Physiology of ghrelin and related peptides.

Growth hormone (GH) released from pituitary under direct control of hypothalamic releasing (i.e., GHRH) and inhibiting (i.e., sst or SRIF) hormones is an anabolic hormone that regulates metabolism of proteins, fats, sugars and minerals in mammals. Cyril Bowers' discovery of GH-releasing peptide (GHRP-6) was followed by a search for synthetic peptide and nonpeptide GH-secretagogues (GHSs) that stimulate GH release, as well as a receptor(s) unique from GHRH receptor. GHRH and GHSs operate through distinct G protein-coupled receptors to release GH. Signal transduction pathways activated by GHS increase intracellular Ca2+ concentration in somatotrophs, whereas GHRH increases cAMP. Isolation and characterization of ghrelin, the natural ligand for GHS receptor, has opened a new era of understanding to physiology of anabolism, feeding behavior, and nutritional homeostasis for GH secretion and gastrointestinal motility through gut-brain interactions. Other peptide hormones (i.e., motilin, TRH, PACAP, GnRH, leptin, FMRF amide, galanin, NPY, NPW) from gut, brain and other tissues also play a role in modulating GH secretion in livestock and lower vertebrate species. Physiological processes, such as neurotransmission, and secretion of hormones or enzymes, require fusion of secretory vesicles at the cell plasma membrane and expulsion of vesicular contents. This process for GH release from porcine somatotrophs was revealed by atomic force microscopy (AFM), transmission electron microscopy (TEM) and immunohistochemical distribution of the cells in pituitary during stages of development.

Alpha-latrotoxin stimulates glutamate release from cortical astrocytes in cell culture.

The mechanism responsible for the ability of bradykinin to cause calcium-dependent release of glutamate from astrocytes in vitro was investigated. The glutamate transport inhibitor, dihydrokainate, did not block bradykinin-induced glutamate release, and bradykinin did not cause cell swelling. These data exclude the involvement of glutamate transporters or swelling mechanisms as mediating glutamate release in response to bradykinin. alpha-Latrotoxin (3 nM), a component of black widow spider venom, stimulated calcium-independent glutamate release from astrocytes. Since alpha-latrotoxin induces vesicle fusion and calcium-independent neuronal neurotransmitter release, our data suggest that astrocytes may release neurotransmitter using a mechanism similar to the neuronal secretory process.

ATP stimulates release of excitatory amino acids from cultured Schwann cells.

The release of excitatory amino acids from Schwann cell cultures in the rat was monitored using high-performance liquid chromatography. The basal concentration of glutamate and aspartate was 33 +/- 4 nM (mean +/- S.E.M., n = 12) and 8 +/- 1 nM (mean +/- S.E.M., n = 12), respectively. ATP (100 microM) caused a receptor-mediated increase in release of glutamate and aspartate from Schwann cell cultures. Bath application of adenosine (100 microM) was without effect on release of excitatory amino acids suggesting involvement of P2 receptors. Suramin, a competitive antagonist at P2 receptors, prevented the response to ATP. The release of excitatory amino acids evoked by ATP was not abolished in calcium-depleted saline. Pretreatment of the Schwann cultures with 50 microM 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetracetic acid-acetoxymethyl ester (BAPTA-AM) abolished the effect of ATP. ATP-evoked release of glutamate from cultured Schwann cells was significantly reduced by thapsigargin (1 microM), an inhibitor of Ca(2+)-ATPase of the Ca2+ pump of internal stores. U73122, a selective inhibitor of receptor-coupled phospholipase C-dependent processes, abolished stimulatory effect of ATP suggesting that ATP's action is mediated through an inositol 1,4,5-triphosphate-sensitive calcium store. The action of ATP was not blocked by L-trans-pyrrolidine-2,4-dicarboxylate, an inhibitor of the electrogenic glutamate transporter, nor was it blocked in Na(+)-free medium, and glutamate release was not stimulated by a depolarizing stimulus, suggesting that ATP-evoked release of glutamate from Schwann cells is not due to the reversal of the glutamate uptake. An anion transport blocker, furosemide, reduced ATP-induced glutamate release. These results suggest that ATP-stimulated glutamate and aspartate release from Schwann cells may be through a calcium-dependent furosemide-sensitive mechanism.

Mechanism of action of the growth hormone secretagogue, L-692,585, on isolated porcine somatotropes.

