Increased capsaicin receptor TRPV1-expressing sensory fibres in irritable bowel syndrome and their correlation with abdominal pain.

OBJECTIVE: The capsaicin receptor TRPV1 (transient receptor potential vanilloid type-1) may play an important role in visceral pain and hypersensitivity states. In irritable bowel syndrome (IBS), abdominal pain is a common and distressing symptom where the pathophysiology is still not clearly defined. TRPV1-immunoreactive nerve fibres were investigated in colonic biopsies from patients with IBS, and this was related to abdominal pain. METHODS: Rectosigmoid biopsies were collected from 23 IBS patients fulfilling Rome II criteria, and from 22 controls. Abdominal pain scores were recorded using a validated questionnaire. TRPV1-, substance P- and neuronal marker protein gene product (PGP) 9.5-expressing nerve fibres, mast cells (c-kit) and lymphocytes (CD3 and CD4) were quantified, following immunohistochemistry with specific antibodies. The biopsy findings were related to the abdominal pain scores. RESULTS: A significant 3.5-fold increase in median numbers of TRPV1-immunoreactive fibres was found in biopsies from IBS patients compared with controls (p<0.0001). Substance P-immunoreactive fibres (p = 0.01), total nerve fibres (PGP9.5) (p = 0.002), mast cells (c-kit) (p = 0.02) and lymphocytes (CD3) (p = 0.03) were also significantly increased in the IBS group. In multivariate regression analysis, only TRPV1-immuno-reactive fibres (p = 0.005) and mast cells (p = 0.008) were significantly related to the abdominal pain score. CONCLUSIONS: Increased TRPV1 nerve fibres are observed in IBS, together with a low-grade inflammatory response. The increased TRPV1 nerve fibres may contribute to visceral hypersensitivity and pain in IBS, and provide a novel therapeutic target.

Burning mouth syndrome as a trigeminal small fibre neuropathy: Increased heat and capsaicin receptor TRPV1 in nerve fibres correlates with pain score.

Burning mouth syndrome (BMS) is often an idiopathic chronic and intractable pain condition, affecting 1.5-5.5% of middle-aged and elderly women. We have studied the heat and capsaicin receptor TRPV1, and its regulator nerve growth factor (NGF), in BMS. Patients with BMS (n=10) and controls (n=10) were assessed for baseline and post-topical capsaicin pain scores, and their tongue biopsies immunostained for TRPV1, NGF, and structural nerve markers neurofilament and peripherin. Nerve fibres penetrating the epithelium were less abundant in BMS (p<0.0001), indicating a small fibre neuropathy. TRPV1-positive fibres were overall significantly increased in BMS (p=0.0011), as were NGF fibres (p<0.0001) and basal epithelial cell NGF staining (p<0.0147). There was a significant correlation between the baseline pain score and TRPV1 (p=0.0143) and NGF fibres (p=0.0252). A significant correlation was observed between baseline and post-capsaicin pain (p=0.0006). Selective TRPV1 and NGF blockers may provide a new therapy for BMS.

Sodium channel Nav1.8 immunoreactivity in painful human dental pulp.

BACKGROUND: The tetrodotoxin-resistant voltage-gated sodium channel Nav1.8 (SNS1/PN3) is expressed by nociceptors and may play a role in pain states. METHODS: Using specific antibodies for immunohistochemistry, we studied Nav1.8 immunoreactivity in human dental pulp in relation to the neuronal marker neurofilament. Human tooth pulp was extracted from teeth harvested from a total of twenty-two patients (fourteen without dental pain, eight patients with dental pain). RESULTS: Fibres immunoreactive for Nav1.8, were significantly increased on image analysis in the painful group: median (range) Nav1.8 to Neurofilament % area ratio, non-painful 0.059 (0.006-0.24), painful 0.265 (0.13-0.5), P = 0.0019. CONCLUSION: Nav1.8 sodium channels may thus represent a therapeutic target in trigeminal nerve pain states.

Distribution of peptide histidine isoleucine in the mammalian respiratory tract and some aspects of its pharmacology.

Peptide histidine isoleucine (PHI), a newly discovered neuropeptide, has been detected by RIA and immunocytochemistry in the upper respiratory tracts of the guinea pig, rat, and cat. HPLC of tracheal extracts showed a single peak of PHI immunoreactivity in each species. The immunoreactive PHI peak found in the guinea pig and rat trachea was eluted earlier than the corresponding peak from the cat, which was coeluted with the porcine PHI standard. Immunocytochemistry showed PHI immunoreactivity to be present within ganglion cells and nerve fibers in the respiratory tracts of all three species. The distribution of PHI was similar to that of vasoactive intestinal polypeptide and ganglion cells were found to contain both PHI and vasoactive intestinal polypeptide immunoreactivities. Pure natural porcine PHI induced a dose-dependent relaxation of isolated guinea pig tracheal muscle which was not blocked by antagonists to catecholamines, histamine, 5-hydroxy-tryptamine, and acetylcholine. PHI may thus be one of the local factors in respiratory control.

