Soluble guanylate cyclase and neuronal nitric oxide synthase colocalize in rat nucleus tractus solitarii.

Nitric oxide has been implicated in transmission of cardiovascular signals in the nucleus tractus solitarii (NTS). Pharmacological studies suggest that activation of neurons by nitric oxide in the NTS may involve soluble guanylate cyclase (sGC). However, anatomical data supporting this suggestion have not been available. In this study, we tested the hypothesis that neurons and fibers containing neuronal nitric oxide synthase (nNOS) lie in close proximity to those containing sGC and the two enzymes colocalize in some neurons and fibers in the NTS. We perfused six rats and obtained brain stem sections for double immunofluorescent staining utilizing antibodies selective for sGC and for nNOS combined with confocal microscopy. The distribution and staining intensity of nNOS-immunoreactivity (IR) was similar to our earlier reports. IR of sGC was present in cell bodies, proximal dendrites and fibers of many brain stem regions. Strong sGC-IR was noted in the hypoglossal, dorsal motor nucleus of vagus and gracilis nuclei. The NTS exhibited moderate sGC-IR. Superimposed images showed that many NTS neurons contained both nNOS-IR and sGC-IR. The percentage of sGC-IR positive cells that were also nNOS-IR positive differed among NTS subnuclei. Similarly, the percentage of nNOS-IR positive cells that were also sGC positive differed among NTS subnuclei. Fibers stained for both nNOS-IR and sGC-IR were also present in NTS subnuclei. In addition, we identified fibers that were stained for nNOS-IR or sGC-IR alone and often found such singly labeled fibers apposed to each other. These data support our hypothesis and provide anatomical support for the suggestion that nitroxidergic activation of the NTS involves sGC.

Nerve plexuses in the trachea and extrapulmonary bronchi of the rat.

Intrinsic nerve plexuses of the rat trachea and extrapulmonary bronchi were examined by immunohistochemistry. Three nerve plexuses--peritracheal and peribronchial, intramuscular, and submucosal--were found in the wall of the trachea and bronchi. Nerve cell bodies were located in the peritracheal and peribronchial nerve plexuses. They occurred singly or formed ganglia in the plexus, and regional differences in cell numbers were found in the cervical and thoracic portions of the trachea and in the extrapulmonary bronchia. In total, 83.5 +/- 28.3 ganglia (mean +/- SD, 57-131, n=5) and 749.8 +/- 221.1 nerve cell bodies (540-1,080, n=5) were found in the nerve plexus. The mean densities of ganglia were 0.31, 0.97 and 1.15/mm2, and the mean densities of the nerve cell bodies were 1.82, 9.26 and 11.54/mm2 in the cervical region, thoracic region of trachea, and extrapulmonary bronchi, respectively. Almost all nerve cell bodies in ganglia were positive for choline acetyltransferase and neuropeptide Y (NPY), and a few cells were positive for vasoactive intestinal peptide (VIP). In addition, in cholinergic nerves, a few nerve fibers in the smooth muscles were positive for substance P (SP), calcitonin gene-related peptide (CGRP), and VIP, and a moderate number of fibers were positive for NPY. Tyrosine hydroxylase-immunoreactive nerve fibers were observed around blood vessels and within nerve bundles in the tunica adventitia. In the epithelium, nerve fibers were positive for SP and CGRP. Our results indicate that postganglionic neurons form three layers of cholinergic plexuses in the rat trachea and extrapulmonary bronchi, and that all of these possess intrinsic and extrinsic peptidergic innervation.

Chronic morphine-induced loss of the facilitative interaction between vasoactive intestinal polypeptide and delta-opioid: involvement of protein kinase C and phospholipase Cbetas.

