MAB350 Sigma-AldrichAnti-Enkephalin Antibody, clone NOC1

Acupuncture or electroacupuncture (EA) potentially offers a nonpharmacological approach to reduce high blood pressure (BP). However, ~70% of the patients and animal subjects respond to EA, while 30% do not. EA acts, in part, through an opioid mechanism in the rostral ventrolateral medulla (rVLM) to inhibit sympathoexcitatory reflexes induced by gastric distention. CCK-8 opposes the action of opioids during analgesia. Therefore, we hypothesized that CCK-8 in the rVLM antagonizes EA modulation of sympathoexcitatory cardiovascular reflex responses. Male rats anesthetized with ketamine and α-chloralose subjected to repeated gastric distension every 10 min were examined for their responsiveness to EA (2 Hz, 0.5 ms, 1-4 mA) at P5-P6 acupoints overlying median nerve. Repeated gastric distension every 10 min evoked consistent sympathoexcitatory responses. EA at P5-P6 modulated gastric distension-induced responses. Microinjection of CCK-8 in the rVLM reversed the EA effect in seven responders. The CCK1 receptor antagonist devazepide microinjected into the rVLM converted six nonresponders to responders by lowering the reflex response from 21 ± 2.2 to 10 ± 2.9 mmHg (first vs. second application of EA). The EA modulatory action in rats converted to responders with devazepide was reversed with rVLM microinjection of naloxone (n = 6). Microinjection of devazepide in the absence of a second application of EA did not influence the primary pressor reflexes of nonresponders. These data suggest that CCK-8 antagonizes EA modulation of sympathoexcitatory cardiovascular responses through an opioid mechanism and that inhibition of CCK-8 can convert animals that initially are unresponsive to EA to become responsive.

Purines such as adenosine 5'-triphosphate (ATP) act as extracellular messengers through specific purinergic receptors. Three different classes of purinergic receptors have been identified and termed P1, P2X, and P2Y. The purinergic receptor subunit P2X2 is a ligand-gated ion channel that is widely expressed by neurons in the CNS. In the brainstem medulla oblongata, the ionotropic P2X2 receptor (P2X2R) is enriched in the area postrema (AP). Two different antisera to P2X2R were used to determine the chemical nature of P2X2R immunoreactive cell bodies in the rat AP, an area lacking a blood-brain barrier. Subcellularly, P2X2R immunoreactivity was located to the periphery of individual cell bodies. The majority of P2X2R-immunoreactive cells were shown to contain tyrosine hydroxylase (TH) (63.5 ± 7.7%) and dopamine β-hydroxylase (61.5 ± 5.1%). Phenylethanolamine N-methyltransferase (PNMT)-containing cells were not detected in the AP, supporting a noradrenergic nature of P2X2R cells in the AP. There were no P2X2R-immunoreactive cells in the AP that contained the GABA-synthesizing enzyme glutamic acid decarboxylase 65. Only single vesicular glutamate transporter 2-immunoreactive cell bodies that were not P2X2R-positive were demonstrated in the AP. Some P2X2R-positive cells in the AP were immunoreactive for the neuropeptides substance P and pituitary adenylate cyclase-activating polypeptide, whereas dynorphin-, enkephalin-, or cholecystokinin-positive cells were not P2X2R-immunoreactive. Presence of P2X2R in a majority of noradrenergic cells of the AP implies that ATP may have a regulatory action on neuronal noradrenaline release from the AP, a circumventricular organ with a strategic position enabling interactions between circulating substances and the central nervous system.

Chemokine receptors are now known to play an important role in cancer growth and metastasis. However, there is little information regarding chemokine expression in gastric cancer. In this study, we examined CXCL12 expression in gastric cancer and also evaluated whether the down-regulation of CXCL12 is due to aberrant methylation of the gene.

Acupuncture regulates autonomic function. Our previous studies have shown that electroacupuncture (EA) at the Jianshi-Neiguan acupoints (P5-P6, underlying the median nerve) inhibits central sympathetic outflow and attenuates excitatory cardiovascular reflexes, in part, through an opioid mechanism. It is unknown if EA at these acupoints influences the parasympathetic system. Thus, using c-Fos expression, we examined activation of nucleus ambiguus (NAmb) neurons by EA, their relation to cholinergic (preganglionic parasympathetic) neurons and those containing enkephalin. To enhance detection of cell bodies containing enkephalin, colchicine (90-100 μg/kg) was administered into the subarachnoid space of cats 30 h prior to EA or sham-operated controls for EA. Following bilateral barodenervation and cervical vagotomy, either EA for 30 min at P5-P6 acupoints or control stimulation (needle placement at P5-P6 without stimulation) was applied. While perikarya containing enkephalin were observed in some medullary nuclei (e.g., raphé), only enkephalin-containing neuronal processes were found in the NAmb. Compared to controls (n=4), more c-Fos immunoreactivity, located principally in close proximity to fibers containing enkephalin was noted in the NAmb of EA-treated cats (n=5; Pless than 0.01). Moreover, neurons double-labeled with c-Fos and choline acetyltransferase in the NAmb were identified in EA-treated, but not control animals. These data demonstrate for the first time that EA activates preganglionic parasympathetic neurons in the NAmb. Because of their close proximity, these EA-activated neurons likely interact with nerve fibers containing enkephalin. These results suggest that EA at the P5-P6 acupoints has the potential to influence parasympathetic outflow and cardiovascular function, likely through an enkephalinergic mechanism.

