Opiate agonist-induced re-distribution of Wntless, a mu-opioid receptor interacting protein, in rat striatal neurons.

Wntless (WLS), a mu-opioid receptor (MOR) interacting protein, mediates Wnt protein secretion that is critical for neuronal development. We investigated whether MOR agonists induce re-distribution of WLS within rat striatal neurons. Adult male rats received either saline, morphine or [d-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO) directly into the lateral ventricles. Following thirty minutes, brains were extracted and tissue sections were processed for immunogold silver detection of WLS. In saline-treated rats, WLS was distributed along the plasma membrane and within the cytoplasmic compartment of striatal dendrites as previously described. The ratio of cytoplasmic to total dendritic WLS labeling was 0.70±0.03 in saline-treated striatal tissue. Morphine treatment decreased this ratio to 0.48±0.03 indicating a shift of WLS from the intracellular compartment to the plasma membrane. However, following DAMGO treatment, the ratio was 0.85±0.05 indicating a greater distribution of WLS intracellularly. The difference in the re-distribution of the WLS following different agonist exposure may be related to DAMGO's well known ability to induce internalization of MOR in contrast to morphine, which is less effective in producing receptor internalization. Furthermore, these data are consistent with our hypothesis that MOR agonists promote dimerization of WLS and MOR, thereby preventing WLS from mediating Wnt secretion. In summary, our findings indicate differential agonist-induced trafficking of WLS in striatal neurons following distinct agonist exposure. Adaptations in WLS trafficking may represent a novel pharmacological target in the treatment of opiate addiction and/or pain.

Amygdalar peptidergic circuits regulating noradrenergic locus coeruleus neurons: linking limbic and arousal centers.

The endogenous opioid peptides, met- or leu-enkephalin, and corticotropin-releasing factor (CRF) regulate noradrenergic neurons in the locus coeruleus (LC) in a convergent manner via projections from distinct brain areas. In contrast, the opioid peptide dynorphin (DYN) has been shown to serve as a co-transmitter with CRF in afferents to the LC. To further define anatomical substrates targeting noradrenergic neurons by DYN afferents originating from limbic sources, anterograde tract-tracing of biotinylated dextran amine (BDA) from the central amygdaloid complex was combined with immunocytochemical detection of DYN and tyrosine hydroxylase (TH) in the same section of tissue. Triple labeling immunocytochemistry was combined with electron microscopy in the LC where BDA was identified using an immunoperoxidase marker, and DYN and TH were distinguished by the use of sequential immunogold labeling and silver enhancement to produce different sized gold particles. Results show direct evidence of a monosynaptic pathway linking amygdalar DYN afferents with LC neurons. To determine whether DYN-containing amygdalar LC-projecting neurons colocalize CRF, retrograde tract-tracing using fluorescent latex microspheres injected into the LC was combined with immunocytochemical detection of DYN and CRF in single sections in the central amygdala. Retrogradely labeled neurons from the LC were distributed throughout the rostro-caudal extent of the central nucleus of the amygdala (CeA) as previously described. Cell counts showed that approximately 42% of LC-projecting neurons in the CeA contained both DYN and CRF. Taken with our previous studies showing monosynaptic projections from amygdalar CRF neurons to noradrenergic LC cells, the present study extends this by showing that DYN and CRF are co-transmitters in monosynaptic projections to the LC and are poised to coordinately impact LC neuronal activity.

Lipopolysaccharide directly stimulates cortisol secretion by human adrenal cells by a cyclooxygenase-dependent mechanism.

Activation of the hypothalamo-pituitary-adrenal axis by bacterial lipopolysaccharide (LPS; endotoxin) is well documented, although there has been uncertainty about whether LPS exerts a direct effect at the level of the adrenal. The present study found that LPS caused a dose-dependent stimulation of basal cortisol secretion by the human adrenocortical cell line, NCI-H295R, without affecting aldosterone. The expression of both Toll-like receptor 2 (TLR2) and TLR4 was demonstrated in these cells, and the specific ligands for TLR4 (purified LPS and lipid A) and TLR2 (Pam3Cys) were found to stimulate cortisol release, suggesting that these receptors may mediate the effects of LPS in adrenal cells, as has been shown in other cell types. LPS was also found to stimulate prostaglandin E2 release by these cells. The effects of LPS on cortisol were attenuated in the presence of both indomethacin and a specific COX-2 inhibitor, but not a COX-1 inhibitor, suggesting an obligatory role for COX-2 activation and prostaglandin synthesis in the adrenal response to LPS.

Bacterial lipopolysaccharide directly stimulates cortisol secretion in human adrenal cells.

Adrenomedullin and pro-adrenomedullin N-terminal 20 peptide (PAMP) are expressed in vascular cells and in the adrenal cortex and medulla. Lipopolysaccharide (LPS), a bacterial product that induces septic shock, is a potent stimulant of adrenomedullin secretion in vascular cell types and is also known to stimulate the hypothalamo-pituitary-adrenal axis (HPA). The present study was designed to investigate the actions of LPS on the human adrenocortical cell line, H295R. Exposure of cells to LPS for 24 hours had no effect on adrenomedullin or PAMP secretion, but was found to significantly and selectively increase cortisol secretion with no effect on aldosterone. Dibutyryl cAMP, however, caused a significant increase in both adrenomedullin and PAMP release over this time period. There are two conclusions which can be drawn from these observations. First that adrenomedullin and PAMP are regulated by different mechanisms in vascular and adrenal cells and second, that LPS is able to directly stimulate cortisol secretion, with implications for the physiological response to septic shock.