Ghrelin Stimulates Porcine Somatotropes

Ghrelin is an endogenous ligand for growth hormone secretagogue receptor (GHS-R) and is predominantly produced by the stomach and lower amounts in the hypothalamus and various peripheral tissues. Ghrelin is a potent stimulator of growth hormone (GH) secretion from the pituitary in vivo and in vitro. GH secretion from the pituitary also is controlled by two hypothalamic peptides: stimulatory GH-releasing hormone (GHRH) and inhibitory somatostatin-14 (SRIH). GH participates in its own rhythmic secretion through feedback action on GHRH and SRIH neurons. The mechanism of action of GHS is not established. The present study examined the signal transduction pathways of ghrelin in isolated porcine somatotropes. The ability of ghrelin to induce an increase in the intracellular Ca2+ concentration – [Ca2+]i – somatotropes was examined in dispersed porcine pituitary cells using a calcium imaging system. Somatotropes were functionally identified by application of human growth hormone releasing hormone (hGHRH). Ghrelin increased the [Ca2+]i in a dose-dependent manner in 98% of the cells that responded to. In the presence of (D-Lys3)-GHRP-6, a specific receptor antagonist of GHS-R, the increase in [Ca2+]i evoked by ghrelin was decreased. Pretreatment of cultures with somatostatin or neuropeptide Y reduced the ghrelin-induced increase of [Ca2+]i. The stimulatory effect of ghrelin on somatotropes was greatly attenuated in lowcalcium saline and blocked by nifedipine, an L-type calcium channel blocker, suggesting involvement of calcium channels. In a zero Na+ solution, the stimulatory effect of ghrelin on somatotropes was decreased, suggesting that besides calcium channels, sodium channels are also involved in ghrelin-induced calcium transients. Either SQ-22536, an adenylyl cyclase inhibitor, or U73122, a phospholipase C inhibitor, decreased the stimulatory effects of ghrelin on [Ca2+]i transiently, indicating the involvement of adenylyl cyclase-cyclic adenosine monophosphate and phospholipase C inositol 1,4,5-trisphosphate pathways. The non-peptidyl GHS, L-692,585 (L-585), induced changes in [Ca2+]i similar to those observed with ghrelin. Application of L-585 after ghrelin did not have additive effects on [Ca2+]i. Preapplication of L-585 blocked the stimulatory effect of ghrelin on somatotropes. Our results suggest that the actions of ghrelin and synthetic GHS closely parallel each other, in a manner that is consistent with an increase of hormone secretion. An understanding of the molecular mechanisms by which ghrelin and GHS modulate GH secretion is of particular interest in the regulation of GH for muscle accretion and somatic growth

Subpopulations of Chicken Somatotropes with Differing Intracellular Calcium Concentrations Responses to Secretagogues

Multiple secretagogues stimulate the release of growth hormone (GH). The present studies examined the ability of chicken somatotropes to respond to GH secretagogues with increased intracellular calcium concentrations ([Ca 2+ ]i ). It was hypothesized that there are subsets of the somatotrope population with different responsiveness to the various secretagogues. Avian somatotropes were identified and distinguished from other anterior pituitary cells, by their unique ability to respond to GH-releasing hormone with increased [Ca 2+ ]i with immunocytochemistry used as a post-hoc confirmatory test. Large increases in [Ca 2+ ]i (222 ± 16 nm) were evoked by thyrotropin-releasing hormone in only 73% of the somatotropes. Similarly, [Ca 2+ ]i was increased by perifusion with pituitary adenylate cyclase-activating peptide in 85% and by leptin but only in 51% of somatotropes. Ghrelin acutely increased [Ca 2+ ]i in only 21% of somatotropes. Perfusion with gonadotropinreleasing hormone elevated [Ca 2+ ]i , but in only 40% of somatotropes. The kinetics of calcium transients and the magnitude of the response differed from those observed in the presumptive gonadotropes. It is concluded that there are subsets of the somatotrope population in the anterior pituitary gland with differences in their ability to respond to various secretagogue

