Neurogenic nitric oxide mediates relaxation of canine corpus cavernosum

PURPOSE: The aim of the present study is to analyze mechanisms underlying neurogenic relaxation of the corpus cavernosum which are believed to participate in penile erection. MATERIALS AND METHODS: Mechanical responses to nerve stimulation by electrical pulses and nicotine were measured in strips of canine corpus cavernosum precontracted with phenylephrine. Cyclic guanosine monophosphate (GMP) contents in the strips were also measured by radioimmunoassay. Immunohistochemistry for nitric oxide synthase (NOS) and vasoactive intestinal polypeptide (VIP) was performed. RESULTS: Transmural electrical stimulation and nicotine produced relaxations in the isolated canine corpus. The neurogenic relaxation was abolished by N omega-nitro-L-arginine, a NOS inhibitor, and the inhibition was reversed by L-arginine. Relaxations induced by nerve stimulation and exogenous nitric oxide (NO) were depressed by oxyhemoglobin and methylene blue. Vasoactive intestinal polypeptide (VIP)-induced relaxations were not influenced by these inhibitors. In the controls strips and those made unresponsive to VIP by its repeated application, the responses to nerve stimulation did not differ. The content of cyclic GMP in the tissue increased in response to nicotine, the effect being abolished by the NO synthase inhibitor. Immunohistochemical study demonstrated neurons containing NOS and VIP. CONCLUSIONS: It appears that the relaxation induced by nerve stimulation is mediated solely by NO liberated from the nerve that activates soluble guanylate cyclase and increases the production of cyclic GMP in smooth muscle, whereas VIP does not play a role in the regulation of muscle tone under the experimental conditions used.     

Interstitial cells of Cajal mediate inhibitory neurotransmission in the stomach

The structural relationships between interstitial cells of Cajal (ICC), varicose nerve fibers, and smooth muscle cells in the gastrointestinal tract have led to the suggestion that ICC may be involved in or mediate enteric neurotransmission. We characterized the distribution of ICC in the murine stomach and found two distinct classes on the basis of morphology and immunoreactivity to antibodies against c-Kit receptors. ICC with multiple processes formed a network in the myenteric plexus region from corpus to pylorus. Spindle-shaped ICC were found within the circular and longitudinal muscle layers (IC-IM) throughout the stomach. The density of these cells was greatest in the proximal stomach. IC-IM ran along nerve fibers and were closely associated with nerve terminals and adjacent smooth muscle cells. IC-IM failed to develop in mice with mutations in c-kit. Therefore, we used W/W(V) mutants to test whether IC-IM mediate neural inputs in muscles of the gastric fundus. The distribution of inhibitory nerves in the stomachs of c-kit mutants was normal, but NO-dependent inhibitory neuro-regulation was greatly reduced. Smooth muscle tissues of W/W(V) mutants relaxed in response to exogenous sodium nitroprusside, but the membrane potential effects of sodium nitroprusside were attenuated. These data suggest that IC-IM play a critical serial role in NO-dependent neurotransmission: the cellular mechanism(s) responsible for transducing NO into electrical responses may be expressed in IC-IM. Loss of these cells causes loss of electrical responsiveness and greatly reduces responses to nitrergic nerve stimulation.     

A new source of cells contributing to the developing gastrointestinal tract demonstrated in chick embryos

BACKGROUND & AIMS: Smooth muscle cells in the walls of the gastrointestinal tract are thought to derive solely from mesoderm surrounding the primitive gut. A population of neuroepithelial cells has recently been shown to migrate from the ventral part of the neural tube in the region joined by the vagus nerve. We sought to determine if these cells contributed to the development of the stomach and intestine. METHODS: Cells of the ventral hindbrain of chick embryos were tagged by replication-deficient retroviral vectors containing the lacZ gene, providing a permanent label that is transmitted without dilution as the cells divide. Embryos were processed for detection of labeled cells. Specific markers were used to determine differentiation of progeny in the gastrointestinal tract. RESULTS: Cells labeled in the ventral neural tube migrate in association with the vagus nerve. Labeled cells are found in the intestine and stomach after time for further migration and differentiation. Using a specific marker, they were clearly identified as smooth muscle cells. CONCLUSIONS: Some of the smooth muscle cells of the gastrointestinal tract are derived from precursor cells that originate in the ventral part of the hindbrain neural tube. Their developmental importance and functional significance remain to be determined.     

