help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Xu, R.
Right arrow Articles by Chen, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Xu, R.
Right arrow Articles by Chen, C.
Endocrinology Vol. 143, No. 12 4609-4619
Copyright © 2002 by The Endocrine Society

ARTICLE

Orexin-A Augments Voltage-Gated Ca2+ Currents and Synergistically Increases Growth Hormone (GH) Secretion with GH-Releasing Hormone in Primary Cultured Ovine Somatotropes

Ruwei Xu, Qinling Wang, Ming Yan, Maria Hernandez, Changhong Gong, Wah Chin Boon, Yoko Murata, Yoichi Ueta and Chen Chen

Prince Henry’s Institute of Medical Research (R.X., Q.W., M.Y., M.H., C.G., W.C.B., Y.M., C.C.), Clayton, Victoria 3168, Australia; Department of Physiology, Monash University (R.X., C.C.), Clayton, Victoria 3168, Australia; and Department of Physiology, University of Occupational and Environmental Health (Y.U.), Yahatanishi-ku, 807-8555, Kitakyushu, Japan

Address all correspondence and requests for reprints to: Dr. Chen Chen, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail: chen.chen{at}med.monash.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Orexins are recently discovered neuropeptides that play an important role in the regulation of hormone secretion, and their receptors have been recently demonstrated in the pituitary. The effects of orexin-A on voltage-gated Ca2+ currents and GH release in primary cultured ovine somatotropes were examined. The expression of orexin-1 receptor was demonstrated by RT-PCR in ovine somatotropes, from which Ca2+ currents were also isolated as L, T, and N currents. Application of orexin-A (100 nM) significantly and reversibly increased only the L current, and coadministration of orexin-A and GHRH (10 nM) showed an additive effect on this current, but no effect of orexin-A was observed on either T or N current. Furthermore, the orexin-A-induced increase in the L current was completely abolished by the inhibition of protein kinase C (PKC) activity using calphostin C (100 nM), phorbal 12,13-dibutyrate pretreatment (0.5 µM) for 16 h or specific PKC inhibitory peptide PKC19–36 (1 mM). However, the increase in L current by orexin-A was sustained when cells were preincubated with a specific protein kinase A blocker H89 (1 µM) or a specific intracellular Ca2+ store depleting reagent thapsigargin (1 µM). Finally, orexin-A alone did not significantly increase GH release, but coadministration of orexin-A and GHRH showed a synergistic effect on GH secretion in vitro. Our results therefore suggest that orexin-A may play an important role in regulating GHRH-stimulated GH secretion through the enhancement of the L-type Ca2+ current and the PKC-mediated signaling pathway in ovine somatotropes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OREXIN-A AND -B (also known as hypocretin-1 and hypocretin-2) are two novel hypothalamic peptides identified by two independent groups that act through two subtypes of receptors (orexin 1-R and orexin 2-R) belonging to the G protein-coupled receptor superfamily (1, 2). Orexin-A is a peptide with 33 amino acids; orexin-B has 28 amino acids. There is 46% sequence homology between the two. Orexin 1-R is strongly selective for orexin-A, whereas orexin 2-R has equal affinity for orexin-A and orexin-B (2). It has been well established that orexins play a significant role in the control of energy homeostasis, e.g. food intake and sleep-wake cycle (3, 4). The wide distribution of orexin peptides, receptors, and their neuronal projections in the central nervous system and pituitary revealed in recent studies has strongly suggested that the physiological functions of orexins reach much beyond the above activities (4, 5, 6). One of such important features of orexins is the control of hormonal secretion. Published studies have shown that orexins strongly and specifically activate both hypothalamus-pituitary-adrenal and hypothalamus-pituitary-gonadal axes and therefore influence the secretion of a number of hormones, including ACTH, LH, corticosterone, and PRL, both in vivo and in vitro (3, 7, 8, 9, 10, 11). In addition, the stimulatory effects of orexins on the secretion of insulin and TSH have been reported (12, 13).

The presence of orexin-A neuronal projections and its receptors in the pituitary in different species, including humans, indicates that orexins may regulate pituitary hormone secretion (14, 15). However, the role of orexin-A in the regulation of an important anabolic hormone in the pituitary, GH, is not clear. There have been studies of the possible regulatory effects of orexins on GH secretion in vivo and in vitro, but the results are still limited and controversial (3, 16). Given that GH is integrally linked to energy homeostasis and metabolism and is closely related to the sleep-wake cycle, we proposed that orexins may play a role in the regulation of GH secretion by modifying the membrane electrical properties, e.g. ion channels, in somatotropes. In fact, modification of ion channels in somatotropes is one of the most important mechanisms by which GH secretion is regulated by hypothalamic peptides, including GHRH, GH-releasing peptide (GHRP), and somatostatin (17, 18). This hypothesis is also strongly supported by several lines of evidence. First, orexins and their receptors were recently discovered in the anterior lobe of the pituitary in both rats and humans, indicating a possible paracrine or autocrine effect on pituitary hormones, including GH (14, 15, 19). Second, orexins have been shown to change the electrophysiological activities in a number of neuronal and nonneuronal cells (20, 21). For example, orexins have been recently demonstrated to induce depolarization in vagus neurons and reduce the K+ currents in macrophages (22, 23). Finally, activation of orexin receptors may cause Ca2+ influx, and orexins may regulate hormone secretion through the adenylate cyclase signaling pathway (24, 25, 26).

The existence of Ca2+ channels is ubiquitous in all neuroendocrine cells. Upon appropriate stimulation, the movement of Ca2+ ions across the cell membrane through Ca2+ channels not only transfers depolarizing charge into excitable cells, but also initiates specific intracellular signaling to be decoded by Ca2+-binding proteins, which subsequently leads to the operation of their diverse functions. It has long been recognized that voltage-gated Ca2+ channels play a key role in the control of GH secretion from the pituitary (11, 17, 27). Previous studies have shown that the modification of other channels, including K+, Na+, Cl-, and cation channels, may also contribute to the depolarization of the somatotrope in different species (28, 29, 30). Among these channels, voltage-gated Ca2+ channels are particularly important, because the Ca2+ influx through these channels contributes predominantly to the elevation of the intracellular calcium concentration ([Ca2+]i) in somatotropes, leading to GH release (29, 31, 32). In ovine somatotropes, it has been shown that GHRH and GHRPs significantly influence GH secretion by modifying T- and L-type currents through activation of the adenylate cyclase-dependent cascade or protein kinase C (PKC) (17, 33).

In present study we tested the effect of orexin-A because it exhibits a stronger influence on the biosynthesis and secretion of anterior pituitary hormones than orexin-B (2, 7). To investigate the possible role of orexin-A in the regulation of GH secretion and the correlated mechanisms, we first studied the effects of orexin-A on the L-type Ca2+ current. After the response was confirmed, the interaction between orexin-A and GHRH in the modification of L current was examined. We also assessed the influence of these two peptides on GH secretion by measuring the GH concentration after cultured somatotropes were treated with GHRH, orexin-A, or a combination of both. Furthermore, the involvement of second messenger systems in orexin-A-induced effects on Ca2+ channels was determined.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
Orexin-A was purchased from American Peptide Co. (Sunnyvale, CA) and Peptide Institute (Osaka, Japan). GHRH1–44 was obtained from Auspep (Parkville, Australia). DMEM, HEPES, and carbohydrate solutions were purchased from Trace Biosciences Pty. Ltd. (Noble Park, Australia). Medium 199 and collagenase type I were obtained from Worthington Biochemical Corp. (Freehold, NJ). Tissue culture reagents (deoxyribonuclease, hyaluronidase, trypsin inhibitor, and pancreatin), sera, ATP, creatine phosphokinase, phosphocreatine, nefidipine (NFD), {omega}-conotoxin (CTX), and all general salts for recording solutions and molecular biological studies were purchased from Sigma (St. Louis, MO) or as otherwise specified in the text. Tetrodotoxin (TTX) was purchased from Alomone Laboratories (Jerusalem, Israel). L-Glutamine was purchased from Life Technologies, Inc. (Gaithersburg, MD). PKC inhibitory peptides (PKC19–36) and phorbal 12,13-dibutyrate (PDBu) were obtained from Research Chemicals International (Natick, MA). H89 and calphostin C (Cal-C) were obtained from Calbiochem (Alexandria, Australia).