Urocortin 3 (Ucn 3) is a corticotrophin-releasing factor related neuropeptide highly expressed in the brain. Ucn 3 nerve fibers heavily innervate the hypothalamic ventromedial nucleus (VMH), and Ucn 3 injection into the VMH suppresses feeding. Currently, the origin of the Ucn 3 afferent input into the VMH is unknown. In the present study, anatomical tracing shows that the major Ucn 3 afferent input to the VMH resides in the anterior parvicellular part of the paraventricular nucleus of the hypothalamus (PVHap) and the adjacent posterior part of the bed nucleus of stria terminalis (pBNST). VMH also receives moderate Ucn 3 input from the medial amygdala. Ucn 3 neurons located immediately caudal to the PVHap/pBNST in the rostral perifornical hypothalamic area (rPFH) provide only minimal input. The paucity of rPFH-VMH Ucn 3 projection is consistent with the finding that only Ucn 3 neurons in the rPFH co-expressed enkephalin (Enk), and Ucn 3/Enk double-labeled nerve fibers and terminals were observed predominately in the lateral septum (LS), whereas only a few double-labeled fibers were found in other brain areas including the VMH. Furthermore, retrograde tracing demonstrates that Ucn 3 neurons in the rPFH project to the LS. In conclusion, the present study determines that the major Ucn 3 afferent into the VMH originates from the PVHap/pBNST. Moreover, anatomical heterogeneity is observed in the hypothalamic Ucn 3 neuron population as the rostral part (PVHap/pBNST) of the population projects to the VMH and the caudal part (rPFH) co-localizes with Enk and provides major afferent input to the LS.

In thrombotic processes, during the association of leukocytes with platelets and endothelial cells, P-selectin glycoprotein ligand-1 (PSGL-1) binds to P-selectin, expressed on activated platelets and endothelial cells. Our aim was to establish the role of PSGL-1 in thrombus formation by evaluating the response to thrombotic stimuli in wild type and PSGL-1 knockout mice.

Chemokine receptor CXCR4 is known to be crucially involved in tumor progression, but the role of its ligand, stromal cell-derived factor-1 (SDF-1), remains unclear. The present study was conducted to clarify the clinicopathological and prognostic impact of SDF-1 expression in invasive breast cancers. Expression of SDF-1 mRNA and protein was examined in five breast cancer cell lines with or without estradiol treatment. In 52 surgically resected breast cancers, the level of SDF-1 mRNA in frozen samples and the pattern of SDF-1 protein immunoreactivity in formalin-fixed paraffin-embedded tissue sections were compared. In another cohort of 223 breast cancers, the correlation between SDF-1 immunoreactivity and clinicopathological parameters was examined using a tissue microarray. Estradiol treatment markedly increased the expression of SDF-1 mRNA and protein in the estrogen receptor (ER)-positive cell lines, MCF-7 and T47D. Among the 52 resected breast cancers, those with a cytoplasmic-dominant pattern of SDF-1 expression showed higher SDF-1 mRNA levels (median 27.4) than those with a membrane-dominant or negative pattern (median 13.6, P = 0.0017). Accordingly, the cytoplasmic-dominant pattern was defined as high SDF-1 expression, and other patterns were defined as low SDF-1 expression. Among the cohort of 223 tumors, high SDF-1 expression was detected in 158 (70.9%) and was significantly correlated with ER positivity (P < 0.0001), HER2 negativity (P = 0.021), and lower grade (P < 0.0001). Univariate analysis demonstrated that high SDF-1 expression was a significant indicator of better clinical outcome in both the entire patient cohort (P = 0.017) and the 133 patients with ER-positive tumors (P = 0.036), but not in the 90 patients with ER-negative tumors. Multivariate analysis showed that SDF-1 status was an independent factor related to overall survival in patients with ER-positive tumors (P = 0.046). SDF-1 status is a significant prognostic factor and may be clinically useful for assigning adjuvant therapy to patients with ER-positive invasive breast cancers.