Further studies concerning possible transmitters from NANC nerves in the circular muscle of the rat stomach fundus

We examined the characteristics of the non-adrenergic, non-cholinergic (NANC) inhibitory response of the circular muscle of the rat stomach fundus to transmural nerve stimulation or high K+. Treatments with isotonic high K+ (20 mM), nitric oxide (NO) and sodium nitroprusside (SNP) all elevated cyclic GMP levels in the rat stomach fundus in the presence of atropine and guanethidine. Isotonic high K+-induced formation of cyclic GMP was completely inhibited by tetrodotoxin (TTX) or NG-nitro-L-arginine (L NNA). The K+ also increased cyclic AMP levels and this response was completely inhibited by TTX. Dose-dependent relaxation of the fundus in response to SNP was shifted to the right by a prior incubation with high concentration of SNP (10(-4) M) for 2 hrs. Incubating the fundus with SNP for 2 hrs significantly inhibited NO induced cyclic GMP formation. Relaxation responses to transmural stimulation (1 Hz or 30 Hz), isotonic high K+ and NO were significantly reduced by a prior incubation with SNP. Isotonic high K+ (20 mM)-induced relaxation of circular muscle strips was not completely inhibited by combined treatment with 10(-5)M L-NNA, 5 x 10(-5)M oxyhemoglobin and anti VIP (1:200). These results suggest that NO as well as VIP is possible transmitter from NANC nerves in the circular muscle of the rat stomach fundus and there should be one or more inhibitory mediators other than VIP and NO.     

Nerve stimulation and denervation induce differential patterns of immediate early gene mRNA expression in skeletal muscle

Many properties of skeletal muscle cells are closely regulated by motor nerves. Neuromuscular synaptic transmission (including the 'activity' it triggers) mediates many of these effects, while denervation results in a different spectrum of muscle cell changes. However, little is known about the early regulatory events that occur in mature muscle cells in response to muscle activity or denervation. We have examined the effects of motor nerve stimulation and denervation on the expression of 4 immediate early genes (IEGs)--c-jun, junB, zif268, and nur77--in mature mouse gastrocnemius muscle. Electrical stimulation of the sciatic nerve in a pattern of brisk intermittent exercise induced a marked rise in zif268 and c-jun mRNA levels within 45 min, a minimal rise in junB, and no change in nur77 mRNA levels. By contrast, surgical denervation resulted in a marked increase of c-jun, a slight rise in junB, and no change in nur77 or zif268 mRNA levels. These findings show that neural stimulation and denervation lead to differential patterns of IEG expression. The selectivity of these patterns suggests that differential IEG expression may play an important role in regulating the specific phenotypic changes in skeletal muscles that result from denervation, innervation, and various patterns of stimulation.     

Responses to electrical stimulation, noradrenaline, serotonin, and vasopressin in the isolated ear artery of the developing lamb and ewe

Responses of isolated helical strips of ovine ear artery to electrical stimulation of postganglionic adrenergic neurons and exogenous agonists were studied at various stages of development from 110 days of gestation through to adulthood. Only rudimentary responses were observed at 110-115 days of gestation. A parallel development of responses to noradrenaline (NA), serotonin, and lysine vasopressin began sometime after 110-115 days of gestation and continued until 133-137 days of gestation but there was little development of the latter responses until more than 3-5 days post partum. Development of responses to exogenous agonists was incomplete 2-3 weeks post partum. The development of postganglionic adrenergic responses lagged behind those to exogenous NA. Two to three weeks post partum the NA maximal response was one-third that of adult tissue whereas the response to 16 Hz (highest frequency used) was one-sixth that of adult tissue. The NA threshold concentration was lower in arterial strips of adult animals than it was in those of younger animals. The data suggest that development of functional post-ganglionic adrenergic innervation of vascular smooth muscle begins late in gestation and continues well after birth; this development is preceded by development of vascular mechanisms involved in the response to several agonists.     