Preparation and culture of ovine somatotropes
Adult sheep pituitary glands were collected from a local abattoir and then subjected to collagenase/pancreatin treatments to dissociate the cells as described previously (34). Briefly, whole pituitaries were divested of encapsulating neurohypophysis and pituitary stalk tissues. The anterior pituitaries were then minced and placed in calcium-free PBS with BSA. The tissue fragments were gently washed and incubated with deoxyribonuclease, hyaluronidase, trypsin inhibitor, pancreatin and collagenase (~3 mg/ml) for 30–40 min at 37 C in a shaking bath. After centrifugation at 1500 rpm, the cells were suspended in the medium 199 and optimally counted under a microscope. Cell yield was normally 3 x 107/pituitary gland, with more than 90% viability (trypan blue exclusion test). The cell suspension (3–5 ml) was placed, under sterile conditions, above a layer of column of increasing density Percoll solutions. In our experimental conditions, seven Percoll dilutions were prepared for a discontinuous density gradient as follows: 1.10, 1.074, 1.071, 1.068, 1.063, 1.058, 1.040, and 1.029 g/ml. This Percoll gradient was further calibrated using density marker beads (Pharmacia Biotech, Uppsala, Sweden). Finally, a top layer of suspended cells was loaded gently over the Percoll gradient. Tubes then were centrifuged (J6-HC centrifuge, Beckman, Palo Alto, CA) at 2500 rpm for 30 min at 4 C without a brake, and it was found that about 80% of cells in fractions 2 and 3, with density ranging from 1.063–1.071, were somatotropes after immunocytochemistry studies were performed (34). Cells were then removed from each layer, and the fractions with the most somatotropes were subsequently seeded into 35-mm culture dishes for electrophysiological studies. Six- and 48-well plates were used to culture cells for molecular biological studies (RNA extraction and further RT-PCR) and incubation experiments (GH RIA). Cells were grown in DMEM supplemented with 10% fetal calf serum and 1% (vol/vol) L-glutamine (200 mM) in a humidified incubator (37 C, 5% CO2). The culture medium was changed every 2–3 d, and electrophysiological recordings were performed after 4–10 d in culture.

RNA isolation and RT-PCR
As the expression of orexin receptors was high in most regions in the brain (35), we chose rat brain tissue as a positive control. Sprague Dawley rats were purchased from Monash University Central Animal Services. All animal experiments were approved by the ethical committee of Monash Medical Center (Melbourne, Australia). A total of two rats were anesthetized with pentobarbital (100 mg/kg, ip) and killed by decapitation. Brains were removed immediately and frozen quickly in liquid nitrogen. The total RNA from rat brain was extracted using RNeasy Mini Kits fitted with a built-in column (QIAGEN, Hilden, Germany). Total RNA from cells cultured in petri dishes (~2 x 106 cells/dish) was isolated using the methods previously described (36). Briefly, culture medium from the culture dishes was removed, and cells were washed with ice-cold PBS. Lysis buffer containing 2% sodium dodecyl sulfate, 200 mM Tris-Cl, and 0.5 mM EDTA was added at about 50 µl/cm2. After incubation for approximately 2 min, the lysate was transferred to a sterile microfuge tube, and 150 µl potassium acetate solution (50 g potassium acetate, 11 ml glacial acetic acid, and water to 100 ml) were added. The tube was incubated on ice for 3–5 min and centrifuged at maximum speed in a microcentrifuge. The supernatant containing the RNA was further extracted with a mixture of chloroform/isomyl alcohol (24:1). The upper phase was removed and reextracted. The RNA was finally precipitated with an equal volume of ice-cold isopropanol on ice for 20–30 min and then pelleted after centrifugation and washing with 70% ethanol. One microgram of total RNA extracted from each cell culture well was treated with deoxyribonuclease I (Roche, Indianapolis, IN) to eliminate possible contamination of genomic DNA.

One microgram of RNA from rat brain or ovine somatotropes was then reverse transcribed to cDNA in a 20-µl reverse transcription (RT) reaction system containing random primers and avian myeloblastosis virus reverse transcriptase (Roche). The RT reactions were carried out at 46 C for 2 h. One microliter of the RT reaction products was used for subsequent PCR amplification for 35 cycles. Primer for ovine orexin-1 receptor was designed according to the rat cDNA templates with an expected size of 310 bp (37). The set of primers for the amplification of the orexin-1 receptor was 5'-AATCGCACACGGCTCTTCTCTGTC (sense) and 5'-CACCATCAGCATCTTGGCAGTC (antisense). We used a denaturation step at 95 C for 1 min, an annealing step at 56 C for 1 min, and an extension step at 72 C for 1 min for a total of 35 cycles, followed by an additional extension step at 72 C for 10 min. Detection of PCR amplification products was carried out by electrophoresis (1.5% agarose gel containing ethidium bromide). The PCR products were extracted from the gel and sequenced in an automated DNA sequencer. Finally, the sequenced data were analyzed using Blast Nucleic Acid Database Searches from the National Center for Biotechnology Information.

Electrophysiological recording
On the day of the recording, culture medium was replaced by patch-clamp bath solution through a gravity pressure perfusion system at least 10 min before recording. Transmembrane Ca2+ currents were recorded using the gigaseal patch clamp technique in classic whole cell recording configuration. Electrodes were pulled by a Sutter P-87 microelectrode puller (Sutter Instrument Co., Novato, CA) from borosilicate micropipettes with inner filament and had an initial input resistance of 2–5 M{Omega}. All recordings were made using the Axopatch 200A amplifier (Axon Instruments, Foster City, CA). The bath solution was composed of the following: 40 mM tetraethyl ammonium chloride, 90 mM NaCl, 5 mM CaCl2, 0.5 mM MgCl2, 10 mM glucose, and 10 mM HEPES (pH 7.4, adjusted with NaOH; osmolarity of 300 mosmol/liter with sucrose). To exclude the contamination of Na+ current, TTX was added to the bath solution with a final concentration of 1 µM on the day of experimentation. The pipette solution was composed of the following: 120 mM CsAsp, 20 mM tetraethyl ammonium chloride, 10 mM EGTA, 10 mM glucose, and 10 mM HEPES. Just before recording, an ATP regenerative system (2 mM ATP, 5 mM Na2-phosphocreatine, and 20 U/ml creatine phosphokinase) plus 0.1 mM GTP were added to the pipette solution (pH adjusted to 7.4 and osmolarity to 300 mosmol/liter). PKC19–36 was also included in the pipette solution in studies of the involvement of the PKC pathway in orexin-A-induced effects in ovine somatotropes.