Complete thoracic (T) spinal cord injury (SCI) above the T6 level typically results in autonomic dysreflexia, an abnormal hypertensive condition commonly triggered by nociceptive stimuli below the level of SCI. Overexpression of nerve growth factor in the lumbosacral spinal cord induces profuse sprouting of nociceptive pelvic visceral afferent fibers that correlates with increased hypertension in response to noxious colorectal distension. After complete T4 SCI, we evaluated the plasticity of propriospinal neurons conveying visceral input rostrally to thoracic sympathetic preganglionic neurons. The anterograde tracer biotinylated dextran amine (BDA) was injected into the lumbosacral dorsal gray commissure (DGC) of injured/nontransected rats immediately after injury (acute) or 2 weeks later (delayed). At 1 or 2 weeks after delayed or acute injections, respectively, a higher density (P < 0.05) of BDA(+) fibers was found in thoracic dorsal gray matter of injured vs. nontransected spinal cords. For corroboration, fast blue (FB) or cholera toxin subunit beta (CTb) was injected into the T9 dorsal horns 2 weeks postinjury/nontransection. After 1 week transport, more retrogradely labeled (P < 0.05) DGC propriospinal neurons (T13-S1) were quantified in injured vs. nontransected cords. We also monitored immediate early gene c-fos expression following colorectal distension and found increased (P < 0.01) c-Fos(+) cell numbers throughout the DGC after injury. Collectively, these results imply that, in conjunction with local primary afferent fiber plasticity, injury-induced sprouting of DGC neurons may be a key constituent in relaying visceral sensory input to sympathetic preganglionic neurons that elicit autonomic dysreflexia after high thoracic SCI.

The distribution of substance P (SP) immunoreactivity and the colocalization of SP with other bioactive substances in chromaffin cells and nerve fibers were investigated in the rat adrenal gland at the light microscopic level. In the capsule and cortex, SP immunoreactivity was seen in some nerve fibers around blood vessels and in thick nerve bundles passing through the cortex directly into the medulla. In the medulla, the SP immunoreactivity was observed in a small number of chromaffin cells; these SP-immunoreactive chromaffin cells were either phenylethanolamine N-methyltransferase (PNMT) immunoreactive or immunonegative, indicating that they were either adrenaline cells or noradrenaline (NA) cells. SP-immunoreactive varicose nerve fibers were also found in the medulla and were in contact with a cluster of the NA cells showing catecholamine fluorescence, which suggests that SP from medullary nerve fibers may regulate the secretory activity of the NA cells. Because no SP-immunoreactive ganglion cell was present in the rat adrenal gland, the intra-adrenal nerve fibers were considered to be extrinsic in origin. The double-immunostaining method further revealed that the SP-immunoreactive chromaffin cells also exhibit immunoreactivities for calcitonin gene-related peptide (CGRP), and neuropeptide tyrosine (NPY), suggesting that these peptides can also be released from the chromaffin cells by certain stimuli. The intra-adrenal nerve fibers in the medulla were composed of SP-single immunoreactive, and SP/CGRP-, SP/choline acetyltransferase (ChAT)-, SP/nitric oxide synthase (NOS)-, SP/pituitary adenylate cyclase activating polypeptide (PACAP)-, ChAT/NOS-, and ChAT/PACAP-immunoreactive nerve fibers, which may affect the secretory activity of the NA cells. In the adrenal capsule, the nerve fibers were present around blood vessels and showed immunoreactivities for SP/ CGRP, SP/NPY, SP/NOS, and SP/vasoactive intestinal polypeptide, suggesting that the origin of nerve fibers in the capsule may differ from those in the medulla.

Group I metabotropic glutamate receptors (mGluRIs) can be colocalized with ionotropic glutamate receptors in postsynaptic membranes. We have investigated whether mGluRIs alter the gene transcription induced by N-methyl-D-aspartate (NMDA) and (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolpropionic acid (AMPA) receptors in rat neocortical gamma-aminobutyric acid (GABA) interneurons. In cultures of dissociated interneurons, the mGluRI antagonists LY367385 and MPEP reduced the increase in phosphorylation of the transcription factor CREB induced by NMDA as well as the expression of the proenkephalin (PEnk) gene. In contrast, they enhanced the AMPA-induced CREB phosphorylation and PEnk gene expression. Stimulation of the mGluRIs was due to network activity that caused the release of endogenous glutamate and could be blocked by tetrodotoxin. In organotypic cultures of neocortex, endogenous glutamate enhanced the PEnk gene expression by acting on NMDA and AMPA receptors. These effects were modulated via mGluRIs. In patch-clamp experiments and in biochemical studies on receptor density, stimulation of mGluRIs acutely affected NMDA receptor currents but had no long-term effect on NMDA receptor density at the cell surface. In contrast, stimulation of mGluRIs decreased the density of AMPA receptors located at the cell surface. Our results suggest that mGluRIs regulate the glutamate-induced gene expression in neocortical interneurons in a physiologically relevant manner.