The effects of a GH secretagogue, L-692,585 (L-585), and human GH-releasing hormone (hGHRH) on calcium transient and GH release were investigated in isolated porcine pituitary cells using calcium imaging and the reverse hemolytic plaque assay (RHPA). Somatotropes were functionally identified by the application of hGHRH. All cells that responded to hGHRH responded to L-585 application. Perfusion application of 10 microM hGHRH and L-585 for 2 min resulted in an increase in intracellular calcium concentrations ([Ca(2+)](i)) of 53+/-1 nM (mean+/-S.E.M.) (P < 0.01) and 68+/-2 nM (P < 0.01) respectively. The L-585 response was characterized by an initial increase in [Ca(2+)](i) followed by a decline to a plateau level above the baseline. Concurrent calcium imaging with RHPA indicated that the L-585-evoked increase in [Ca(2+)](i) coincided with GH release. L-585 significantly increased the percentage of plaque-forming cells (24+/-3 vs 40+/-6%; P < 0.05) and mean area of plaques (1892+/-177 vs 3641+/-189 micro m(2); P < 0.01) indicating increased GH release. Substance P (SP) analogue ([d -Arg(1),d -Phe(5),d -Trp(7,11)]-SP) blocked, and the hGHRH receptor antagonist ((Phenylac-Tyr(1),d -Arg(2), p-chloro-Phe(6), Homoarg(9), Tyr (Me)(10), Abu(15), Nle(27),d -Arg(28), Homoarg(29))-GRF (1-29) amide) decreased the stimulatory effect of hGHRH. These failed to block the stimulatory effect of L-585, suggesting a different receptor for L-585 from the GHRH receptor. The hGHRH-induced calcium transients and initial peak increase induced by L-585 were significantly decreased by removal of calcium from the bathing medium or the addition of nifedipine, an L-calcium channel blocker. The plateau component of L-585-induced calcium change was abolished by removal of calcium and nifedipine. These results suggest an involvement of calcium channels in GH release. Either SQ-22536, an adenylate cyclase inhibitor, or U73122, a phospholipase C (PLC) inhibitor, blocked the stimulatory effects of hGHRH and L-585 on [Ca(2+)](i) transient, indicating the involvement of adenylate cyclase-cAMP and PLC-inositol triphosphate pathways. These results further suggested that calcium mobilization from internal stores during the first phase of the L-585 response induced an increase in [Ca(2+)](i) whereas calcium influx during the second phase is a consequence of somatotrope depolarization.

Effect of capsaicin and resiniferatoxin on peptidergic neurons in cultured dorsal root ganglion.

The neurotoxic effect of capsaicin has been shown to be selective on a subpopulation of small dorsal root ganglion neurons in newborn animals. The aim of this study was to provide evidence of the long lasting effect of capsaicin and its ultrapotent analog resiniferatoxin (RTX) on sensory peptidergic neurons maintained in organotypic cultures. The effects of the two irritants were examined on neurons that contained substance P (SP) and calcitonin gene-related peptide (CGRP). Exposure of the cultures to 10 microM capsaicin and 100 nM RTX for periods of 2 days or longer resulted in almost complete elimination of SP-immunoreactive (IR) neurites and reduction, but not elimination, of CGRP-IR neurites. In addition, both 10 microM capsaicin and 100 nM RTX significantly reduced the number of SP- and CGRP-IR cell bodies within DRG explants. Capsaicin in 100 microM concentration produced complete elimination of SP-IR fibers and a greater decrease in the number of CGRP-IR fibers, but failed to completely eliminate IR cell bodies. Exposure of the cultures to the irritants in the same concentrations for 90 min did not produce a measurable effect on SP- or CGRP-IR in neurites or cell bodies. It is important to establish that the effect of capsaicin and RTX on cultured neurons was of long duration (longer than 4 days) and is therefore different from depletion of peptides. These findings demonstrate that processes of cultured sensory neurons are much more sensitive to capsaicin and RTX than cell bodies. Furthermore, our results show that SP-IR neuronal elements are more sensitive to capsaicin than CGRP-IR elements. These data suggest that cultured sensory neurons express the functional properties of differentiated sensory neurons in vivo.

Calcitonin gene-related peptide immunoreactivity in neuronal perikarya in dorsal root.

