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 voltagegated 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.Although anterior pituitary secretory cells are derived from the same progenitor cells, they differ with respect to their secretory patterns in vitro and in vivo. In vitro, basal prolactin (PRL) 1 and growth hormone (GH) secretion from pituitary fragments, dispersed pituitary cells, and immortalized lacto-somatotrophs is high and is dependent on the extracellular calcium concentration (1-4). In contrast, basal luteinizing hormone (LH) secretion is low and not dependent on the extracellular calcium concentration (1). In vivo, animals bearing ectopic pituitary grafts release high levels of PRL and low levels of LH for a prolonged period, leading to pseudo-pregnancy (5). Because of the high levels of basal GH and PRL secretion, it is not surprising that lactotrophs and somatotrophs are under negative hypothalamic control by G i/o -coupled dopamine and somatostatin receptors, in addition to positive control by Ca 2ϩ -mobilizing and G s -coupled receptors, such as GH-releasing hormone and thyrotropin-releasing hormone receptors. On the other hand, LH secretion from gonadotrophs is under positive hypothalamic control by Ca 2ϩ -mobilizing receptors, including gonadotropin-releasing hormone (GnRH) and endothelin-A, but no inhibitory hypothalamic factor has been identified (6, 7).It is not known what endows lactotrophs and somatotrophs, but not gonadotrophs, with the ability to secrete high levels of hormone in the absence of any stimuli. One possibility is that lactotrophs and somatotrophs fire spontaneous action potentials (APs) that are capable of driving ...

Introduction We examined the participation of a membrane form of estrogen receptor (mER)-α in the activation of mitogenactivated protein kinases (extracellular signal-regulated kinase [ERK]1 and ERK2) related to cell growth responses in MCF-7 cells.

Introduction 17β-estradiol (E 2 ) can rapidly induce cAMP production, but the conditions under which these cAMP levels are best measured and the signaling pathways responsible for the consequent proliferative effects on breast cancer cells are not fully understood. To help resolve these issues, we compared cAMP mechanistic responses in MCF-7 cell lines selected for low (mER low ) and high (mER high ) expression of the membrane form of estrogen receptor (mER)-α, and thus addressed the receptor subform involved in cAMP signaling.

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.

To characterize the contribution of interleukin-6 (IL-6) to spinal cord injury pain (SCIP), we employed a clinically relevant rat contusion model of SCIP. Using Western blots, we measured IL-6 levels in lumbar segments (L1-L5), at the lesion site (T10), and in the corresponding lumbar and thoracic dorsal root ganglia (DRG) in 2 groups of similarly injured rats: (a) SCI rats that developed hind-limb mechanical allodynia (SCIP), and (b) SCI rats that did not develop SCIP. Only in SCIP rats did we find significantly increased IL-6 levels. Immunocytochemistry showed elevated IL-6 predominantly in reactive astrocytes. Our data also showed that increased production of IL-6 in hyperreactive astrocytes in SCIP rats may explain still-poorly understood astrocytic contribution to SCIP. To test the hypothesis that IL-6 contributes to mechanical allodynia, we treated SCIP rats with neutralizing IL-6 receptor antibody (IL-6-R Ab), and found that one systemic injection abolished allodynia and associated weight loss; in contrast to gabapentin, the analgesic effect lasted for at least 2weeks after the injection, despite the shorter presence of the Ab in the circulation. We also showed that IL-6-R Ab partially reversed SCI-induced decreases in the protein levels of the glutamate transporter GLT-1 12hours and 8days after Ab injection, which may explain the lasting analgesic effect of the Ab in SCIP rats. A link between reactive astrocytes IL-6-GLT-1 has not been previously shown. Given that the humanized IL-6-R Ab tocilizumab is Food and Drug Administration-approved for rheumatoid arthritis, we are proposing tocilizumab as a novel and potentially effective treatment for SCIP.