Distribution of immunoreactive growth hormone-releasing hormone in the human brain and intestine and its production by tumors.

A recently developed RIA for human pancreatic tumor GH-releasing hormone (hpGRH1-44NH2) was used to investigate its distribution in the human gastrointestinal tract and brain, and to determine the incidence of its production by tumors. GRH-immunoreactivity (GRH-IR) was present in several regions of the human brain, the highest concentration occurring in the hypothalamus, septum, and substantia inominata. In the gastrointestinal tract GRH-IR was present in the upper intestine, where it was confined to the epithelial mucosa. Approximately one third of all tumors examined (35 out of 97) contained significant amounts of GRH-IR. Gel chromatography of brain and intestinal extracts, and of several tumors, revealed the presence of two molecular forms of GRH-IR, one coeluting with the synthetic hpGRH1-44 amide standard (and also hpGRH1-40) and another eluting significantly later. The earlier eluting GRH-IR peak was found to be similar to the synthetic hpGRH1-44 NH2 and hpGRH1-40 on high pressure liquid chromatography analysis.

Peptide histidine-methionine immunoreactivity in plasma and tissue from patients with vasoactive intestinal peptide-secreting tumors and watery diarrhea syndrome.

The presence of peptide histidine-methionine (PHM)-like peptides has been determined in plasma and tumor specimens from patients with vasoactive intestinal peptide (VIP)-secreting tumors and the watery diarrhea syndrome. All patients had strikingly elevated plasma concentrations of PHM immunoreactivity (median, 1800; range, 500-6800 pmol/liter; n = 12), which were higher than those of VIP (median, 235; range, 50-580 pmol/liter). In patients with other endocrine and nonendocrine pancreatic tumors, plasma PHM concentrations were not significantly different from normal (median, 20; range, 5-60 pmol/liter; n = 28). Plasma samples from patients with diarrhea due to other illnesses also had PHM concentrations that were not significantly different from normal (median, 40; range, 10-80 pmol/liter; n = 23). The gel chromatographic profiles of plasma and tumor extracts from patients with VIP-secreting tumors revealed the presence of at least two molecular forms that reacted with an antiserum directed to the N-terminus of PHM (SY1). The later peak (Kav, 0.50-0.53) corresponded in position to synthetic PHM and also reacted with the PHM-specific antiserum (SY2). The earlier peak (Kav, 0.30-0.37), not reactive with antiserum SY2, corresponded to a large molecular form of PHM-like immunoreactivity previously identified as the predominant form in normal human stomach and plasma, though not in the rest of the intestinal tract. The neuroendocrine nature of the tumors was confirmed by the demonstration of immunostaining with a battery of antisera to neuroendocrine markers. Immunocytochemistry revealed the presence of both VIP and PHM in tumor cells. The presence of high circulating concentrations of PHM-like immunoreactivity in patients with VIP-secreting tumors, as measured with a PHM N-terminus-directed antiserum, SY1, suggests that use of this type of antiserum may provide valuable information in the diagnosis of such tumors. The contribution of the PHM-like peptides to the features of this syndrome is not known.

SNS/PN3 and SNS2/NaN sodium channel-like immunoreactivity in human adult and neonate injured sensory nerves.

Two tetrodotoxin-resistant voltage-gated sodium channels, SNS/PN3 and SNS2/NaN, have been described recently in small-diameter sensory neurones of the rat, and play a key role in neuropathic pain. Using region-specific antibodies raised against different peptide sequences of their alpha subunits, we show by Western blot evidence for the presence of these channels in human nerves and sensory ganglia. The expected fully mature 260 kDa component of SNS/PN3 was noted in all injured nerve tissues obtained from adults; however, for SNS2/NaN, smaller bands were found, most likely arising from protein degradation. There was increased intensity of the SNS/PN3 260 kDa band in nerves proximal to the site of injury, whereas it was decreased distally, suggesting accumulation at sites of injury; all adult patients had a positive Tinel's sign at the site of nerve injury, indicating mechanical hypersensitivity. Injured nerves from human neonates showed similar results for both channels, but neonate neuromas lacked the SNS2/NaN 180 kDa molecular form, which was strongly present in adult neuromas. The distribution of SNS/PN3 and SNS2/NaN sodium channels in injured human nerves indicates that they represent targets for novel analgesics, and could account for some differences in the development of neuropathic pain in infants.