After obtaining the high resistance seal, the pipette potential was held to -80 mV, and voltage pulses (10 mV, 200-msec duration) were delivered periodically to monitor access resistance. Access to the cell interior was judged by the appearance of a membrane capacitative transient current after a gentle, but quick, suction under our experimental condition. Whole cell capacitance (7.9 ± 0.3 pF; n = 79) and series resistance were compensated (~80%) before experimentation, and leak current was routinely subtracted using the option offered by the Clampex 7.0 program (Axon Instruments). We also monitored the change in series resistance over the course of each experiment, and recordings with significant change in series resistance were terminated or excluded from the final data analysis. The signals were filtered at 2 kHz, and the sweeps were sampled at 1-msec intervals in our recording protocols.

Cell culture dishes were fixed on the stage of an Olympus Corp. inverted microscope (New Hyde Park, NY), and a gravity pressure system was used to perfuse the cells at a rate of approximately 1 ml/min. Protein kinase A (PKA)-cAMP and PKC blockers, including H89 and Cal-C, were added by hand to the culture dishes containing the cells to be recorded. Recordings started at least 5 min after these blockers were added to achieve an even distribution of these chemicals (the concentrations cited in Results are the final concentrations when diluted in the bath solution). Orexin-A was mixed with bath solution and applied by gravity pressure perfusion system. Application of vehicle from the same system did not change Ca2+ currents. All experiments were performed at room temperature (20–22 C).

Incubation procedures and RIA
Before each incubation experiment, the cells were washed three times with incubation medium (medium 199 containing 0.1% BSA) and then preincubated for 1 h with incubation medium as during the equilibration period. The medium was subsequently discarded after 1-h incubation, and 2 ml/well fresh incubation medium, with or without the test substances, were added for 30 min. Parallel incubations containing a solvent of drugs alone were included in the incubation as a control. At the end of the incubation period, the conditioned medium was collected and stored at –20 C before the GH RIA.

The concentration of GH in the incubation medium was measured in a double antibody RIA using kits provided by the National Hormone and Pituitary Program, NIH (ovine GH and ovine GH antisera). All samples were assayed in duplicate. The sensitivity of the assay was 0.3 ng/ml. The inter- and intraassay coefficients of variation were less than 15% and 8%, respectively (n = 4). All samples from one experiment were measured in the same assay, and GH values were expressed as nanogram equivalents of ovine GH standard.

Data analysis
A pCLAMP 7.0 software (Axon Instruments) was used to acquire and analyze the data. A paired t test was used as appropriate to evaluate the statistical significance of differences between two group means of currents, and the effects were considered significant at P < 0.05. Group data are expressed as the mean ± SEM in Results. The traces in the figures are representative of at least four recordings under the same experimental conditions or as indicated otherwise in the text. GH data in incubation studies are also presented as the mean ± SEM. Statistical comparisons were made by t test, and significance was taken as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Presence of orexin-1 receptor in purified ovine pituitary cells
RT-PCR and sequence analysis demonstrated that orexin 1-R was strongly expressed in both ovine somatotropes and rat brain tissue. As shown in Fig. 1Go, no amplification was detected in the negative control, i.e. without RT, hence confirming that the PCR product did not come from genomic DNA contamination. The PCR product was extracted, and a partial sequence for ovine pituitary cells was obtained. The ovine sequence showed 87% and 89% homology to rat and human orexin-1 receptor genes, respectively.



View larger version (55K):
[in this window]
[in a new window]
  		Figure 1. Expression of orexin-1 receptor in ovine somatotropes. Ethidium bromide-stained 1.5% agarose gel showing cDNA amplified with orexin 1-R receptor primer from RNA of ovine anterior pituitary cells (predominantly somatotropes) and rat brain tissue. The left lane was loaded with molecular marker (Roche). The size of amplified fragments was 310 bp. No amplification was detectable in the negative control, i.e. when avian myeloblastosis virus reverse transcriptase was omitted in the RT reaction mix.

 
Characterization of Ca2+ currents in ovine somatotropes
Voltage-gated calcium channels have been reported as L, T, N, P/Q, and R subtypes in different cell types (38). In ovine somatotropes, a significant part of L-type Ca2+ and a small part of transient Ca2+ currents were demonstrated when the cells were recorded in the bath solution containing 2.5 mM Ca2+, 1 µM TTX, and internal CsCl (39). In this study we used an L-type current blocker NFD and a specific N-type Ca2+ current blocker CTX to systemically examine the components of voltage-gated Ca2+ currents under different holding potentials. Figure 2Go shows a group of recordings from one cell for such a characterization. The total Ca2+ currents shown in Fig. 2AaGo were evoked by depolarizing test pulses from –50 mV up to +20 mV with a holding potential of –80 mV. NFD (10 µM) reduced about 70% of the total Ca2+ current (Fig. 2AbGo), suggesting the presence of a large portion of L-type current (comparing the difference between Fig. 2AGo, a and b). The rest of the Ca2+ currents were further suppressed by addition of the N current blocker CTX (1 µM; comparing the difference between Fig. 2AGo, b and c). The current remaining was mostly the T-type according to its specific activation and inactivation characteristics (Fig. 2AcGo). This T current is quickly activated and inactivated within 100 msec, a kinetic characteristic allowing it to be easily distinguished from P/Q currents, whose inactivation is much slower (40). While using a holding potential of –30 mV to exclude T and N currents, the total currents were predominantly the L-type. Addition of NFD suppressed almost all of the total current (comparing Fig. 2BGo, a and b). Further addition of CTX exerted only a very minor effect on the current (comparing Fig. 2BGo, b and c). In comparison, the NFD-sensitive L currents under the two holding potentials were similar (127 pA in Fig. 2AGo vs. 124 pA in Fig. 2BGo).



View larger version (18K):
[in this window]
[in a new window]
  		Figure 2. Characterization of Ca2+ currents in ovine somatotropes using different holding potentials and specific Ca2+ blockers. A, Voltage-gated Ca2+ currents were evoked by depolarizing test pulses from a holding potential of –80 mV with a 10-mV interval. Aa, Ca2+ currents evoked by depolarizing test pulses up to +20 mV (as indicated in the lower panel). Ab, Ca2+ currents evoked by depolarizing test pulses up to +20 mV in the presence of the L-type Ca2+ current blocker NFD (10 µM). Ac, Ca2+ currents evoked by depolarizing test pulses up to +20 mV in the presence of both NFD and the N-type Ca2+ current blocker CTX (1 µM). B, Voltage-gated Ca2+ currents were evoked by depolarizing test pulses with a 10-mV interval from a holding potential of –30 mV. Ba, Ca2+ currents evoked by depolarizing test pulses up to +20 mV (as indicated in the lower panel). Bb, Ca2+ currents evoked by depolarizing test pulses to +20 mV in the presence of NFD. Bc, Ca2+ currents evoked by depolarizing test pulses to +20 mV in the presence of both NFD and CTX.

 
The current-voltage (I-V) curves revealed three distinctive subtypes of Ca2+ currents recorded under holding potentials of –80 and –30 mV, respectively (Fig. 3Go, A and B). These curves were made using the peak values measured from each of the Ca2+ current traces. The currents were determined manually using a mobile cursor, because the time points where the peak values of the currents were taken in different test potentials were changing. Under the holding potential of –80 mV, the largest amplitudes of total currents were detected at +10 mV. These total currents were significantly reduced and shifted 10–20 mV toward left upon inhibition of L or L and N currents (Fig. 3AGo). However, the peak values of the isolated L current were obtained at the depolarizing pulse of +20 mV under the holding potential of –30 mV (Fig. 3BGo).



