A rat knockout model implicates TRPC4 in visceral pain sensation.

Acute and chronic pain resulting from injury, surgery, or disease afflicts >100 million Americans each year, having a severe impact on mood, mental health, and quality of life. The lack of structural and functional information for most ion channels, many of which play key roles in the detection and transmission of noxious stimuli, means that there remain unidentified therapeutic targets for pain management. This study focuses on the transient receptor potential canonical subfamily 4 (TRPC4) ion channel, which is involved in the tissue-specific and stimulus-dependent regulation of intracellular Ca²âº signaling. Rats with a transposon-mediated TRPC4-knockout mutation displayed tolerance to visceral pain induced by colonic mustard oil (MO) exposure, but not somatic or neuropathic pain stimuli. Moreover, wild-type rats treated with a selective TRPC4 antagonist (ML-204) prior to MO exposure mimicked the behavioral responses observed in TRPC4-knockout rats. Significantly, ML-204 inhibited visceral pain-related behavior in a dose-dependent manner without noticeable adverse effects. These data provide evidence that TRPC4 is required for detection and/or transmission of colonic MO visceral pain sensation. In the future, inhibitors of TRPC4 signaling may provide a highly promising path for the development of first-in-class therapeutics for this visceral pain, which may have fewer side effects and less addictive potential than opioid derivatives.

Changes of atrial natriuretic peptide in rat supraoptic neurones during pregnancy.

To better understand the role of atrial natriuretic peptide (ANP) in the central regulation of hydro-mineral homeostasis, we analysed its expression in rat hypothalamic neurones during gestation and postpartum. These physiological events are characterized by opposing body fluid regulations. Quantitative in situ hybridization analysis showed that starting from mid-pregnancy, ANP mRNA declined in neurones of the preoptic area, periventricular area, lateral hypothalamus and endorhinal nucleus, and remained low at postpartum. By contrast, magnocellular cells in the supraoptic nucleus (SON) showed four- and 10-fold more ANP mRNA in sections from preterm and postpartum rats, respectively, compared to nonpregnant controls (P < 0.001). Oxytocin mRNA paralleled ANP mRNA expression in the SON, whereas vasopressin mRNA rose in early pregnancy and declined thereafter. High hypothalamic ANP concentration at day 21 of gestation versus nonpregnant rats (3.1 +/- 0.5 versus 1.8 +/- 0.4 ng/mg protein, P < 0.05) suggested that ANP transcript accumulation in the SON is associated with increased utilization of the peptide. The elevation of hypothalamic ANP (two-fold) and ANP receptors by treatment of ovariectomized rats with 17beta-oestradiol (25 micro g/rat, 10 days) was abolished by coadministration of progesterone. Thus, we concluded that elevated oestradiol at term stimulates ANP synthesis and paracrine ANP activation in the hypothalamus. Overall, we provide experimental, anatomical and molecular evidence for ANP regulation in hypothalamic neurones at preterm and after 17beta-oestradiol stimulation. Our study supports the concept that ANP expressed in the SON acts as a peptidergic neurotransmitter involved in water and salt regulation during pregnancy and postpartum.

Coordinated expression of beta-amyloid precursor protein and the putative beta-secretase BACE and alpha-secretase ADAM10 in mouse and human brain.

To define the enzymes involved in the etiology of Alzheimer's disease, we compared in mouse and human brain the mRNA levels and cellular localization of the ubiquitous beta-amyloid precursor protein (beta-APP) with those of the putative alpha-secretases ADAM10 and ADAM17 and the beta-secretases BACE and BACE2. In situ hybridization performed in mice during prenatal and postnatal development and in adulthood revealed the coexpression of beta-APP, BACE, and ADAM10. The patterns of BACE2 and ADAM17 only partially overlapped with that of beta-APP. beta-APP, BACE, and ADAM10 mRNAs have also been detected by northern blot in human brain cortex of normal subjects and in Alzheimer's disease subjects. In situ hybridization performed using combined biotin- and radiolabeled riboprobes provided evidence for the coexpression of beta-APP with BACE and ADAM10 in human cortical neurons. Our data provide cytochemical evidence supporting the role of ADAM10 and BACE as authentic alpha- and beta-secretases.

Inhibitory effects of MLDG-containing heterodimeric disintegrins reveal distinct structural requirements for interaction of the integrin alpha 9beta 1 with VCAM-1, tenascin-C, and osteopontin.