Ghrelin Stimulates Porcine Somatotropes

Ghrelin is an endogenous ligand for growth hormone secretagogue receptor (GHS-R) and is predominantly produced by the stomach and lower amounts in the hypothalamus and various peripheral tissues. Ghrelin is a potent stimulator of growth hormone (GH) secretion from the pituitary in vivo and in vitro. GH secretion from the pituitary also is controlled by two hypothalamic peptides: stimulatory GH-releasing hormone (GHRH) and inhibitory somatostatin-14 (SRIH). GH participates in its own rhythmic secretion through feedback action on GHRH and SRIH neurons. The mechanism of action of GHS is not established. The present study examined the signal transduction pathways of ghrelin in isolated porcine somatotropes. The ability of ghrelin to induce an increase in the intracellular Ca2+ concentration – [Ca2+]i – somatotropes was examined in dispersed porcine pituitary cells using a calcium imaging system. Somatotropes were functionally identified by application of human growth hormone releasing hormone (hGHRH). Ghrelin increased the [Ca2+]i in a dose-dependent manner in 98% of the cells that responded to. In the presence of (D-Lys3)-GHRP-6, a specific receptor antagonist of GHS-R, the increase in [Ca2+]i evoked by ghrelin was decreased. Pretreatment of cultures with somatostatin or neuropeptide Y reduced the ghrelin-induced increase of [Ca2+]i. The stimulatory effect of ghrelin on somatotropes was greatly attenuated in lowcalcium saline and blocked by nifedipine, an L-type calcium channel blocker, suggesting involvement of calcium channels. In a zero Na+ solution, the stimulatory effect of ghrelin on somatotropes was decreased, suggesting that besides calcium channels, sodium channels are also involved in ghrelin-induced calcium transients. Either SQ-22536, an adenylyl cyclase inhibitor, or U73122, a phospholipase C inhibitor, decreased the stimulatory effects of ghrelin on [Ca2+]i transiently, indicating the involvement of adenylyl cyclase-cyclic adenosine monophosphate and phospholipase C inositol 1,4,5-trisphosphate pathways. The non-peptidyl GHS, L-692,585 (L-585), induced changes in [Ca2+]i similar to those observed with ghrelin. Application of L-585 after ghrelin did not have additive effects on [Ca2+]i. Preapplication of L-585 blocked the stimulatory effect of ghrelin on somatotropes. Our results suggest that the actions of ghrelin and synthetic GHS closely parallel each other, in a manner that is consistent with an increase of hormone secretion. An understanding of the molecular mechanisms by which ghrelin and GHS modulate GH secretion is of particular interest in the regulation of GH for muscle accretion and somatic growth.</p

Subpopulations of Chicken Somatotropes with Differing Intracellular Calcium Concentrations Responses to Secretagogues

Multiple secretagogues stimulate the release of growth hormone (GH). The present studies examined the ability of chicken somatotropes to respond to GH secretagogues with increased intracellular calcium concentrations ([Ca 2+ ]i ). It was hypothesized that there are subsets of the somatotrope population with different responsiveness to the various secretagogues. Avian somatotropes were identified and distinguished from other anterior pituitary cells, by their unique ability to respond to GH-releasing hormone with increased [Ca 2+ ]i with immunocytochemistry used as a post-hoc confirmatory test. Large increases in [Ca 2+ ]i (222 ± 16 nm) were evoked by thyrotropin-releasing hormone in only 73% of the somatotropes. Similarly, [Ca 2+ ]i was increased by perifusion with pituitary adenylate cyclase-activating peptide in 85% and by leptin but only in 51% of somatotropes. Ghrelin acutely increased [Ca 2+ ]i in only 21% of somatotropes. Perfusion with gonadotropinreleasing hormone elevated [Ca 2+ ]i , but in only 40% of somatotropes. The kinetics of calcium transients and the magnitude of the response differed from those observed in the presumptive gonadotropes. It is concluded that there are subsets of the somatotrope population in the anterior pituitary gland with differences in their ability to respond to various secretagogues</p

Acute death of astrocytes in blast-exposed rat organotypic hippocampal slice cultures.

Blast traumatic brain injury (bTBI) affects civilians, soldiers, and veterans worldwide and presents significant health concerns. The mechanisms of neurodegeneration following bTBI remain elusive and current therapies are largely ineffective. It is important to better characterize blast-evoked cellular changes and underlying mechanisms in order to develop more effective therapies. In the present study, our group utilized rat organotypic hippocampal slice cultures (OHCs) as an in vitro system to model bTBI. OHCs were exposed to either 138 ± 22 kPa (low) or 273 ± 23 kPa (high) overpressures using an open-ended helium-driven shock tube, or were assigned to sham control group. At 2 hours (h) following injury, we have characterized the astrocytic response to a blast overpressure. Immunostaining against the astrocytic marker glial fibrillary acidic protein (GFAP) revealed acute shearing and morphological changes in astrocytes, including clasmatodendrosis. Moreover, overlap of GFAP immunostaining and propidium iodide (PI) indicated astrocytic death. Quantification of the number of dead astrocytes per counting area in the hippocampal cornu Ammonis 1 region (CA1), demonstrated a significant increase in dead astrocytes in the low- and high-blast, compared to sham control OHCs. However only a small number of GFAP-expressing astrocytes were co-labeled with the apoptotic marker Annexin V, suggesting necrosis as the primary type of cell death in the acute phase following blast exposure. Moreover, western blot analyses revealed calpain mediated breakdown of GFAP. The dextran exclusion additionally indicated membrane disruption as a potential mechanism of acute astrocytic death. Furthermore, although blast exposure did not evoke significant changes in glutamate transporter 1 (GLT-1) expression, loss of GLT-1-expressing astrocytes suggests dysregulation of glutamate uptake following injury. Our data illustrate the profound effect of blast overpressure on astrocytes in OHCs at 2 h following injury and suggest increased calpain activity and membrane disruption as potential underlying mechanisms