L-arginine deficiency causes suppression of nonadrenergic noncholinergic nerve-mediated smooth muscle relaxation: role of L-citrulline recycling

Studies were performed on the internal anal sphincter (IAS) smooth muscle strips obtained from opossums (Didelphis virginiana). Isometric tension and L-arginine levels of the tissues were measured under basal conditions, in the presence of electrical field stimulation (EFS) and after treatment with different concentrations of arginase. For the nonadrenergic noncholinergic nerve stimulation, short trains (4 sec) as well as continuous EFS were used. During continuous EFS, after the initial IAS relaxation, the response began to fade within several min to approximately 80% recovery of the basal tone. We also examined the influence of L-arginine and L-citrulline on these responses. For some studies, the tissues were pretreated with L-glutamine (an inhibitor of L-citrulline uptake), L-glutamate or N(G)-hydroxy-L-arginine (an inhibitor of arginase). Interestingly, the basal levels of L-arginine were found to be significantly higher in the IAS (tonic smooth muscle) than in the rectal (phasic smooth muscle) smooth muscle. Arginase caused a concentration-dependent attenuation of the IAS relaxation caused by EFS. L-Citrulline and L-arginine were equipotent in reversing the attenuation. Both arginase (60 min pretreatment) and continuous EFS (tissues collected at the time of maximal recovery of the basal IAS tone after the initial relaxation) caused significant decreases in L-arginine levels. The decreases in the levels of L-arginine were restored by the exogenous administration of either L-arginine or L-citrulline. The restoration of L-arginine levels by L-citrulline but not by L-arginine was selectively blocked by L-glutamine. Furthermore, the IAS relaxation, attenuated by arginase was unaffected by L-glutamine but was restored by N-hydroxy-L-arginine pretreatment. The studies suggest that L-citrulline-L-arginine recycling may play a significant role in the maintenance of IAS relaxation in response to nonadrenergic noncholinergic nerve stimulation.     

Development of nicotinic receptor clusters and innervation accompanying the change in muscle phenotype in the mouse esophagus

During development, the external muscle of the mouse esophagus undergoes a transdifferentiation from smooth to striated muscle (Patapoutian et al. [1995] Science 270:1818-1821). We now report on the development of the innervation accompanying the change in phenotype of the external muscle of the mouse esophagus. The phenotype of the muscle was monitored by using light and electron microscopy. Nicotinic acetylcholine receptors were localised by using a fluorescence conjugate of alpha-bungarotoxin, and neural elements were localised by using antisera to synaptophysin (a synaptic vesicle protein that was used to label all nerve terminals), the vesicular acetylcholine transporter (VAChT), calcitonin gene-related peptide (CGRP), nitric oxide synthase (NOS), and vasoactive intestinal peptide (VIP). CGRP and VAChT were co-localised in the terminals of vagal motoneurons that innervate the external muscle, and NOS and VIP were co-localised in intrinsic (enteric) neurons, which provide some terminals that are associated with motor endplates. Cells exhibiting striations were first observed in the outer layers of the most rostral regions of the esophagus of embryonic day 15 (E15) mice. Clusters of nicotinic acetylcholine receptors were also first observed at the rostral end of the esophagus of E15 mice, and developed in a rostrocaudal progression that coincided with the appearance of striations within the muscle cells. Synaptophysin-, VAChT- and NOS-immunoreactive nerve terminals were present within the external muscle prior to the formation of receptor clusters, and their appearance did not follow any apparent rostrocaudal sequence. Surprisingly, not all of the receptor clusters at E15 had synaptophysin- and VAChT-immunoreactive nerve terminals closely associated with them. However, from E18 on, almost all of the clusters had synaptophysin-immunoreactive nerve terminals in close association. At late embryonic and early postnatal stages, there was a rostrocaudal gradient in the proportion of receptor clusters having VAChT-immunoreactive nerve terminals associated with them. Nerve terminals associated with nicotinic receptor clusters did not show detectable CGRP-immunoreactivity until one to two weeks after the appearance of synaptophysin- and VAChT-immunoreactivity. The NOS-immunoreactive neurons did not show detectable VIP-immunoreactivity until three days after NOS could be detected. These results show that the appearance of clusters of nicotinic receptors in the external muscle of the esophagus coincides with the expression of a striated muscle phenotype, but not with the presence of ingrowing nerve terminals. However, many of the receptor clusters that were observed first were apparently uninnervated.     