The integrin alpha9beta1 is expressed on epithelial cells, smooth muscle cells, skeletal muscle, and neutrophils and recognizes at least three distinct ligands: vascular cell adhesion molecule 1 (VCAM-1), tenascin-C, and osteopontin. The alpha9 subunit is structurally similar to the integrin alpha4 subunit, and alpha9beta1 and alpha4beta1 both recognize VCAM-1 as a ligand. We therefore examined whether the disintegrin EC3, which we have recently shown specifically inhibits the binding of alpha4 integrins to ligands, would also be a functional inhibitor of alpha9beta1. EC3 and a novel heterodimeric disintegrin that we identified, EC6, both were potent inhibitors of alpha9beta1-mediated adhesion to VCAM-1 and of neutrophil migration across tumor necrosis factor-activated endothelial cells. A peptide containing a novel MLDG motif shared by both of these disintegrins also inhibited alpha9beta1- and alpha4beta1-mediated adhesion to VCAM-1. Surprisingly though, concentrations of EC3 that completely inhibited adhesion of alpha9-transfected cells to VCAM-1 had little or no effect on adhesion to either of the other alpha9beta1 ligands, osteopontin and tenascin-C. Furthermore, peptides AEIDGIEL and SVVYGLR, which we have previously shown inhibit binding of alpha9beta1-expressing cells to tenascin-C and osteopontin, respectively, had no effect on adhesion to VCAM-1. These data suggest that there are structurally distinct requirements for interactions of the alpha9beta1 integrin with VCAM-1 and the extracellular matrix ligands osteopontin and tenascin-C.

Developmental expression and tissue distribution of Phex protein: effect of the Hyp mutation and relationship to bone markers.

Mutations in PHEX, a phosphate-regulating gene with homology to endopeptidases on the X chromosome, are responsible for X-linked hypophosphatemia (XLH). The murine Hyp homologue has the phenotypic features of XLH and harbors a large deletion in the 3' region of the Phex gene. We characterized the developmental expression and tissue distribution of Phex protein, using a monoclonal antibody against human PHEX, examined the effect of the Hyp mutation on Phex expression, and compared neprilysin (NEP), osteocalcin, and parathyroid hormone/parathyroid hormone-related protein (PTH/PTHrP) receptor gene expression in bone of normal and Hyp mice. Phex encodes a 100- to 105-kDa glycoprotein, which is present in bones and teeth of normal mice but not Hyp animals. These results were confirmed by in situ hybridization (ISH) and ribonuclease protection assay. Phex protein expression in femur and calvaria decreases with age, suggesting a correlation between Phex expression and bone formation. Immunohistochemical studies detected Phex protein in osteoblasts, osteocytes, and odontoblasts, but not in osteoblast precursors. In contrast to Phex, the abundance of NEP messenger RNA (mRNA) and protein is not significantly altered in Hyp bone. Similarly, osteocalcin and PTH/PTHrP receptor gene expression are not compromised in bone of Hyp mice. Our results are consistent with the hypothesis that loss of Phex function affects the mineralizing activity of osteoblasts rather than their differentiation.

Cellular localization of neprilysin in mouse bone tissue and putative role in hydrolysis of osteogenic peptides.

The regulation of osteoblast and osteoclast metabolism is mediated by both hormones and local bone peptide factors. Peptides and hormones are under control of membrane peptidases such as Neprilysin (NEP). NEP is a widely distributed cell-surface zinc-metallopeptidase that is involved in the regulation of several important physiological processes by controlling the half-life of bioactive peptides. Although NEP is known to be present in skeletal tissues, neither its cellular localization nor its function have been established. To address this question, we examined NEP distribution in bones of postnatal mouse. In situ hybridization (ISH) and immunohistochemistry showed that NEP messenger RNA (mRNA) and protein are associated with bone-forming cells including presumptive osteoblast precursors, preosteoblasts, osteoblasts, and osteocytes. NEP levels in newborn and adult mice bones also were compared by immunoblotting. Higher amounts of NEP immunoreactivity were observed in newborn as compared with adult bones, suggesting a relationship between NEP expression and bone growth. To further explore this hypothesis, we monitored in vitro NEP proteolytic activity using a series of synthetic osteogenic peptides such as parathyroid hormone-related peptide 1-43 (PTHrP1-34), osteostatin (PTHrP107-139), osteogenic growth peptide (OGP), calcitonin, alpha-calcitonin gene-related peptide (alpha-CGRP), and PTH1-34. Except for PTH1-34, all peptides were found to be NEP substrates.

Molecular cloning and biochemical characterization of a new mouse testis soluble-zinc-metallopeptidase of the neprilysin family.

