were characterized using tight-seal whole-cell recordings in dispersed goldfish pituitary cells. Cultured goldfish pituitary cells had an average membrane potential of -36 +/- 3 mV. When held at membrane potentials more negative than -60 mV, these cells were excitable, responding to depolarizing current pulses or anode break with the firing of single action potentials. Results from total current voltage-clamp recordings suggested that all goldfish pituitary cells possess voltage-dependent Na+, Ca2+, and K+ currents. These currents were further characterized independently under isolated current recording conditions. The rapid, transient Na+ current activated at voltages more positive than -40 mV and was sensitive to tetrodotoxin. The steady state inactivation of this Na+ current was also voltage-dependent; at the measured resting potential, > or = 50% of the Na+ current was not available for activation. The voltage-dependence and activation kinetics of the tetraethylammonium-sensitive K+ current resembled those of the delayed rectifier K+ current. The K+ current activated slowly at potentials more positive than -40 mV, and showed little inactivation over the duration of a 1-sec depolarizing pulse. Steady-state inactivation characteristics indicated that < 50% of the K+ current was inactivated at resting potentials. Experiments with 4-aminopyridine indicated the presence of an early transient K+ current that activated in a similar voltage range as the delayed rectifier current. Using barium as the charge carrier to measure Ca2+ currents, a high-voltage activated, long-lasting Ca2+ current was revealed. This "L-type" Ca2+ current activated at potentials more positive than -30 mV and was inhibited by verapamil and nifedipine. This study indicates that goldfish pituitary cells possess the electrophysiological properties required for the participation of voltage-sensitive ion channels in the regulation of hormone release.

4-Aminopyridine↗