Identification of two Y-box binding proteins that interact with the promoters of columbid annexin I genes

The aim of this study was to investigate the role of noradrenergic descending nervous pathways in external anal sphincter motility. For this purpose, the effects of intravenously injected adrenoceptor antagonist and agonist on the tonic electrical activity of this sphincter were studied in anesthetized cats. The effects of stimulating the region of the locus coeruleus and the effects of intravenous, intracerebroventricular and intrathecal injection of the above drugs on the electromyographic responses of this muscle to pudendal nerve stimulation were also investigated. The tonic sphincteric activity and the reflex response triggered by electrically stimulating pudendal afferent nerve fibers were inhibited by alpha1-adrenoceptor antagonist nicergoline and enhanced by alpha1-adrenoceptor agonist phenylephrine. Stimulation of the locus coeruleus area either inhibited or enhanced the reflex responses. Intracerebroventricular and intrathecal injection of the alpha2-adrenoceptor agonists, morphine and leu-enkephalin decreased the amplitude of these reflex responses. All the effects of opioids were blocked by naloxone and by spinalization performed at the cervical and lumbar levels. The direct response elicited by stimulating the sphincteric motor axons was not affected either by these drugs or by the brainstem stimulation. These results suggests the existence of a pontine neuronal network controlling the motility of the external anal sphincter via noradrenergic and opioid neurons.     

Hyperactivation may assist human spermatozoa to detach from intimate association with the endosalpinx

The behaviour of human spermatozoa was observed during incubation with epithelial cells isolated from the isthmic and ampullary sections of human uterine (Fallopian) tubes. During incubation, spermatozoa were observed to bind to the epithelial cells of the tube (the endosalpinx), and individual spermatozoa attached and detached at intervals. The kinematic characteristics of spermatozoa during these behaviour patterns were determined. The results showed that detached spermatozoa typically had an increased curvilinear velocity and amplitude of lateral head displacement, accompanied by a decrease in their linearity. Significantly (P < 0.01) more of the detaching spermatozoa were hyperactivated than were spermatozoa prior to attachment for both isthmic (35.3 +/- 5.5 versus 4.0 +/- 3.3%; mean +/- SEM) and ampullary (26.0 +/- 7.0 versus 2.0 +/- 1.4%) regions. Incubation with epithelial cells from either region produced no differences in any category of sperm behaviour. Furthermore, there was no significant difference between regions in the amount of time spermatozoa spent bound (33.6 +/- 12.9 and 20.6 +/- 3.0 s for isthmic and ampullary tissue respectively). These results support the hypothesis that hyperactivation may assist spermatozoa in breaking connections with epithelial cells.     

Site-specific mutations of the N-terminal threonines in the D1 protein affects photoautotrophic growth but not D1 protein stability in Synechocystis 6803

OBJECT: The goal of this study was to identify the neurological characteristics of patients with poststroke pain who show a favorable response to motor cortex (MC) stimulation used to control their pain. METHODS: The neurological characteristics of 31 patients treated by MC stimulation were analyzed. In 15 patients (48%), excellent or good pain control (pain reduction > 60%) was achieved for follow-up periods of more than 2 years by using MC stimulation at intensities below the threshold for muscle contraction. Satisfactory pain control was achieved in 13 (73%) of 18 patients in whom motor weakness in the painful area was virtually absent or mild, but in only two (15%) of the 13 patients who demonstrated moderate or severe weakness in the painful area (p < 0.01). Muscle contraction was inducible in the painful area in 20 patients when stimulated at a higher intensity. No such muscle response was inducible in the remaining 11 patients, no matter how extensively the authors attempted to determine appropriate stimulation sites. Satisfactory pain control was achieved in 14 (70%) of the 20 patients in whom muscle contraction was inducible, but in only one (9%) of the 11 patients in whom muscle contraction was not inducible (p < 0.01). No significant relationship was observed between pain control and various sensory symptoms, including the presence of hypesthesia, spontaneous dysesthesia, hyperpathia, and allodynia, or the disappearance of the N20 component of the median nerve-evoked somatosensory scalp potential. No significant relationship existed between the effect of MC stimulation on the pain and stimulation-induced phenomena, including paresthesia, improvement in motor performance, and attenuation of involuntary movements. CONCLUSION: These findings suggest that the pain control afforded by MC stimulation requires neuronal circuits that are maintained by the presence of intact corticospinal tract neurons originating from the MC. Preoperative evaluation of motor weakness of the painful area appears to be useful for predicting a favorable response to MC stimulation in the control of poststroke pain.     