Because of their roles in controlling the activity of several bio-active peptides, members of the neprilysin family of zinc metallopeptidases have been identified as putative targets for the design of therapeutic agents. Presently, six members have been reported, these are: neprilysin, endothelin-converting enzyme (ECE)-1 and ECE-2, the Kell blood group protein, PHEX (product of the phosphate-regulating gene with homologies to endopeptidase on the X chromosome) and X-converting enzyme (XCE). In order to identify new members of this important family of peptidases, we designed a reverse transcriptase-PCR strategy based on conserved amino acid sequences of neprilysin, ECE-1 and PHEX. We now report the cloning from mouse testis of a novel neprilysin-like peptidase that we called NL1. NL1 is a glycoprotein that, among the members of the family, shows the strongest sequence identity with neprilysin. However, in contrast with neprilysin and other members of the family which are type II integral membrane proteins, NL1 was secreted when expressed in cultured mammalian cells, likely due to cleavage by a subtilisin-like convertase at a furin-like site located 22 amino acid residues in the C-terminus of the transmembrane domain. The recombinant enzyme exhibited neprilysin-like peptidase activity and was efficiently inhibited by phosphoramidon and thiorphan, two inhibitors of neprilysin. Northern blot analysis and in situ hybridization showed that NL1 mRNA was found predominantly in testis, specifically in round and elongated spermatids. This distribution of NL1 mRNA suggests that it could be involved in sperm formation or other processes related to fertility.

Study of salivary response to continuous infusion of cerulein and secretin in healthy subjects.

OBJECTIVE: The effects of intravenous infusion of cerulein and secretin on the secretion and biochemical composition of whole saliva in humans were studied. STUDY DESIGN: A total of 33 subjects were divided into 3 groups, which underwent intravenous infusion with saline solution (NaCl 0.15 mol/L(-1)); saline solution, cerulein (1.25 x 10(-3) microg kg(-1) min(-1)), and cerulein supplemented with secretin (2.5 x 10(-2) clinical units kg(-1) min(-1)); and saline solution, secretin (2.5 x 10(-2) clinical units kg(-1) min(-1)), and secretin supplemented with cerulein (1.25 x 10(-3) microg kg(-1) min(-1)). RESULTS: Cerulein reduced salivary flow rate, bicarbonate concentration and output, and protein output and increased amylase activity. The inhibitory effect of cerulein on salivary flow rate and bicarbonate concentration and output prevailed when an infusion of cerulein was supplemented with secretin. Cerulein and secretin acting together increased protein concentration. Secretin alone decreased salivary flow rate and bicarbonate concentration, whereas secretin supplemented with cerulein not only decreased salivary flow rate, bicarbonate concentration, and bicarbonate output but also increased protein concentration. CONCLUSION: The effect of secretin and cerulein on salivary secretion and its composition is quite different from that observed in the pancreas.

Structural requirements of echistatin for the recognition of alpha(v)beta(3) and alpha(5)beta(1) integrins.

There are key differences between the amino acid residues of the RGD loops and the C termini of echistatin, a potent antagonist of alpha(IIb)beta(3), alpha(v)beta(3) and alpha(5)beta(1), and eristostatin, a similar disintegrin selectively inhibiting alpha(IIb)beta(3). In order to identify echistatin motifs required for selective recognition of alpha(v)beta(3) and alpha(5)beta(1) integrins, we expressed recombinant echistatin, eristostatin, and 15 hybrid molecules. We tested them for their ability to inhibit adhesion of different cell lines to fibronectin and von Willebrand factor and to express ligand-induced binding site epitope. The results showed that Asp(27) and Met(28) support recognition of both alpha(v)beta(3) and alpha(5)beta(1). Replacement of Met(28) with Asn completely abolished echistatin's ability to recognize each of the integrins, while replacement of Met(28) with Leu selectively decreased echistatin's ability to recognize alpha(5)beta(1) only. Eristostatin in which C-terminal WNG sequence was substituted with HKGPAT exhibited new activity with alpha(5)beta(1), which was 10-20-fold higher than that of wild type eristostatin. A hypothesis is proposed that the C terminus of echistatin interacts with separate sites on beta(1) and beta(3) integrin molecules.

EC3, a novel heterodimeric disintegrin from Echis carinatus venom, inhibits alpha4 and alpha5 integrins in an RGD-independent manner.