This laboratory recently demonstrated a multiplicative interaction between the pelvic visceral afferent transmitter vasoactive intestinal polypeptide (VIP) and the delta-opioid receptor (DOR)-selective agonist [D-Pen2,5] enkephalin (DPDPE) to regulate cAMP levels in spinal cord [Brain Res. 959 (2003) 103]. Although DOR activation is required for the manifestation of the VIP-DPDPE facilitative interaction, its relevance to opioid antinociception remains unclear. The current study investigates whether or not the VIP-DPDPE facilitation of cAMP formation is subject to tolerance formation, a hallmark characteristic of opioid antinociception. Chronic morphine exposure abolishes the VIP-DPDPE facilitative interaction, consistent with its relevance to DOR antinociception. However, acute in vitro inhibition of protein kinase C (PKC) reinstates the VIP-DPDPE multiplicative interaction characteristic of opioid naïve spinal tissue. This suggests that its chronic morphine-induced loss requires a PKC phosphorylation. PKC phosphorylation negatively modulates phospholipase C (PLC)beta, enzymes intimately associated with phosphoinositide turnover and calcium trafficking. These are essential determinants of acute and chronic opioid effects. Accordingly, the effect of chronic morphine on their state of phosphorylation was also investigated. Central nervous system opioid tolerance is associated with the reciprocal phosphorylation (regulation) of two PLCbeta isoforms, PLCbeta1 and PLCbeta3. However, although chelerythrine reinstates the chronic morphine-induced loss of the multiplicative VIP-DPDPE interaction, it does not alter the associated changes in PLCbeta phosphorylation, possibly indicating different time courses of restitution of function and/or involvement of different kinases for different components of tolerance. These results could provide a mechanistic rubric for understanding positive modulation of opioid antinociception by afferent transmission.

Modulation of visceral pain and inflammation by protease-activated receptors.

The gastrointestinal (GI) tract is exposed to a large array of proteases, under both physiological and pathophysiological conditions. The discovery of G protein-coupled receptors activated by proteases, the protease-activated receptors (PARs), has highlighted new signaling functions for proteases in the GI tract, particularly in the domains of inflammation and pain mechanisms. Activation of PARs by selective peptidic agonists in the intestine or the pancreas leads to inflammatory events and changes in visceral nociception, suggesting that PARs could be involved in the modulation of visceral pain and inflammation. PARs are present in most of the cells that are potentially actors in the generation of irritable bowel syndrome (IBS) symptoms. Activation of PARs interferes with several pathophysiological factors that are involved in the generation of IBS symptoms, such as altered motility patterns, inflammatory mediator release, altered epithelial functions (immune, permeability and secretory) and altered visceral nociceptive functions. Although definitive studies using genetically modified animals, and, when available, pharmacological tools, in different IBS and inflammatory models have not yet confirmed a role for PARs in those pathologies, PARs appear as promising targets for therapeutic intervention in visceral pain and inflammation processes.

Immunocytochemical localization of extracellular signal-regulated kinases 1 and 2 phosphorylated neurons in the brainstem of rat following visceral noxious stimulation.

The aim of the present study was to determine the activation of the extracellular signal-regulated kinases (ERKs) 1 and 2 in brainstem neurons following noxious visceral stimulation. Ether and urethane anaesthetized rats received an intraperitoneal injection of acetic acid (ENS, UNS) or were left untreated (ECT, UCT). Paraffin embedded brain sections immunoreacted with an antibody specific for phosphorylated ERKs. In noxious stimulated rats ERKs activated neuron profiles in the periaqueductal gray matter, parabrachial, dorsal raphe, solitary tract nucleus, area postrema and superior colliculus suggest that ERKs activation takes place mainly in brainstem nuclei in which nociception and visceral activities interact. The comparison between ENS and UNS rats shows that the long acting anaesthetic urethane attenuates the number of the ERKs activated neurons compared to the short acting ether.

TRPC6 immunoreactivity is colocalized with neuronal nitric oxide synthase in extrinsic fibers innervating guinea pig intrinsic cardiac ganglia.