Calcitonin gene-related peptide (CGRP) has been localized in neuronal cell bodies in dorsal root ganglia (DRG) and in fibers of the dorsal horn (DH), where it may be involved in transmission of a sensory signal. The capacity of neurons from DRG and dorsal roots (DR) to produce CGRP in culture was examined in this investigation. CGRP-like immunoreactivity was observed in cultured neuronal cell bodies in the DRG and DR. CGRP-positive cell bodies in the DR explains why extirpation of the DRG fails to eliminate CGRP-positive fibers from the DH.

Cultured astrocytes express proteins involved in vesicular glutamate release.

Bradykinin induces receptor-mediated calcium-dependent release of glutamate from cultured astrocytes through a mechanism that is neither due to cell-swelling mechanism nor due to the reversal of the glutamate transporter. Astrocytes may thus release glutamate using a mechanism resembling the neuronal vesicular release of neurotransmitters. Synaptobrevin is a vesicular protein that together with plasma membrane proteins syntaxin and SNAP-25 participate in formation of the anchoring core complex required for initiation of exocytosis. Here, we demonstrate that synaptobrevin II is present in cultured astrocytes. Furthermore, we demonstrate that botulinus toxin type B and tetanus toxin cause a decrease in synaptobrevin II immunoreactivity and abolish bradykinin-induced release of glutamate from cultured astrocytes. While we were not able to demonstrate the presence of SNAP-25 or syntaxin immunoreactivity in cultured astrocytes, pretreatment with BoTx-A (which cleaves SNAP-25) and BoTx-C (which cleaves syntaxins) result in a decrease in the baseline release of glutamate and diminish the bradykinin-evoked release of glutamate from cultured astrocytes. These findings strongly support the notion that astrocytes may release neurotransmitters using a mechanism similar to the neuronal secretory process.

Excitatory amino acids are released from rat primary afferent neurons in vitro.

Multiple lines of evidence implicate the excitatory amino acids (EAAs) (L-aspartate (L-Asp) and L-glutamate (L-Glu) as excitatory transmitters in the spinal cord. The specific objective of this study was to determine whether the EAAs are released from primary afferents. Dorsal root ganglia (DRG) from 2 to 18-day-old rats dissected and cultured for 1-2 weeks were washed in modified Ringers recording solution for a period of 1 h to allow equilibration. The mean +/- S.E.M. baseline concentrations of EAAs recovered during a 5 min interval were 533.29 +/- 65.59 nmol for L-Glu and 106.67 +/- 14.05 nmol for L-Asp. Stimulation of DRG organotypic cultures with potassium resulted in a significant concentration-dependent increase in the release of both EAAs. The concentration of Asp increased to 166 +/- 17% and 203 +/- 13% in response to 5 min exposure of the culture to 25 and 50 mM potassium, respectively. The concentration of Glu increased to 155 +/- 12% and 226 +/- 18% of control in response to the same stimuli. In response to application of 50 mM potassium for 25 min, peak concentrations increased to 465 +/- 53% for Asp and 312 +/- 51% for Glu of the basal concentration. Exposure of the cultures to 1 or 10 microM capsaicin also caused release of both EAAs. The concentrations of Asp and Glu significantly increased to 204 +/- 11% and 165 +/- 15% of basal concentrations, respectively, in response to a 5 min exposure to 1 microM capsaicin. High [K+]e failed to increase the release of EAAs from cultures where DRG cell bodies were removed 72 h prior to release experiments. These results confirm results demonstrating release of EAA from mammalian spinal cord tissues and directly demonstrate for the first time that primary afferent fibers are specifically involved in this release.

Glutamate-mediated astrocyte-neuron signalling.

Neurotransmitter released from neurons is known to signal to neighbouring neurons and glia. Here we demonstrate an additional signalling pathway in which glutamate is released from astrocytes and causes an NMDA (N-methyl-D-aspartate) receptor-mediated increase in neuronal calcium. Internal calcium was elevated and glutamate release stimulated by application of the neuroligand bradykinin to cultured astrocytes. Elevation of astrocyte internal calcium was also sufficient to induce glutamate release. To determine whether this released glutamate signals to neurons, we studied astrocyte-neuron co-cultures. Bradykinin significantly increased calcium levels in neurons co-cultured with astrocytes, but not in solitary neurons. The glutamate receptor antagonists D-2-amino-5-phosphonopentanoic acid and D-glutamylglycine prevented bradykinin-induced neuronal calcium elevation. When single astrocytes were directly stimulated to increase internal calcium and release glutamate, calcium levels of adjacent neurons were increased; this increase could be blocked by D-glutamylglycine. Thus, astrocytes regulate neuronal calcium levels through the calcium-dependent release of glutamate.