View larger version (18K):
[in this window]
[in a new window]
  		Figure 3. Current-voltage (I-V) relationships of three subtypes of Ca2+ currents in ovine somatotropes. Currents measured at the peak of each trace. I-V curves with different holding potentials correspond to the recording conditions in Fig. 2Go, A and B, respectively (mean ± SEM; n = 4). Note that under the holding potential of –80 mV (A), total Ca2+ current ({diamondsuit}), T-plus N-type ({blacksquare}), and T-type currents (indicated as {blacktriangleup}) were recorded, whereas under –30 mV (B), the majority of the current was L-type ({diamondsuit}).

 
Effect and time course of orexin-A on Ca2+ currents
To obtain a stable Ca2+ current lasting for at least 15 min for the following designed experimentation, an ATP regenerative system comprised of ATP (2 mM), Na2-phosphocreatine (5 mM), creatine phosphokinase (20 U/ml), and GTP (0.1 mM) was included in the pipette solution, and it was demonstrated that this inclusion did not alter Ca2+ or K+ currents (41, 42). We first examined the effect of orexin-A on the total Ca2+ current. Figure 4AGo demonstrates the peak Ca2+ currents in controls (a) and after addition of orexin-A for 5 min (b), and the recovery of Ca2+ currents after orexin-A was washed away (c). Application of orexin-A significantly and reversibly increased voltage-gated Ca2+ currents. The stimulatory effect of orexin-A on total Ca2+ currents was also demonstrated in the I-V curves (Fig. 4BGo). To exclude the systemic variations, we performed the entire procedure using bath solution as a placebo. The statistical data indicate that orexin-A significantly and reversibly increased Ca2+ currents, whereas no influence from the perfusion system was evident (Fig. 4CGo). The time course of the response to orexin-A is also shown in Fig. 5Go. The stimulatory effect of orexin-A on the Ca2+ currents took place gradually and reached a maximum in about 4 min. The complete recovery of Ca2+ currents required approximately 4–6 min after the removal of orexin-A. Meanwhile, no significant "rundown" of Ca2+ currents was observed during the experiment (Fig. 5Go, control).



View larger version (26K):
[in this window]
[in a new window]
  		Figure 4. Effect of orexin-A on Ca2+ currents in ovine somatotropes. A, Data are shown for a representative cell. The total Ca2+ current was measured following voltage steps to +20 mV at the holding potential of –80 mV. Traces show the Ca2+ current in controls (a), 5 min after the application of orexin-A (100 nM; b), and approximately 10 min after removal of orexin-A (c). B, Statistical data of the current-voltage relationship curves obtained from a group of four recordings. Orexin-A significantly and reversibly increased the amplitude of the voltage-gated Ca2+ current (mean ± SEM; n = 4). C, Statistical data demonstrate that orexin-A significantly increases voltage-gated calcium currents, whereas there is no change in the current amplitude when vehicle (bath solution only) is used.

 


View larger version (18K):
[in this window]
[in a new window]
  		Figure 5. Time-response relationship of the effect of orexin-A on total Ca2+ currents. Voltage-gated total Ca2+ currents were recorded every 2 min with a depolarizing pulse to +20 mV from a holding potential of –80 mV. The period in which orexin-A (100 nM) or vehicle was applied to recorded cells is indicated (filled bar; mean ± SEM; n = 6).

 
Effect of orexin-A on the L-type Ca2+ current
As orexin-B increases only the L current in ovine somatotropes (Xu, R., manuscript in preparation), we extended our studies by examining the effect of orexin-A on this current. To isolate the L-type Ca2+ current, the N-type Ca2+ current blocker CTX (10 µM) was added to bath solution when the cells were recorded with a holding potential of –30 mV to exclude the T current. The isolated L-type current was the largest portion of the total currents (~60% of the amplitude of the total currents) in ovine somatotropes, and orexin-A (100 nM) significantly and reversibly increased the L-type current (Fig. 6Go). No effect was observed in the N or T current from the addition of orexin-A at the same concentration (data not shown). As only the L-type current was altered by orexin-A, the total Ca2+ current using the holding potential of –80 mV was used in the following experiments. Meanwhile, the Ca2+ currents recorded under this holding potential of –80 mV were found to be more stable than those under the holding potential of –30 mV.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 6. Effect of orexin-A on the L-type Ca2+ current. The L-type current was isolated using a holding potential of –30 mV in the presence of the specific N-type current blocker CTX. Statistical data (mean ± SEM; n = 4) demonstrate that orexin-A significantly and reversibly increases this current.

 
Orexin-A and GHRH additively increase Ca2+ currents
As Ca2+ currents have been previously demonstrated to be increased by GHRH in ovine somatotropes, we tested the interactions between orexin-A and GHRH (100 nM) in the modification of the Ca2+ current. To exclude the possible cross-effect between the receptors of orexin-A and GHRH, we administered orexin-A (100 nM) and GHRH in different orders. In the first series of experiments GHRH was delivered 8 min before orexin-A. GHRH increased the Ca2+ current significantly, and subsequent addition of orexin-A further elevated the level of Ca2+ currents (Fig. 7AGo). When orexin-A was added 8 min before GHRH, orexin-A alone significantly augmented the Ca2+ currents, and addition of GHRH further enhanced the Ca2+ currents (Fig. 7BGo). Complete recovery was achieved in both experimental groups. The total increase in Ca2+ currents produced by orexin-A and GHRH was similar between the two sets of experiments.



View larger version (11K):
[in this window]
[in a new window]
  		Figure 7. Orexin-A and GHRH additively increase the voltage-gated Ca2+ currents in ovine somatotropes. A, Statistical analysis (mean ± SEM; n = 6) of the effects of GHRH, followed by coadministration of GHRH and orexin-A, on voltage-gated Ca2+ currents. B, Statistical analysis (mean ± SEM; n = 5) of the effects of orexin-A, followed by coadministration of orexin-A and GHRH, on voltage-gated Ca2+ currents.

 
Involvement of the PKA-cAMP system in the Ca2+ current response to orexin-A
The involvement of second messenger systems in somatotropes was investigated using specific PKA-cAMP blockers. These blockers have been previously successfully employed to examine the effect of a GHRH-induced change in the Ca2+ and K+ currents in somatotropes (43, 44, 45). The specific blocking effects of H89 and other inhibitors on the PKA signaling pathway have been examined by us in a recent study of the effect of GHRP-2 on inward rectifying K+ currents (Xu, R., unpublished data) and by others (44). In this study a cell-permeable, specific PKA inhibitor, H89, was subsequently used. Cells were incubated in the presence of H89 (1 µM) in the bath solution for 5 min to completely inhibit PKA activity. The amplitude of the Ca2+ currents was increased significantly and reversibly by orexin-A in the presence of H89 (Fig. 8Go). These results thus indicate that the PKA-cAMP system may not be involved in the orexin-A-induced increase in the Ca2+ current.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 8. Involvement of the PKA-cAMP system in the Ca2+ current response to orexin-A. In the presence of a specific PKA blocker H89 (1 µM), orexin-A increased Ca2+ currents in ovine somatotropes.