Tachykinins depolarize guinea pig intracardiac neurons by activating nonselective cationic channels. Recently, members of the transient receptor potential family of membrane channels (TRPC) have been implicated in the generation of G protein-coupled receptor-activated nonselective cationic currents. We have investigated whether guinea pig cardiac neurons exhibit immunoreactivity to TRPC. Our results showed that nerve fibers within guinea pig intrinsic cardiac ganglia exhibited immunoreactivity to TRPC6. After culture of cardiac ganglia whole-mount explants for 72 hours, the TRPC6-IR fiber networks were absent. Therefore, the TRPC6-IR fibers were derived from sources extrinsic to the heart. A small percentage ( approximately 3%) of intracardiac neurons also exhibited TRPC6 immunoreactivity in control preparations, and the percentage of cells exhibiting TRPC6 immunoreactivity was not changed following explant culture for 72 hours. The few intrinsic TRPC6-IR neurons also exhibited nitric oxide synthase (NOS) immunoreactivity, indicating that they were nitrergic as well. We compared the immunohistochemical staining patterns of TRPC6-IR fibers with the staining patterns of a number of other neurotransmitters or neurotransmitter synthetic enzymes that mark specific extrinsic inputs to the intrinsic cardiac ganglia. The TRPC6-IR fibers were not immunoreactive for choline acetyltransferase, tyrosine hydroxylase, or substance P. However, the TRPC6-IR fibers exhibited immunoreactivity to neuronal NOS. Therefore, we propose that the TRPC6-IR fibers within the guinea pig intrinsic cardiac ganglia are vagal sensory fibers that also contain NOS. We found, in support of this conclusion, that TRPC6-IR cells were also present in sections of nodose ganglia.

Neutral endopeptidase knockout induces hyperalgesia in a model of visceral pain, an effect related to bradykinin and nitric oxide.

Neutral endopeptidase (EC3.4.24.11, NEP, enkephalinase) is a zinc-metalloendopeptidase, cleaving a variety of substrates like enkephalins, substance P, and bradykinin. In the brain, NEP is a key enzyme in the degradation of enkephalins. Pharmacological inhibition of NEP-activity causes analgesia resulting from enhanced extracellular enkephalin concentrations. Recently, transgenic mice lacking the enzyme NEP have been developed (Lu, 1995). The present study was designed to investigate the nociceptive behavior of these NEP-knockout mice. Interestingly, NEP-deficient mice did not respond with decreased pain perception, but exhibited hyperalgesia in the hot-plate jump, warm-water tail-withdrawal, and mostnotablyin theacetic-acid writhing test. Inhibition of aminopeptidase N by bestatin reduced writhing in both strains, whereas NEP-inhibition by thiorphan reduced writhing selectively in wild-type mice. Naloxone increased writhing in wild-type but not in knockouts, whereas the bradykinin B2-receptor antagonist HOE140 reduced writhing selectively in NEP-knockouts. Similarly, the nitric oxide synthase inhibitor L-NAME reduced writhing in NEP-knockouts. These results indicate that genetic elimination of NEP, in contrast to pharmacological inhibition, leads to bradykinin-induced hyperalgesia instead of enkephalin-mediated analgesia. Nitric oxide (NO) is suggested to be involved in this process.

Association of extracellular acetylcholinesterase with gustatory nerve terminal fibers in the nucleus of the solitary tract.

Acetylcholinesterase (AChE) staining is associated with terminal fields of the glossopharyngeal and chorda tympani nerves in the nucleus of the solitary tract (NST). To address AChE function at these sites, the location of the staining was examined at the fine structural level in combination with the labeling of chorda tympani nerve fibers with biotinylated dextran in golden Syrian hamsters. AChE staining was located in the endoplasmic reticulum of geniculate ganglion neuronal somata, and extracellularly, surrounding labeled chorda tympani terminal fibers and boutons in the NST. Neuronal profiles adjacent to these labeled fibers were stained less intensely, whereas most non-adjacent profiles were unstained. The location of staining is consistent with the secretion of AChE into the extracellular space by primary afferent chorda tympani fibers. AChE staining was reduced in the dextran-labeled chorda tympani fibers and terminals as well as adjacent non-labeled profiles 2 weeks following nerve transection and dextran application. The distribution of staining outside synapses and the loss of staining following denervation is suggestive of a non-cholinergic role for AChE in the intact gustatory system.