ATP stimulates calcium-dependent glutamate release from cultured astrocytes.

ATP caused a dose-dependent, receptor-mediated increase in the release of glutamate and aspartate from cultured astrocytes. Using calcium imaging in combination HPLC we found that the increase in intracellular calcium coincided with an increase in glutamate and aspartate release. Competitive antagonists of P(2) receptors blocked the response to ATP. The increase in intracellular calcium and release of glutamate evoked by ATP were not abolished in low Ca(2+)-EGTA saline, suggesting the involvement of intracellular calcium stores. Pre-treatment of glial cultures with an intracellular Ca(2+) chelator abolished the stimulatory effects of ATP. Thapsigargin (1 microM), an inhibitor of Ca(2+)-ATPase from the Ca(2+) pump of internal stores, significantly reduced the calcium transients and the release of aspartate and glutamate evoked by ATP. U73122 (10 microM, a phospholipase C inhibitor, attenuated the ATP-stimulatory effect on calcium transients and blocked ATP-evoked glutamate release in astrocytes. Replacement of extracellular sodium with choline failed to influence ATP-induced glutamate release. Furthermore, inhibition of the glutamate transporters p-chloromercuri-phenylsulfonic acid and Ltrans-pyrolidine-2,4-dicarboxylate failed to impair the ability of ATP to stimulate glutamate release from astrocytes. However, an anion transport inhibitor, furosemide, and a potent Cl(-) channel blocker, 5-nitro-2(3-phenylpropylamino)-benzoate, reduced ATP-induced glutamate release. These results suggest that ATP stimulates excitatory amino acid release from astrocytes via a calcium-dependent anion-transport sensitive mechanism.

Neuroligand-evoked calcium-dependent release of excitatory amino acids from Schwann cells.

Bradykinin caused a receptor-mediated increase in release of the excitatory amino acids (EAAs) glutamate and aspartate from Schwann cell cultures obtained from dorsal root ganglia (DRG) together with an increase in the cytoplasmic level of free calcium. Perturbations which inhibited brady-kinin-induced calcium mobilization prevented the release of EAAs from glia. The addition of ionomycin caused a calcium-dependent release of EAAs. Therefore, bradykinin causes calcium dependent-release of EAAs from DRG Schwann cells. Bradykinin did not cause cell swelling and p-chloromercuriphenylsulfonic acid, an inhibitor of the electrogenic glutamate transporter, did not reduce bradykinin-induced EAA release. Therefore, bradykinin stimulates EAA release from Schwann cells through a mechanism that is neither the previously described volume regulated release mechanism nor due to the reversal of the glutamate transporter.

Neuroligand-evoked calcium-dependent release of excitatory amino acids from cultured astrocytes.

The release of excitatory amino acids (EAAs) from neuron-free cultures of neocortical astrocytes was monitored using HPLC. The neuroligand bradykinin caused a dose-dependent receptor-mediated increase in release of the EAAs glutamate and aspartate from type 1 astrocyte cell cultures obtained from rat cerebral cortex. Removal of calcium from the extracellular fluid prevented the bradykinin-induced release of EAAs from astrocytes. The addition of the calcium ionophore ionomycin caused a calcium-dependent release of EAAs. Inhibitors of the glutamate transporters p-chloromercuriphenylsulfonic acid, L-trans-pyrrolidine-2,4-dicarboxylate, and dihydrokainate failed to impair the ability of bradykinin to stimulate glutamate release from astrocytes. alpha-Latrotoxin, an active compound of black widow spider venom, caused a significant increase of the release of glutamate in calcium-containing saline. In calcium-depleted saline, alpha-latrotoxin produced an initial increase in the concentration of glutamate followed by a decline in the concentration of glutamate indicating stimulation of exocytosis coupled with low calcium-induced inhibition of endocytosis. Taken together, these data suggest that astrocytes may release neurotransmitter through a mechanism that is similar to the neuronal secretory process. Given the important role of glutamate in the induction of long-term potentiation, learning, memory, and excitotoxicity, it will be important to determine external signals that control both the uptake and release of glutamate by astrocytes.