 
Effect of PKC system on the Ca2+ current response to orexin-A
To study the involvement of the PKC pathway in the orexin-A-induced action on Ca2+ channels, we used Cal-C (a PKC inhibitor), PDBu (a short-term pretreatment as a stimulator and a long-term pretreatment for the down-regulation of PKC), and PKC19–36 (a PKC inhibitory peptide) to quench the intracellular PKC activity before the application of orexin-A. All of these chemicals have been used effectively in a previous study (43). Incubation of the cells with Cal-C at a concentration of 1 µM for 5 min did not change the basal levels of Ca2+ currents. The increase in Ca2+ currents induced by orexin-A was totally abolished, however, by this PKC inhibitor (Fig. 9AGo). Furthermore, we microdialyzed the cells with PKC19–36, a specific PKC inhibitory peptide, to test its influence on the effect of orexin-A. Again, the increase in Ca2+ currents produced by orexin-A was completely inhibited by this peptide (Fig. 9BGo). When the cells were stimulated with PDBu (10 µM) for 5 min (stimulation of PKC activity), Ca2+ currents increased significantly, mimicking the effect of orexin-A (Fig. 9CGo). When cells were pretreated with PDBu (0.5 µM) for 16 h to down-regulate PKC activity, the orexin-A-mediated increase in Ca2+ current was completely suppressed (Fig. 9DGo). Therefore, these data strongly suggest that the PKC pathway is required for the increase in Ca2+ currents induced by orexin-A in ovine somatotropes.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 9. Involvement of the PKC system in the Ca2+ current response to orexin-A in ovine somatotropes. A, Statistical data show that Cal-C (100 nM) totally abolished the orexin-A-induced increase in Ca2+ current (mean ± SEM; n = 4). B, Statistical data show that PKC inhibitory peptide PKC19–36 completely abolished the orexin-A-induced increase in Ca2+ current. As PKC19–36 is not permeable through the cell membrane, it was included in pipette solution to be introduced into the recorded cells (mean ± SEM; n = 4). C, Statistical data (mean ± SEM; n = 5) show the acute application of PDBu (0.5 µM; for 5 min) on the Ca2+ current. D, Statistical data (mean ± SEM; n = 5) show the effect of long-term pretreatment of PDBu (16 h) on the orexin-A-induced increase in the Ca2+ current.

 
Involvement of intracellular Ca2+ store in orexin-A induced Ca2+ current increase
To rule out the possible involvement of intracellular Ca2+ store in the orexin-A-mediated effect, we depleted the Ca2+ store by pretreatment of somatotropes with thapsigargin (10 µM) for 30 min. Statistical data (Fig. 10Go) show that after such a pretreatment, orexin-A still elevated the Ca2+ current significantly, suggesting that the intracellular Ca2+ store does not contribute to the orexin-A-induced increase in Ca2+ currents.



View larger version (12K):
[in this window]
[in a new window]
  		Figure 10. Involvement of intracellular Ca2+ store in the orexin-A-induced increase in the Ca2+ current. Statistical data show that thapsigargin (1 µM for ~30 min) does not prevent orexin-A from increasing the voltage-gated Ca2+ current in ovine somatotropes (mean ± SEM; n = 4).

 
Orexin-A and GHRH have a synergistic effect on GH release in vitro
Finally, we examined the direct effect of orexin-A on GH secretion in somatotropes in vitro. Cells were incubated with orexin-A (100 nM), GHRH (1 or 10 nM), or their combination for 30 min. GHRH dose-dependently increased GH release, whereas orexin-A alone did not increase GH secretion. Coadministration of GHRH and orexin-A synergistically augmented GH release (Fig. 11Go).



View larger version (10K):
[in this window]
[in a new window]
  		Figure 11. Orexin-A and GHRH have a synergistic effect on GH release in vitro. Effects of orexin-A, different concentrations of GHRH, and the coadministration of orexin-A and GHRH on GH release in cultured ovine somatotropes were observed. The columns show GH release (mean ± SEM; n = 4) in response to the individual peptides or the combination of both peptides indicated at the bottom of the figure (also see Results).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present studies we have shown the strong expression of orexin-1 receptor in ovine somatotropes by RT-PCR, suggesting that orexins may exert biological actions in the anterior pituitary. Subsequent electrophysiological studies have demonstrated that orexin-A significantly increases the L-type Ca2+ current via a PKC-mediated signal transduction pathway. Functionally, we revealed that GHRH and orexin-A not only have an additive effect on the augmentation of L-type current, but also have a synergistic effect on GH release in primary cultured ovine somatotropes.

Our hypothesis was that orexin-A increases GH release by acting on pituitary somatotropes. This was based on the observations that orexins influenced the secretion of a number of pituitary hormones and energy balance or homeostasis (see introduction). Immunohistochemical studies of the structure of orexins, their accumulation with the vesicles at axon terminals, as well as their strong expression within the hypothalamus have strongly suggested that orexins modulate neuronal network signaling, including pituitary function (46). As Ca2+ oscillation is one of the most important events closely associated with the release of GH in somatotropes (32, 47), we first investigated the effect of orexin-A on the membrane voltage-gated Ca2+channel, which is a major determinant of [Ca2+]i. Three subtypes of Ca2+ currents in ovine somatotropes were isolated using specific Ca2+ channel blockers and special holding potentials, among which the L current (60–70%) is much larger than the T or N current. Using whole cell, patch-clamp techniques, we demonstrated that orexin-A significantly and reversibly increases only the L-type Ca2+ current in ovine somatotropes, indicating that orexin-A may be coupled to Ca2+ channels via the orexin-1 receptor on the cell membrane. In fact, the stimulating effect of orexins on [Ca2+]i and other biophysical properties of ion channels have been demonstrated previously in neurons and other cells (1, 3, 22, 23, 48).

Using specific PKA and PKC pathway blockers, we further examined the involvement of second messenger systems in this orexin-A-induced increase in Ca2+ currents. In somatotropes, we and others have previously showed that the K+, Na+, and Ca2+ channels were modified by GHRH, GHRP, and somatostatin (17, 44, 45), and both PKA-cAMP and PKC systems were involved in the intracellular signal transduction employed by these peptides (44, 49). We therefore tried to elucidate the possible intracellular signaling pathways by which orexin-A induced the increase in the L current in ovine somatotropes. As the PKA-cAMP pathway was not implicated in the orexin-B-induced Ca2+ increase (Xu, R., unpublished data), we believed that this was very likely the case for orexin-A. We therefore used one of the specific PKA inhibitors, H89, in our investigations. As expected, the orexin-A-induced increase in the Ca2+ currents was not affected in the presence of H89. It is therefore implied that the cAMP-PKA system may not be involved in the effect of orexin-A on the L-type Ca2+ current.

PKC and calcium have long been recognized as important messengers mediating the effects of orexins in neurons and pheochromocytoma cells (1, 11, 50), but no study has been undertaken in somatotropes. We used several approaches to stimulate or block intracellular PKC activity. Acute stimulation of the intracellular PKC activity by PDBu mimicked the effect of orexin-A on the Ca2+ current, suggesting the involvement of the PKC signaling pathway. Meanwhile, we showed that the effect of orexin-A on Ca2+ currents was completely abolished by pretreatment of the cells with PDBu for 16 h (down-regulation of the PKC system); a PKC blocker, Cal-C; or the specific PKC inhibitory peptide, PKC19–36. These results strongly indicate that orexin-A augments the L-type Ca2+ current through a PKC-dependent pathway. Our observations were in agreement with previous findings that orexin-A increased [Ca2+]i via the PKC pathway, leading to elevated activities of the neurons in arcuate nuclei and the ventral tegmental area (50, 51). These findings are also in line with the report that PKC signaling cascade was implicated in orexin-A-stimulated catecholamine secretion in human adrenal cells (11). As PKA-cAMP and PKC signal transduction pathways are involved in the modification of K+ and Ca2+ currents by GHRH in somatotropes (31, 43, 44, 52), orexin-A may interact with GHRH through these intracellular second messenger systems to increase the Ca2+ current.