PHI-like immunoreactivity in the gallbladder and in vitro effect of porcine PHI on smooth muscle of the gallbladder.

The occurrence and distribution of PHI-like immunoreactivity in the guinea pig gallbladder has been analysed by radioimmunoassay and immunocytochemistry. Chromatography of gallbladder extracts by gel permeation and high-performance liquid chromatography revealed that guinea pig PHI-like immunoreactivity is of a similar size to that of porcine PHI but may differ in its amino acid sequence. Immunocytochemistry showed PHI-immunoreactivity to be localised to nerves found predominantly in the ganglionated plexus and the mucosal plexus of the gallbladder. Pure natural porcine PHI induced a dose-dependent relaxation of the isolated guinea pig gallbladder muscle which was not blocked by antagonists to acetylcholine, catecholamines, histamine, and 5-hydroxytryptamine. PHI may thus be one of the local factors involved in controlling gallbladder function.

Isolation and characterisation of GLP-1 7-36 amide from rat intestine. Elevated levels in diabetic rats.

Glucagon-like peptide-1 (GLP-1) was purified to homogeneity by HPLC and anion-exchange chromatography. A molecular mass of 3297.4 Da was obtained by FAB mass spectrometry which corresponded exactly to GLP-1 7-36 NH2, providing evidence that amidation occurs at an arginine residue during the post-translational processing of GLP-1. The distribution of GLP-1 7-36 NH2-like immunoreactivity (GLP-1 7-36 NH2 IR) was determined in the rat gastrointestinal tract. Highest concentrations were found in terminal ileum and colon. Streptozocin-induced diabetic rats, who showed a significant increase in food intake, had a significant increase of GLP-1 7-36 NH2 IR in the colon.

Effects of prepro-vasoactive intestinal peptide-derived peptides on the murine immune response.

We have studied the effects of prepro-vasoactive intestinal polypeptide-derived peptides on lectin-induced lymphocyte proliferation and natural killer cell (NK) activity in cells from murine spleen, mesenteric lymph nodes, Peyer's patches, thymus and peripheral blood mononuclear lymphocytes (PBL). These peptides (vasoactive intestinal peptide (VIP), peptide histidine methionine (PHM-27) and peptide histidine valine (PHV-42)) showed differential effects in their immune response in a dose- and tissue-dependent manner. All peptides significantly decreased DNA synthesis in spleen (range: 45-30%), lymph nodes (range: 30-0%), Peyer's patches (range: 30-4%) and PBL (range: 30-16%). In these tissues there was no significant difference in their potency. In the thymus, however, PHM-27 (range: 27-15%) was significantly more potent (p less than 0.001) in inhibiting DNA synthesis than either VIP (range: 6-0%) or PHV-42 (range: 20-8%). The modulatory effects on NK activity by these peptides also showed an inhibitory effect. The order of potency was: VIP (range: 40-27%), PHV-42 (range: 22-11%) and PHM-27 (range: 20-8%). The presence of VIP inhibitor [( D-p-chloro-Phe6,Leu17]-VIP) at 10(-8) M in both functional assays caused a significant antagonism of the effects of VIP but not PHM-27 or PHV-42. Our results suggest the existence on lymphocytes of different receptors for prepro-VIP-derived peptides, and that they may be considered as important immunoregulatory molecules. Their mechanism of interaction, however, is not clearly understood.

P2X3 receptor in injured human sensory neurons.

The ATP-gated cation channel P2X3 is expressed selectively by rat sensory neurones, and may play a role in nociception by binding ATP released from damaged or inflamed tissues. However, the distribution of this channel in human sensory neurons is not known. Using a specific antibody, we have demonstrated intense P2X3 immunoreactivity within a subset (60%) of small/medium diameter sensory neurones and fine nerve fibres in intact post-mortem human dorsal root ganglia (DRG). Co-localization studies showed < 15% overlap with the trkA immunostaining in DRG, indicating that P2X3 was expressed predominantly in sensory neurons that are also isolectin B4 positive. There was a significant decrease in numbers of P2X3-like immunoreactive neurons in human DRG after central axotomy (to 36%), similar to the decrease in rat DRG after peripheral axotomy. However, Western blotting demonstrated a specific 66 kDa band in human DRG and peripheral organs, including intestine, where histochemistry showed P2X3 immunoreactivity in myenteric plexus neurons. Thus P2X3 antagonists may be analgesic, but are unlikely to have a selective effect on pain in humans.