Limited early apoptotic death of astrocytes following blast exposure.

<p>At 2 h following injury, Annexin V conjugated to Alexa 488 (green; A, E, I) was used to identify apoptotic cells in sham control (A-D), low-blast (E-H), and high-blast (I-L) groups. Samples were additionally labeled with the cell death marker PI (red; B, F, J), an antibody against GFAP (gray; C, G, K), and DAPI (blue). Overlay of Annexin V, PI, GFAP, and DAPI staining (D, H, L). Annexin V positive cells (arrow) were infrequent in all three experimental groups. Almost none of the observed Annexin V positive cells were co-labeled with GFAP. Scale bars 20 μm.</p

Blast-induced loss of GLT-1-expressing astrocytes.

<p>At 2 h following injury, sham control (A), low-blast (B), and high-blast (C) OHCs were stained using antibodies against GLT-1 (green), GFAP (gray), PI (red), and DAPI (blue). Dead astrocytes, identified by co-labeling of GFAP and PI (arrows) were also positive for GLT-1. (D) Representative immunoblot analyses of GLT-1 protein expression in sham control (Sham) and OHCs exposed to blast overpressure (L-Blast and H-Blast). (E) Densitometry analysis of GLT-1/GAPDH ratio for 3 independent experiments revealed no significant (ns) differences between sham control, low-blast, and high blast groups. Scale bars 20 μm.</p

Acute morphological changes and demise of astrocytes following blast exposure.

<p>Representative confocal images acquired in the CA1 hippocampal region from sham controls (A, D), low-blast (B, E), and high-blast (C, F) OHCs that were fixed at 2 h following injury and stained with an anti-GFAP antibody (green), PI (red), and DAPI (blue). Shearing of the astrocytes (thin arrows) was detected in OHCs exposed to blast overpressure (B, C) while it was absent in the sham controls (A). Clasmatodendrosis (arrowheads) was also observed in the low- (E) and high-blast (F) groups, but it was very infrequent in the sham control group (D). At the same time point, only a few dead astrocytes were present in sham control OHCs (D) while significant number of dead astrocytes (thick arrows) was revealed in the low- (E) and high-blast (F) groups. (G) Schematic diagram of OHC, indicating approximate locations in the CA1 region (boxes) where images for quantification of dead astrocytes were taken. (H) Number of dead astrocytes per counting area in the CA1 hippocampal region at 2 h following injury was significantly higher in both the low- (*; P< 0.05; n = 5) and high-blast groups (*; P < 0.05; n = 5) compared to the sham control group (n = 5). Scale bars (A-C) 50 μm (D-F) 20 μm.</p

Increased astrocytic membrane permeability at 2 h post injury.

<p>Dex10 (green) and PI (red) labeled sham control (A), low-blast (B), or high-blast (C) OHCs were fixed 2 h following injury and further stained with an anti-GFAP antibody (gray). Dead astrocytes with increased membrane permeability, identified by overlap of GFAP, PI, and Dex10 staining (arrows), were only present in the low-blast (B) and high-blast (C) OHCs but not in the sham controls (A). Scale bars 20 μm.</p

Calpain-mediated degradation of GFAP at 2 h following blast injury.

<p>Proteins were isolated from sham control (Sham), low-blast (L-Blast), and high-blast (H-Blast) OHCs at 2 h post-injury and analyzed via western blot for expression of GFAP (A, B) or calpain (C). (A) The 38 kDa calpain associated GFAP-BDP was present in blast-injured OHCs, but not in corresponding sham control OHCs. (B) Following inhibition of calpain via calpeptin treatment, this GFAP-BDP at 38 kDa was not observed. (C) Calpain expression in OHCs exposed to blast overpressure compared to sham controls was not changed at this time point. (D) Densitometry analysis of GFAP 50 kDa/GAPDH ratio for 3 independent experiments revealed no significant (ns) differences among control and blast-exposed OHCs without or with calpeptin treatment.</p