It is well established that orexin receptors are coupled to GTP-binding proteins (2). However, the possible involvement of specific G protein subunits is unknown. As orexins (10 nM to 10 µM) failed to directly increase cAMP levels, and coadministration of orexin and vasoactive intestinal peptide or forskolin did not affect the vasoactive intestinal peptide-evoked cAMP rise in primary cultured hypothalamic neurons, orexin receptors may not activate heteromeric Gi or Gs proteins (20). The G protein most likely involved in this orexin-A-evoked effect on the L current in ovine somatotropes is Gq, as observed in neurons (20, 50). It is possible that activation of Gq triggers the stimulation of PKC, which then leads to the phosphorylation of Ca2+ channels, which subsequently increases Ca2+ conductance.

PLC pathway may play a role in the functional regulation of somatotropes (53). GHRP-6 receptor was coupled to inositol trisphosphate (InsP3) in triggering the release of Ca2+ from the intracellular store and activating the PKC system in rat somatotropes (54). It has been recently demonstrated that the effect of orexin-A is mediated by both Ca2+ influx and InsP3 production in Chinese hamster ovary cells expressing the orexin-1 receptor (25, 26). It is, however, unclear whether InsP3 is implicated in the effect of orexin-A on the membrane Ca2+ current in ovine somatotropes. In our experimental conditions, thapsigargin (10 µM) was used to deplete the intracellular Ca2+ store before orexin-A was applied, and we found that the increase in the L-type current produced by orexin-A was not affected. These results suggest that the increase in the Ca2+ current induced by orexin-A was not dependent on calcium release from the intracellular calcium store. This is not surprising given that receptor-operated Ca2+ entry may not be the consequence of intracellular Ca2+ release in several cell types (55, 56).

Finally, we studied the interactions between orexin-A and GHRH in the increase in Ca2+ current and GH secretion. Orexin-A or GHRH individually induced a significant increase in the L-type current, and an additive effect on the current was observed when they were coadministered. This can be explained by the fact that orexin-A and GHRH modify the L-type current through different signaling pathways, e.g. orexin-A mainly activates the PKC system, whereas GHRH predominantly employs PKA-cAMP to increase the L-type current. These data prompted us to study further the direct stimulatory effects of these two peptides on GH secretion in vitro. Cells were subsequently incubated individually or in combination with orexin-A and GHRH, and GH concentrations after these treatments were measured by RIA. It was expected that GHRH dose-dependently increased GH secretion. Interestingly, a synergistic effect on GH release was evident when orexin-A and GHRH were coadministered, although orexin-A by itself did not significantly increase GH release. This synergistic effect corresponded to the findings of a previous electrophysiological study that an additive effect on the L-type current was generated by the coadministration of orexin-A and GHRH. Therefore, the major function of orexin-A in the regulation of GH secretion may be to prime the somatotrope for further action by GHRH. This action of orexin-A significantly increases the susceptibility and excitability of the somatotrope, so that GHRH subsequently generates a much stronger effect on GH release in, specifically, a normal pulsatile pattern. Meanwhile, the L-type Ca2+ current may exert a pivotal effect on the enhanced GH secretion, as both GHRH and orexin-A activate this current.

It should be emphasized, however, that the possible role of orexins in the control of GH secretion is still controversial. It has been reported that GH secretion may or may not be affected by orexin-A in different experimental conditions (3, 16). In this study the orexin-A-induced increase in the L-type Ca2+ current is a modest modification rather than a marked enlargement, which makes it difficult to obtain a dose-response curve. The release and regulatory effect of orexin-A in vivo may reflect the actions of a number of other factors from the hypothalamus, such as leptin or neuropeptide Y. GH release may be significantly influenced by hypothalamic peptides and their complex interaction (57, 58, 59). Furthermore, the distribution of orexin-1 receptor among pituitary cells is, in fact, largely unknown, and the difference between species cannot be ruled out either. We thus proposed that, at least in our experimental conditions, orexin-A may prime the somatotropes so that both L current and GH secretion are increased significantly when GHRH and orexin-A are applied in combination. Hence, orexin-A may play a complementary role in the control of GH release. In the light of our current results, further studies of interactions between orexin-A and other GH secretagogues in GH control are warranted.

In conclusion, we have presented data demonstrating that the L-type current is significantly increased by orexin-A via the PKC signaling pathway in ovine somatotropes. GHRH and orexin-A exhibit an additive and a synergistic effect in stimulating the L current and GH release, respectively. The functions of orexin-A, therefore, may include regulation of GH at the pituitary level by priming the somatotrope for further stimulation by GHRH. This is the first demonstration that orexin-A is able to directly modulate the Ca2+ currents in somatotropes. Augmentation of the L-type Ca2+ currents by orexin-A is linked to an enhancement of GHRH-induced GH secretion in ovine somatotropes.


   Acknowledgments
 
We thank Ms. S. Panckridge for preparing the graphics, and Dr. A. F. Parlow, NIDDK National Hormone and Pituitary Program, for the ovine GH RIA kits.


   Footnotes
 
This work was supported by Australian National Health and Medical Research Council. R.X. was the recipient of a Monash graduate scholarship from Monash University (Melbourne, Australia).

Abbreviations: [Ca2+]i, Intracellular calcium concentration; Cal-C, calphostin C; CTX, {omega}-conotoxin; GHRP, GH-releasing peptide; InsP3, inositol trisphosphate; NFD, nefidipine; orexin 1-R, orexin 1 receptor; orexin 2-R, orexin 2 receptor; PDBu, phorbal 12,13-dibutyrate; PKA, protein kinase A; PKC, protein kinase C; RT, reverse transcription; TTX, tetrodotoxin.

Received May 13, 2002.