Neuronal nitric oxide synthase activation by vasoactive intestinal peptide in bovine cerebral arteries

The participation of nitric oxide and vasoactive intestinal peptide (VIP) in the neurogenic regulation of bovine cerebral arteries was investigated. Nitrergic nerve fibers and ganglion-like groups of neurons were revealed by NADPH-diaphorase staining in the adventitial layer of bovine cerebral arteries. NADPH diaphorase also was present in endothelial cells but not in the smooth muscle layer. Double immunolabeling for neuronal nitric oxide synthase and VIP indicated that both molecules co-localized in the same nerve fibers in these vessels. Transmural nerve stimulation (200 mA, 0.2 milliseconds, 1 to 8 Hz) of endothelium-denuded bovine cerebral artery rings precontracted with prostaglandin F2 alpha, produced tetrodotoxin-sensitive relaxations that were completely suppressed by NG-nitro-L-arginine methyl ester (L-NAME) and by the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline (ODQ), but were not affected by the adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536), nor by VIP tachyphylaxis induced by pretreatment with 1 mumol/L VIP. Transmural nerve stimulation also elicited increases in intracellular cyclic GMP concentration, which were prevented by L-NAME, and small decreases in intracellular cyclic AMP concentration. Addition of VIP to bovine cerebral artery rings without endothelium produced a concentration-dependent relaxation that was partially inhibited by L-NAME, ODQ, and SQ 22,536. The effects of L-NAME and SQ 22,536 were additive. VIP induced a transient increase in intracellular cyclic GMP concentration, which was maximal 1 minute after VIP addition, when the highest relaxation rate was observed, and which was blocked by L-NAME. It is concluded that nitric oxide produced by perivascular neurons and nerve fibers fully accounts for the experimental neurogenic relaxation of bovine cerebral arteries and that VIP, which also is present in the same perivascular fibers, acts as a neuromodulator by activating neuronal nitric oxide synthase.     

Heart rate variability: significance and clinic application

In vitro effects of two bioactive forms of pituitary adenylate cyclase activating polypeptide (PACAP): PACAP-38 and PACAP-27 were studied on rabbit vascular and non-vascular smooth muscle. Segments of the ovarian artery and muscle strips from the fallopian tube were used. Two series of experiments were performed on vessels: the dose-response relationship of PACAP-38 (10(-10)-10(-7) M) was established on noradrenaline- (NA, 10(-6) M) contracted vessels. In the other set of experiments the contractile effect of 10(-8)-10(-4) M NA added cumulatively, was studied on arterial segments incubated with PACAP-38 (10(-7) M), PACAP-27 (10(-7) M) or VIP (10(-7) M). The effect of PACAP-38, PACAP-27 and VIP (10(-10)-10(-6) M) was investigated on spontaneously contracting smooth muscle of the fallopian tube. Longitudinally as well as transversally cut specimens were investigated. PACAP-38 produced a significant dose-related relaxation on the NA-precontracted vessels. However, pre-incubation of the vessels with 10(-7) M PACAP-38, PACAP-27 and vaso active intestinal polypeptide (VIP) did not induce a general rightward shift of the NA concentration-response curves, although a tendency to inhibition in the low-dose interval was observed. The peptides caused a significant, dose-dependent inhibition of both frequency and amplitude on the fallopian tube smooth muscle activity. The effects of the three peptides on longitudinally as well as transversally cut specimens were alike.     

Functional changes in nonadrenergic, noncholinergic inhibitory neurons in ileal circular smooth muscle after small bowel transplantation in rats

This experiment was designed to determine mechanisms of change in nonadrenergic, noncholinergic (NANC) inhibitory neurons in the ileum after small bowel transplantation (SBT) in the rat and whether nitric oxide (NO) serves as an important NANC inhibitory neurotransmitter in the rat ileum. Eight groups of rats (N > or =8 rats/group) were studied: neurally intact unoperated controls; rats one week after anesthesia and sham celiotomy; and separate groups one and eight weeks after either 40 min of cold ischemia of the jejunoileum, combined jejunal and ileal intestinal transection/reanastomosis, or orthotopic SBT of the entire jejunoileum. Contractile activity was evaluated in full-thickness ileal circular muscle strips under isometric conditions. Spontaneous activity did not differ among groups. In all groups, exogenous NO, NG-monomethyl-L-arginine (L-NMMA, an NO synthase inhibitor), and methylene blue (soluble guanylate cyclase inhibitor) had no effect on spontaneous activity, while 8-bromocyclic guanosine monophosphate (8Br-cGMP) inhibited contractile activity in all groups. Low frequency (2-10 Hz) electrical field stimulation (EFS) inhibited contractile activity only in control and SBT groups; L-NMMA and methylene blue did not alter the response to EFS in any group. These results suggest that each aspect of the SBT procedure, ischemia/reperfusion injury, disruption of enteric neural continuity by intestinal transection, and extrinsic denervation, alter function of enteric ileal inhibitory neurons separately early (one week) after operation. NO, a known inhibitory neurotransmitter in other gut regions, does not affect ileal circular muscle in neurally intact tissue nor mediate functional changes in inhibitory nerve function nor smooth muscle contractility after SBT.     