In excitable cells, oscillations in intracellular free

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 2ϩ ] 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 2ϩ ] i and growth hormone (GH) secretion. The Ca 2ϩ -mobilizing action of ET-1 lasted for 2-3 min and was followed by an inhibition of action potential-driven Ca 2ϩ 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 2ϩ 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 2ϩ channels. During prolonged agonist stimulation a gradual recovery of Ca 2ϩ influx and GH secretion occurred. In somatotrophs treated with pertussis toxin overnight, the ET-1-induced Ca 2ϩ -mobilizing phase was preserved, but it was followed immediately by facilitated Ca 2ϩ 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 2ϩ -mobilizing ET A receptors to the G i /G o pathway in somatotrophs provides an effective mechanism to change the rhythm of [Ca 2ϩ ] i signaling and GH secretion during continuous agonist stimulation. Key words: somatotrophs; growth hormone; calcium; endothelin; somatostatin; electrical activityAction potential (AP)-driven C a 2ϩ influx through voltage-gated calcium channels (VGCC s) is operative in various endocrine and neuroendocrine cells, including pituitary somatotrophs. Because many different ionic channels act in concert to control AP firing, this pathway is referred to as the membrane potential (V m )-dependent pathway for C a 2ϩ signaling (Stojilkovic, 1998). Several of the ionic channels contributing to the V m pathway in somatotrophs have been identified. These include VGCCs, voltage-gated sodium channels, pacemaker and ATP-gated cationic channels, and several types of potassium channels, including inwardly rectif ying potassium channels (K ir ) (Lewis et al., 1988;Sims et al., 1991;Brake et al., 1994;Koshimizu et al., 1998). Although resting somatotrophs have a fluctuating V m , Kwiecien and colleagues (1997) have found that these pacemaker fluctuations were unable to initiate spontaneous APs in a majority of cultured somatotrophs. Consequently, they suggested that somatotrophs behave as "conditional pacemakers," because the activation of adenylyl cyclase-coupled receptors is required for AP generation. However, others have observed spontaneous APs (Sims et al., 1991) a...

Although malfunction of spinal cord water channels (aquaporins, AQP) likely contributes to severe disturbances in ion/water homeostasis after spinal cord injury (SCI), their roles are still poorly understood. Here we report and discuss the potential significance of changes in the AQP4 expression in human SCI that generates GFAP-labeled astrocytes devoid of AQP4, and GFAP-labeled astroglia that overexpress AQP4.We used a rat model of contusion SCI to study observed changes in human SCI. AQP4-negative astrocytes are likely generated during the process of SCI-induced replacement of lost astrocytes, but their origin and role in SCI remains to be investigated. We found that AQP4-overexpression is likely triggered by hypoxia. Our transcriptional profiling of injured rat cords suggests that elevated AQP4-mediated water influx accompanies increased uptake of chloride and potassium ions which represents a protective astrocytic reaction to hypoxia. However, unbalanced water intake also results in astrocytic swelling that can contribute to motor impairment, but likely only in milder injuries. In severe rat SCI, a low abundance of AQP4-overexpressing astrocytes was found during the motor recovery phase. Our results suggest that severe rat contusion SCI is a better model to analyze AQP4 functions after SCI. We found that AQP4 increases in the chronic post-injury phase are associated with the development of pain-like behavior in SCI rats, while possible mechanisms underlying pain development may involve astrocytic swelling-induced glutamate release. In contrast, the formation and size of fluid-filled cavities occurring later after SCI does not appear to be affected by the extent of increased AQP4 levels. Therefore, the effect of therapeutic interventions targeting AQP4 will depend not only on the time interval after SCI or animal models, but also on the balance between protective role of increased AQP4 in hypoxia and deleterious effects of ongoing astrocytic swelling.Corresponding Author: Olivera Nesic -Taylor, Ph.D., Department of BMB, University of Texas Medical Branch, MRB 7.138G, 301 University Blvd., Galveston, TX, 77555-1072, Tel: (409) Fax: (409) 772-8028, Olnesic@utmb.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptNeuroscience. Author manuscript; available in PMC 2011 July 28. Published in final edited form as:Neuroscience. I. Why study Aquaporins (AQPs) in spinal cord injury (SCI)?It is estimated that there are 2.5 million people worldwide living with a SCI, and that 130,000 new injuries occur each year (Thuret et al., 2006...