Accepted for publication August 20, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. de Lecea L, Kilduff TS, Peyron C, Gao X, Foye PE, Danielson PE, Fukuhara C, Battenberg EL, Gautvik VT, Bartlett FS, Frankel WN, van den Pol AN, Bloom FE, Gautvik KM, Sutcliffe JG 1998 The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity. Proc Natl Acad Sci USA 95:322–327[Abstract/Free Full Text]
  2. Sakurai T, Amemiya A, Ishii M, Matsuzaki I, Chemelli RM, Tanaka H, Williams SC, Richardson JA, Kozlowski GP, Wilson S, Arch JR, Buckingham RE, Haynes AC, Carr SA, Annan RS, McNulty DE, Liu WS, Terrett JA, Elshourbagy NA, Bergsma DJ, Yanagisawa M 1998 Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell 92:573–585[CrossRef][Medline]
  3. Hagan JJ, Leslie RA, Patel S, Evans ML, Wattam TA, Holmes S, Benham CD, Taylor SG, Routledge C, Hemmati P, Munton RP, Ashmeade TE, Shah AS, Hatcher JP, Hatcher PD, Jones DN, Smith MI, Piper DC, Hunter AJ, Porter RA, Upton N 1999 Orexin A activates locus coeruleus cell firing and increases arousal in the rat. Proc Natl Acad Sci USA 96:10911–10916[Abstract/Free Full Text]
  4. Mondal MS, Nakazato M, Matsukura S 2000 Orexins (hypocretins): novel hypothalamic peptides with divergent functions. Biochem Cell Biol 78:299–305[CrossRef][Medline]
  5. Date Y, Ueta Y, Yamashita H, Yamaguchi H, Matsukura S, Kangawa K, Sakurai T, Yanagisawa M, Nakazato M 1999 Orexins, orexigenic hypothalamic peptides, interact with autonomic, neuroendocrine and neuroregulatory systems. Proc Natl Acad Sci USA 96:748–753[Abstract/Free Full Text]
  6. Mazzocchi G, Malendowicz LK, Aragona F, Rebuffat P, Gottardo L, Nussdorfer GG 2001 Human pheochromocytomas express orexin receptor type 2 gene and display an in vitro secretory response to orexins A and B. J Clin Endocrinol Metab 86:4818–4821[Abstract/Free Full Text]
  7. Pu S, Jain MR, Kalra PS, Kalra SP 1998 Orexins, a novel family of hypothalamic neuropeptides, modulate pituitary luteinizing hormone secretion in an ovarian steroid-dependent manner. Regul Pept 78:133–136[CrossRef][Medline]
  8. Russell SH, Kim MS, Small CJ, Abbott CR, Morgan DG, Taheri S, Murphy KG, Todd JF, Ghatei MA, Bloom SR 2000 Central administration of orexin A suppresses basal and domperidone stimulated plasma prolactin. J Neuroendocrinol 12:1213–1218[CrossRef][Medline]
  9. Jaszberenyi M, Bujdoso E, Pataki I, Telegdy G 2000 Effects of orexins on the hypothalamic-pituitary-adrenal system. J Neuroendocrinol 12:1174–1178[CrossRef][Medline]
  10. Irahara M, Tamura T, Matuzaki T, Saito S, Yasui T, Yamano S, Kamada M, Aono T 2001 Orexin-A suppresses the pulsatile secretion of luteinizing hormone via beta-endorphin. Biochem Biophys Res Commun 281:232–236[CrossRef][Medline]
  11. Mazzocchi G, Malendowicz LK, Gottardo L, Aragona F, Nussdorfer GG 2001 Orexin A stimulates cortisol secretion from human adrenocortical cells through activation of the adenylate cyclase-dependent signaling cascade. J Clin Endocrinol Metab 86:778–782[Abstract/Free Full Text]
  12. Mitsuma T, Hirooka Y, Mori Y, Kayama M, Adachi K, Rhue N, Ping J, Nogimori T 1999 Effects of orexin A on thyrotropin-releasing hormone and thyrotropin secretion in rats. Horm Metab Res 31:606–609[Medline]
  13. Nowak KW, Mackowiak P, Switonska MM, Fabis M, Malendowicz LK 2000 Acute orexin effects on insulin secretion in the rat: in vivo and in vitro studies. Life Sci 66:449–454[CrossRef][Medline]
  14. Date Y, Mondal MS, Matsukura S, Ueta Y, Yamashita H, Kaiya H, Kangawa K, Nakazato M 2000 Distribution of orexin/hypocretin in the rat median eminence and pituitary. Brain Res Mol Brain Res 76:1–6[Medline]
  15. Blanco M, Lopez M, GarcIa-Caballero T, Gallego R, Vazquez-Boquete A, Morel G, SenarIs R, Casanueva F, Dieguez C, Beiras A 2001 Cellular localization of orexin receptors in human pituitary. J Clin Endocrinol Metab 86:1616–1619[Abstract/Free Full Text]
  16. Samson WK, Taylor MM 2001 Hypocretin/orexin suppresses corticotroph responsiveness in vitro. Am J Physiol 281:R1140–R1145
  17. Chen C, Vincent JD, Clarke IJ 1994 Ion channels and signal transduction pathways in the regulation of growth hormone secretion. Trends Endocrinol Metab 5:227–233[Medline]
  18. Chen C, Clarke IJ 1995 Modulation of Ca2+ influx in the ovine somatotroph by growth hormone-releasing factor. Am J Physiol 268:E204–E212
  19. Johren O, Neidert SJ, Kummer M, Dendorfer A, Dominiak P 2001 Prepro-orexin and orexin receptor mRNAs are differentially expressed in peripheral tissues of male and female rats. Endocrinology 142:3324–3331[Abstract/Free Full Text]
  20. van den Pol AN, Gao XB, Obrietan K, Kilduff TS, Belousov AB 1998 Presynaptic and postsynaptic actions and modulation of neuroendocrine neurons by a new hypothalamic peptide, hypocretin/orexin. J Neurosci 18:7962–7971[Abstract/Free Full Text]
  21. Grudt TJ, van den Pol AN, Perl ER 2002 Hypocretin-2 (orexin-B) modulation of superficial dorsal horn activity in rat. J Physiol 538:517–525[Abstract/Free Full Text]
  22. Hwang LL, Chen CT, Dun NJ 2001 Mechanisms of orexin-induced depolarizations in rat dorsal motor nucleus of vagus neurones in vitro. J Physiol 537:511–520[Abstract/Free Full Text]
  23. Ichinose M, Asai M, Sawada M, Sasaki K, Oomura Y 1998 Induction of outward current by orexin-B in mouse peritoneal macrophages. FEBS Lett 440:51–54[CrossRef][Medline]
  24. Malendowicz LK, Tortorella C, Nussdorfer GG 1999 Orexins stimulate corticosterone secretion of rat adrenocortical cells, through the activation of the adenylate cyclase-dependent signaling cascade. J Steroid Biochem Mol Biol 70:185–188[CrossRef][Medline]
  25. Lund PE, Shariatmadari R, Uustare A, Detheux M, Parmentier M, Kukkonen JP, Akerman KE 2000 The orexin OX1 receptor activates a novel Ca2+ influx pathway necessary for coupling to phospholipase C. J Biol Chem 275:30806–30812[Abstract/Free Full Text]
  26. Kukkonen JP, Akerman KE 2001 Orexin receptors couple to Ca2+ channels different from store-operated Ca2+ channels. Neuroreport 12:2017–2020[CrossRef][Medline]
  27. Holl RW, Thorner MO, Mandell GL, Sullivan JA, Sinha YN, Leong DA 1988 Spontaneous oscillations of intracellular calcium and growth hormone secretion. J Biol Chem 263:9682–9685[Abstract/Free Full Text]
  28. Acs Z, Zsom L, Mergl Z, Makara GB 1993 Significance of chloride channel activation in the {gamma}-aminobutyric acid induced growth hormone secretion in the neonatal rat pituitary. Life Sci 52:1733–1739[CrossRef][Medline]
  29. Naumov AP, Herrington J, Hille B 1994 Actions of growth-hormone-releasing hormone on rat pituitary cells: intracellular calcium and ionic currents. Pflugers Arch 427:414–421[CrossRef][Medline]
  30. Kato M 1996 Growth hormone-releasing hormone augments voltage-gated Na+ current in cultured rat pituitary cells. Am J Physiol 270:C125–C130