The distributions of PHI and VIP in porcine gut and their co-localisation to a proportion of intrinsic ganglion cells.

VIP and PHI share sequence homology and certain biological actions. Immunocytochemistry and radioimmunoassay were used to see if the two peptides also have similar distributions in the gut of the pig. PHI-immunoreactive fibres were found, like those containing VIP, in all layers of the bowel wall but in lesser numbers. Unlike VIP-immunoreactive nerves, however, which are ubiquitous in the gastrointestinal tract, PHI-containing neurons were numerous in all areas except the fundus, where only few fibres and no ganglion cells were found to be reactive to PHI antibodies. PHI and VIP immunoreactive materials were also quantified by specific radioimmunoassay of tissue extracts. The concentrations of PHI and VIP were similar in all regions of the gut, except in the fundus where the quantities of VIP-immunoreactivity far exceeded those of PHI. The presence of both VIP- and PHI-immunoreactivities in ganglion cells of the sub-mucous plexus allowed investigation of the co-localisation of the peptides. Serial sections through ganglion cells revealed that a major proportion contain both PHI- and VIP-immunoreactivity. Some cells contained VIP alone, or VIP and weak, equivocal immunostaining of PHI, and a sub-population contained no peptide-immunoreactivity. The presence of both VIP- and PHI-immunoreactivities in the same ganglion cell supports the recent reports of the isolation and characterisation, using genetic technology, of their common precursor molecule. The finding of VIP and not PHI in the fundic region suggests the differential expression of the two peptides.

A VIP/PHI-containing pathway links urinary bladder and sacral spinal cord.

Nerve fibres containing VIP and the co-produced PHI are found in the dorsal horn and autonomic centres of the sacral spinal cord and in pelvic organs. We have investigated the origin of these nerve fibres and a possible peptide-containing pathway linking pelvic viscera with the spinal cord of the cat and rat using neurochemical and neurosurgical procedures, retrograde tracing and immunocytochemistry. Cell bodies were located in the dorsal root ganglia (after colchicine injection), pelvic ganglia and bladder wall. Capsaicin treatment induced a loss of VIP/PHI from the dorsal horn. Retrograde tracing from the bladder revealed True Blue labelled cells in the dorsal root ganglia (L6, S1), parasympathetic nuclei and pelvic ganglia. Labelled cells were sequentially immunostained for VIP/PHI which were numerous in pelvic ganglia and scattered and weak in dorsal root ganglia. Pelvic nerve section induced a decrease of VIP/PHI immunoreactivity from the spinal cord and no change or a minimal increase in immunoreactive nerve fibers of the bladder. Thus pelvic visceral afferents with cell bodies in the dorsal root ganglia are a significant source of VIP/PHI-containing fibres in the sacral dorsal horn.

Peptide histidine methionine (PHM) increases ileostomy output.

The human vasoactive intestinal peptide (VIP) gene also encodes peptides histidine methionine (PHM) which has substantial sequence homology with VIP. Both are present in nerve fibers in the human ileum and circulate in greatly increased concentrations in patients with the watery diarrhoea syndrome. We have infused PHM (23 pmol/kg/min) into 5 patients with ileostomies to determine the effect of PHM on human ileal output. Plasma PHM levels rose from 22 +/- 6 to 6013 +/- 874 pM (mean +/- S.E.M.) during PHM infusions and ileal output rose from 16 +/- 3 to 177 +/- 27 g/30 min (P less than 0.0001). PHM infusions also produced a significant fall in the percentage of solid material and a rise in the concentration of chloride in the ileal effluent. Mean plasma PHM concentrations during PHM infusions were equal to the highest levels seen in patients with the watery diarrhoea syndrome, so PHM may contribute to diarrhoea in this condition. Neuronal PHM may exert physiological control over ileal transport of water and electrolytes.

Heart rate response to peptide histidine valine in human subjects is not mediated through beta receptors.

Peptide histidine valine (PHV) is a 42 amino acid polypeptide closely related to the neuropeptides VIP, PHI and PHM. We have performed a placebo-controlled, double-blind study to assess the hypothesis that the cardiovascular response to PHV infusion may be mediated via the sympathetic nervous system. Four subjects received atenolol or matched placebo 90 min prior to a controlled incremental infusion of PHV, with monitoring of heart rate, blood pressure and skin temperature. Following placebo all subjects showed a dose-related increase in heart rate and skin temperature with no effect on blood pressure during PHV infusion. beta-Blockade had no effect on skin temperature response. Pre-treatment with atenolol reduced the resting blood pressure and the maximum heart rate achieved, but did not affect the percentage increase in heart rate during PHV infusion. This suggests that the action of PHV does not involve beta-receptors. The lack of effect of PHV infusion on blood pressure, despite tachycardia and marked cutaneous vasodilatation, implies that PHV has a different effect on the resistance vessels from that of other peptides such as VIP.