Lateral rectus whole muscle and motor unit contractile measures with the extraocular muscles intact

Both extracellular and intracellular stimulation of single motoneurons were shown to be similarly effective and consistent in eliciting contractile responses in single lateral rectus muscle motor units. The whole muscle was activated by stimulating the sixth nerve in the brain stem. Both whole muscle and motor unit contractile characteristics, under isometric conditions, were found to remain consistent regardless of whether this extraocular muscle was detached or left attached to the globe. In addition, whole muscle twitch and maximum tetanic tension evoked by sixth nerve stimulation was significantly less than would be predicted by the linear summation of individual motor unit twitch and maximum tetanic tensions.     

Ultrastructural studies of PECAM-1/CD31 expression in the developing mouse blood-brain barrier with the application of a pre-embedding technique

Vasoactive intestinal polypeptide (VIP) has been found in pancreatic nerves in several species. Studies were conducted to determine if VIP could be a parasympathetic neurotransmitter in the canine endocrine pancreas. To verify that VIP is localized in pancreatic parasympathetic nerves, sections of canine pancreas were immunostained for VIP. VIP staining was identified in the majority of neuronal cell bodies in intrapancreatic parasympathetic ganglia. In addition. VIP was localized in nerve fibers innervating pancreatic islets in the proximity of alpha cells. Next, to determine if VIP is released during electrical stimulation of parasympathetic nerves, pancreatic spillover of VIP was measured during vagal nerve stimulation (VNS) in anesthetized dogs. VIP spillover increased from a baseline of 630+/-540 pg/min to 2580+/-540 pg/min (delta = +1950+/-490 pg/min, p <0.01). Pancreatic VIP release during VNS was not affected by atropine, whereas ganglionic blockade with hexamethonium nearly abolished the VIP response to VNS (p<0.005 vs control), suggesting that VIP is a postganglionic neurotransmitter in the dog pancreas. To examine the effects of VIP on pancreatic hormone secretion, synthetic VIP was infused locally into the pancreatic artery. VIP, at a low dose (5 pmol/min), increased glucagon secretion from 1750+/-599 to 3800+/-990 pg/min (delta = +2060+/-870 pg/min, p<0.05), but did not affect insulin secretion (delta = -1030+/-760 microU/min, NS). Thus, VIP is contained in and released from pancreatic parasympathetic nerves in proximity to islet alpha cells and exogenous VIP, at a dose which approximates the increase of VIP spillover during VNS, preferentially stimulates glucagon vs insulin secretion. Therefore, VIP is likely to function as a parasympathetic neurotransmitter in the endocrine pancreas in dogs. We hypothesize that VIP could mediate the glucagon response to parasympathetic activation which has been shown to resistant to cholinergic blockade with atropine in several species.     

Inhibition of herpes simplex virus replication by retinoic acid

Superior laryngeal nerve (SLN) stimulation can activate the brainstem swallowing mechanism to produce a complete swallowing sequence consisting of oropharyngeal, oesophageal and lower oesophageal sphincter (LOS) components. However, little is known of the effect of SLN stimulation (peripheral-sensory input from the pharynx) on the characteristics of oesophageal motor activity, especially in the smooth muscle portion. The present study examined the effect of varying stimulus train length and frequency on each of the three components of the reflex. Acute studies were performed in urethane anaesthetized cats. Oesophageal motility was monitored using conventional manometric techniques, and oropharyngeal swallowing by the mylohyoid electromyogram. SLN stimulus train length (1-10 sec) and frequency (5-30 Hz) were varied independently. Increased train length or frequency resulted in (1) an increase in oropharyngeal swallowing and incidence of the complete swallowing response, (2) an increase in latency to onset of the oesophageal peristaltic wave, (3) reduction of the amplitude of the evoked peristaltic contraction in the smooth muscle portion, without altering its velocity, (4) increased LOS relaxation, and increased LOS after-contraction. The LOS contraction was abolished by atropine (100 micrograms kg-1). Therefore, increased SLN stimulation not only results in excitation of the central swallowing program and the oropharyngeal stage of swallowing, but has major effects on the oesophageal and LOS stages of swallowing. Afferent SLN stimuli can impact on the control mechanisms for each stage, to inhibit or excite the stages in different ways.     