  31. Lussier BT, French MB, Moor BC, Kraicer J 1991 Free intracellular Ca2+ concentration and growth hormone (GH) release from purified rat somatotrophs. III. Mechanism of action of GH-releasing factor and somatostatin. Endocrinology 128:592–603[Abstract]
  32. Kwiecien R, Tseeb V, Kurchikov A, Kordon C, Hammond C 1997 Growth hormone-releasing hormone triggers pacemaker activity and persistent Ca2+ oscillations in rat somatotrophs. J Physiol 499:613–623[CrossRef][Medline]
  33. Chen C, Wu D, Clarke IJ 1996 Signal transduction systems employed by synthetic GH-releasing peptides in somatotrophs. J Endocrinol 148:381–386[CrossRef][Medline]
  34. Chen C, Heyward P, Zhang J, Wu D, Clarke IJ 1994 Voltage-dependent potassium currents in ovine somatotrophs and their function in growth hormone secretion. Neuroendocrinology 59:1–9[Medline]
  35. Marcus JN, Aschkenasi CJ, Lee CE, Chemelli RM, Saper CB, Yanagisawa M, Elmquist JK 2001 Differential expression of orexin receptors 1 and 2 in the rat brain. J Comp Neurol 435:6–25[CrossRef][Medline]
  36. Farrell RE 1993 Protocol: rapid isolation of RNA with SDS and potassium acetate reagents. In: Farrell RE, ed. RNA methodologies: a laboratory guide for isolation and characterization. San Diego: Academic Press; 69–70
  37. Lopez M, SenarIs R, Gallego R, GarcIa-Caballero T, Lago F, Seoane L, Casanueva F, Dieguez C 1999 Orexin receptors are expressed in the adrenal medulla of the rat. Endocrinology 140:5991–5994[Abstract/Free Full Text]
  38. Tsien RW, Fox AP, Hess P, McCleskey EW, Nilius B, Nowycky MC, Rosenberg RL 1987 Multiple types of calcium channel in excitable cells. Soc Gen Physiol Ser 41:167–187[Medline]
  39. Chen C, Clarke IJ 1995 Effects of growth hormone-releasing peptide-2 (GHRP-2) on membrane Ca2+ permeability in cultured ovine somatotrophs. J Neuroendocrinol 7:179–186[CrossRef][Medline]
  40. Glassmeier G, Hauber M, Wulfsen I, Weinsberg F, Bauer CK, Schwarz JR 2001 Ca2+ channels in clonal rat anterior pituitary cells (GH3/B6). Pflugers Arch 442:577–587[CrossRef][Medline]
  41. Chen C 1998 Gi-3 protein mediates the increase in voltage-gated K+ currents by somatostatin on cultured ovine somatotrophs. Am J Physiol 275:E278–E284
  42. Chen C, Zhang J, Dayanithi G, Vincent JD, Israel JM 1989 Cation currents on identified rat gonadotroph cells maintained in primary culture. Neurochem Int 15:265–275
  43. Xu R, Roh SG, Loneragan K, Pullar M, Chen C 1999 Human GHRH reduces voltage-gated K+ currents via a non-cAMP-dependent but PKC-mediated pathway in human GH adenoma cells. J Physiol 520:697–707[Abstract/Free Full Text]
  44. Takei T, Takano K, Yasufuku-Takano J, Fujita T, Yamashita N 1996 Enhancement of Ca2+ currents by GHRH and its relation to PKA and [Ca2+]i in human GH-secreting adenoma cells. Am J Physiol 271:E801–E807
  45. Takei T, Yasufuku-Takano J, Takano K, Fujita T, Yamashita N 1998 Effect of Ca2+ and cAMP on capacitance-measured hormone secretion in human GH-secreting adenoma cells. Am J Physiol 275:E649–E654
  46. de Lecea L, Sutcliffe JG 1999 The hypocretins/orexins: novel hypothalamic neuropeptides involved in different physiological systems. Cell Mol Life Sci 56:473–480[CrossRef][Medline]
  47. Kwiecien R, Hammond C 1998 Differential management of Ca2+ oscillations by anterior pituitary cells: a comparative overview. Neuroendocrinology 68:135–151[CrossRef][Medline]
  48. Ivanov A, Aston-Jones G 2000 Hypocretin/orexin depolarizes and decreases potassium conductance in locus coeruleus neurons. Neuroreport 11:1755–1758[Medline]
  49. Adams EF, Lei T, Buchfelder M, Bowers CY, Fahlbusch R 1996 Protein kinase C-dependent growth hormone releasing peptides stimulate cyclic adenosine 3',5'-monophosphate production by human pituitary somatotropinomas expressing gsp oncogenes: evidence for crosstalk between transduction pathways. Mol Endocrinol 10:432–438[Abstract]
  50. Uramura K, Funahashi H, Muroya S, Shioda S, Takigawa M, Yada T 2001 Orexin-a activates phospholipase C- and protein kinase C-mediated Ca2+ signaling in dopamine neurons of the ventral tegmental area. Neuroreport 12:1885–1889[CrossRef][Medline]
  51. van den Pol AN 1999 Hypothalamic hypocretin (orexin): robust innervation of the spinal cord. J Neurosci 19:3171–3182[Abstract/Free Full Text]
  52. Chen C, Wu D, Clarke IJ 1996 Signal transduction systems employed by synthetic GH-releasing peptides in somatotrophs. J Endocrinol 148:381–386
  53. Lei T, Adams EF, Buchfelder M, Fahlbusch R 1996 Relationship between protein kinase C and adenylyl cyclase activity in the regulation of growth hormone secretion by human pituitary somatotrophinomas. Neurosurgery 39:569–575[Medline]
  54. Cheng K, Chan WW, Butler B, Barreto Jr A, Smith RG 1991 Evidence for a role of protein kinase-C in His-D-Trp-Ala-Trp-D-Phe-Lys-NH2-induced growth hormone release from rat primary pituitary cells. Endocrinology 129:3337–3342[Abstract]
  55. Felder CC, Poulter MO, Wess J 1992 Muscarinic receptor-operated Ca2+ influx in transfected fibroblast cells is independent of inositol phosphates and release of intracellular Ca2+. Proc Natl Acad Sci USA 89:509–513[Abstract/Free Full Text]
  56. Zerbes M, Bunn SJ, Powis DA 1998 Histamine causes Ca2+ entry via both a store-operated and a store-independent pathway in bovine adrenal chromaffin cells. Cell Calcium 23:379–386[CrossRef][Medline]
  57. Rauch M, Riediger T, Schmid HA, Simon E 2000 Orexin A activates leptin-responsive neurons in the arcuate nucleus. Pflugers Arch 440:699–703[CrossRef][Medline]
  58. Ida T, Nakahara K, Kuroiwa T, Fukui K, Nakazato M, Murakami T, Murakami N 2000 Both corticotropin releasing factor and neuropeptide Y are involved in the effect of orexin (hypocretin) on the food intake in rats. Neurosci Lett 293:119–122[CrossRef][Medline]
  59. Komaki G, Matsumoto Y, Nishikata H, Kawai K, Nozaki T, Takii M, Sogawa H, Kubo C 2001 Orexin-A and leptin change inversely in fasting non-obese subjects. Eur J Endocrinol 144:645–651[Abstract]



This article has been cited by other articles:


Home page
 J. Neurosci.Home page
J. Nasman, G. Bart, K. Larsson, L. Louhivuori, H. Peltonen, and K. E. O. Akerman
The Orexin OX1 Receptor Regulates Ca2+ Entry via Diacylglycerol-Activated Channels in Differentiated Neuroblastoma Cells.
J. Neurosci., October 18, 2006; 26(42): 10658 - 10666.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
S. Ammoun, L. Johansson, M. E. Ekholm, T. Holmqvist, A. S. Danis, L. Korhonen, O. A. Sergeeva, H. L. Haas, K. E. O. Akerman, and J. P. Kukkonen
OX1 Orexin Receptors Activate Extracellular Signal-Regulated Kinase in Chinese Hamster Ovary Cells via Multiple Mechanisms: The Role of Ca2+ Influx in OX1 Receptor Signaling
Mol. Endocrinol., January 1, 2006; 20(1): 80 - 99.
[Abstract]