PHI stimulates intestinal fluid secretion.

Regulatory peptides are likely to have a role in the control of net intestinal fluid transport. PHI is a peptide recently isolated from porcine duodenum which has been shown to have sequence homologies with other peptides of the glucagon-secretin family. We have studied the effect of intravenous infusion of synthetic PHI on net intestinal fluid transport in the rat small intestine. During PHI infection net absorption was reduced in the duodenum and jejunum and net secretion was observed in the ileum. Thus synthetic PHI appears to be capable of strongly stimulating intestinal secretion in the rat.

Peptide histidine methionine (PHM) and the human male genitalia.

Peptide histidine methionine-like immunoreactivity (PHM-IR) has been demonstrated to be present in the human penis both by radioimmunoassay and immunohistochemistry with particularly high levels in the corpus cavernosum and vas deferens. In the cavernosa, PHM-IR has been localised entirely, in nerves around arteries. High performance liquid chromatography indicated that this PHM-IR co-eluted with synthetic PHM but not porcine PHI. The presence of PHM-IR in the penis suggests that this neuropeptide may play a functional role in penile function.

ATP-gated ion channel P2X(3) is increased in human inflammatory bowel disease.

P2X(3) is a novel ATP-gated cation channel that is selectively expressed by small-diameter sensory neurones in rodents, and may play a role in nociception by binding ATP released from damaged or inflamed tissues. We have studied, for the first time, P2X(3) immunoreactivity in human inflammatory bowel disease, using Western blotting and immunohistochemistry. A major 66-kDa specific protein was found by Western blotting in all colon extracts. In the inflamed group there was a significant two-fold increase in the relative optical density of the 66-kDa band (21.2 +/- 3.1; n=8) compared to controls (11.4 +/- 3.7; n=8; P=0.009). In the control colon, P2X(3)-immunoreactive neurones were scattered throughout the myenteric and submucosal plexuses, with some neurones showing immunopositive axons/dendrites. The pattern of immunostaining was similar to the neuronal marker peripherin. In general, the intensity of the staining was greater in myenteric than submucosal neurones. The number of P2X(3)-immunoreactive neurones was significantly increased in the myenteric plexus of inflamed colon compared to controls (n=13; P=0.01). In humans, unlike rodents, P2X(3) is thus not restricted to sensory neurones. Increased P2X(3) in inflamed intestine suggests a potential role in dysmotility and pain, for which it represents a new therapeutic target.

Ontogeny of PHI in the rat brain.

The regional distribution of immunoreactive PHI (IR-PHI) was investigated in rat brain between postcoitum (pc) and day 60 postpartum (pp). IR-PHI was undetectable in all regions of the foetal brain, and only very small amounts were found at day 7 pp. However, there was a dramatic increase thereafter reaching a peak at day 20 pp (e.g. in the hippocampus there was a 12-fold increase in the PHI concentration). Highest concentrations were found in the cortex (40 +/- 5 pmol/g) and hippocampus (35 +/- 8 pmol/g), with lower concentrations in the diencephalon (11 +/- 4 pmol/g) and mesencephalon (10 +/- 3 pmol/g). The brainstem and cerebellum contained very low amounts of IR-PHI. Permeation analysis of brain extracts, on Sephadex G50-superfine, indicated the presence of one major form of IR-PHI which eluted in a similar position to pure intestinal porcine PHI and human intestinal PHI.

Comparison of neuromedin-N and neurotensin on net fluid flux across rat small intestine.

Neuromedin-N, a hexapeptide recently isolated and purified from porcine spinal cord, has close sequence homology with the C-terminal region of the tridecapeptide neurotensin. Both peptides have a remarkably similar peripheral distribution. Little is known of the biological activity of neuromedin-N. Neurotensin and peptide histidine methionine are known to stimulate net fluid secretion into rat small intestine. We have therefore tested the effect of neuromedin-N and the hexapeptide neurotensin-(8-13), the smallest fully active analogue of neurotensin in this system, compared with that of neurotensin and peptide histidine methionine. All four peptides reduced net absorption in low doses and caused net secretion in larger doses. However, whereas peptide histidine methionine was active in all areas of the small intestine, neurotensin, neurotensin-(8-13) and neuromedin-N were inactive in the duodenum. In the post-duodenal areas neurotensin was approximately 7 times more active than peptide histidine methionine, 21 times more potent than neuromedin-N and 33 times more potent than neurotensin-(8-13).