Role of nitric oxide on cholinergic component of bronchial tone in pig

In vitro studies demonstrated that stimulation of intrinsic nerves of airway smooth muscle results in a predominantly contractile response, followed by a relaxant response which involves cholinergic, adrenergic and non-adrenergic non-cholinergic (NANC) nerve activation. Thus, in this paper it is determined whether endogenous nitric oxide (NO) modulates cholinergic neurotransmission in isolated pig airway smooth muscle. Bronchial rings were suspended in organ baths for isometric measurement of tension and the contractions were induced using electrical field stimulation (EFS) techniques. Then, the effects of L-NG-nitroarginine (L-NOARG, 10 microM), an inhibitor of NO synthase, and L-arginine (L-ARG, 1 mM), a precursor of NO synthesis, were evaluated. The cholinergic contractions induced by electrical field stimulation (EFS: 60 V, 2 ms, 60 Hz) of pig lobar bronchial preparations increased (29%) in the presence of L-NOARG (10 microM). This effect may be released by nerves in pig large airways during EFS.     

Histomorphometric, physical, and mechanical effects of spaceflight and insulin-like growth factor-I on rat long bones

Pituitary adenylate cyclase activating peptide and vasoactive intestinal peptide belong to the same neuropeptide family. Both peptides are present in nerve fibers in the gastric wall and are thought to be involved in the regulation of inflammatory processes. Experimental ulcers were induced in the rat gastric mucosa by local application of acetic acid. During the healing process we examined the PACAP and VIP innervation by means of immunohistochemistry and in situ hybridization. The ulcer area was examined from day 1 to day 15 after ulcer induction. There was a marked depletion of PACAP in nerve fibers at the ulcer margin from day 1 and onwards. On day 10 and day 15, PACAP-immunoreactive nerve fibers could again be visualized at the ulcer margin. In contrast, VIP immunoreactive nerve fibers were present at the ulcer margin at all time points studied. From day 10 following ulcer induction PACAP- and VIP- immunoreactive nerve fibers were increased in frequency in the smooth muscle beneath the ulcer. An upregulation of VIP and PACAP mRNA was also demonstrated in the myenteric ganglia adjacent to ulcer. The present results indicate that neuronal PACAP and VIP react differently to the inflammation at the ulcer margin but similarly in the smooth muscle during the ulcer healing.     

Characterization and functional analysis of the promoter of RAGE, the receptor for advanced glycation end products

The receptor for advanced glycation end products, RAGE, is a member of the immunoglobulin superfamily of cell surface molecules differentially expressed on a range of cell types. Ligation of RAGE perturbs homeostatic mechanisms and, potentially, provides a basis for cellular dysfunction in pathologic situations in which its ligands accumulate. To understand factors underlying RAGE expression, we cloned the 5'-flanking region of the RAGE gene and characterized putative regulatory motifs. Analysis of the putative promoter region revealed the presence of three potential NF-kappaB-like and two SP1 binding sites. Transient transfection of vascular endothelial and smooth muscle cells using chimeric 5'-deletion constructs linked to luciferase reporter revealed that the region -1543/-587 contributed importantly to both basal and stimulated expression of the RAGE gene. This region of the RAGE gene contained three putative NF-kappaB-like binding sites and was responsible for increased luciferase activity observed when endothelial or smooth muscle cells were stimulated with lipopolysaccharide. DNase I footprinting assays and electrophoretic mobility shift assay revealed that two of the three NF-kappaB-like binding sites (1 and 2) were likely functional and responsive to stimuli. Upon simultaneous mutation of NF-kappaB-like sites 1 and 2, both basal promoter expression and response to stimulation with LPS, as measured by relative luciferase activity, were significantly diminished. These results point to NF-kappaB-dependent mechanisms regulating cellular expression of RAGE and suggest a means of linking RAGE to the inflammatory response.