EC3, a heterodimeric disintegrin (Mr = 14,762) isolated from Echis carinatus venom is a potent antagonist of alpha4 integrins. Two subunits called EC3A and EC3B were isolated from reduced and alkylated EC3 by reverse-phase high performance liquid chromatography. Each subunit contained 67 residues, including 10 cysteines, and displayed a high degree of homology to each other and to other disintegrins. EC3 inhibited adhesion of cells expressing alpha4beta1 and alpha4beta7 integrins to natural ligands vascular cell adhesion molecule 1 (VCAM-1) and mucosal addressin cell adhesion molecule 1 (MadCAM-1) with IC50 = 6-30 nM, adhesion of K562 cells (alpha5beta1) to fibronectin with IC50 = 150 nM, and adhesion of alphaIIbbeta3 Chinese hamster ovary cells to fibrinogen with IC50 = 500 nM; it did not inhibit adhesion of alphavbeta3 Chinese hamster ovary cells to vitronectin. Ethylpyridylethylated EC3B inhibited adhesion of Jurkat cells to immobilized VCAM-1 (IC50 = 6 microM), whereas EC3A was inactive in this system. The MLDG motif appeared to be essential for activity of EC3B. Linear MLDG peptide inhibited the adhesion of Jurkat to VCAM-1 in a dose-dependent manner (IC50 = 4 mM), whereas RGDS peptide was not active at the same concentration. MLDG partially inhibited adhesion of K562 cells to fibronectin (5-10 mM) in contrast to RGDS peptide (IC50 = 3 mM), inhibiting completely at 10 mM.

Nerve growth factor and proprotein convertases furin and PC7 in transected sciatic nerves and in nerve segments cultured in conditioned media: their presence in Schwann cells, macrophages, and smooth muscle cells.

Synthesis of proteins such as nerve growth factor (NGF) is induced after nerve lesion. The NGF precursor (pro-NGF) requires a posttranslational processing by proprotein convertases to become active. In this report, we re-examine the localization of NGF protein and mRNA in injured nerve and show that the candidate pro-NGF convertases furin and PC 7 colocalize with NGF in non-neuronal cells in nerve. By Northern blot analysis, 1.5-kb and 1.3-kb NGF mRNAs were shown to be increased in distal and immediately proximal nerve segments on days 1, 4, and 14 after lesion; by Western blot analysis, NGF proteins of high molecular weight were detected after injury. In vivo, two phases of NGF immunopositivity were observed, in macrophages and perivascular cells shortly after lesion and in endoneurial cells on day 1 and 4. To identify the cells containing NGF, nerve segments were incubated in serum-containing medium with or without conditioning by white blood cells isolated from the circulation. Both hybridization and immunoreactivity signals for NGF were elevated after incubation of nerve segments for 4 hours in conditioned media, so that cells with NGF immunoreactivity could be identified by antibodies to specific cell markers. In these nerve fragments, Schwann cells, perivascular smooth muscle cells, and macrophages contained NGF immunoreactivity. The concentration of furin and PC7 mRNA also increased in lesioned nerves. By immunocytochemical investigation of nerve explants, furin and PC7 were detected in endoneurial cells, macrophages and perivascular cells and were colocalized with NGF. These in vitro and in vivo findings suggest that both furin and PC7 are associated with NGF in several cell types of the sciatic nerve and, hence, may be implicated in intracellular processing of pro-NGF.

Interaction of leukocytes with platelet microparticles derived from outdated platelet concentrates.

Platelet microparticles (PMP) were isolated from outdated platelets by a combination of differential centrifugation and gel filtration, and the concentration of PMP was expressed in the equivalent of GPIIb/IIIa complex measured by captured ELISA. PMP bound to isolated neutrophils and macrophages in a dose-dependent manner, but they did not bind to lymphocytes. Incubation of PMP with neutrophils did not activate these cells as measured by up-regulation of Mac-1, release of human granulocyte elastase, and calcium mobilization. Incubation of PMP with macrophages did not enhance IL-8 production and the oxygen burst but slightly and significantly increased production of MCP-1. After 10 min incubation of PMP with macrophages, an increase of GPIIb/IIIa antigen was observed suggesting that PMP may be endocytosed by macrophages. In conclusion, PMP bind to leukocytes, but, in contrast to activated platelets, do not play a significant role in leukocyte activation.

Role of epidermal growth factor (EGF) in oesophageal mucosal integrity.

Oesophageal mucosa has well established protective mechanisms, which operate within pre-epithelial, epithelial and post-epithelial compartments. Since refluxed acid and pepsin always act from the luminal side of the mucosa, protective factors like EGF, operating as a part of pre-epithelial defence, are thought to be pivotal in the maintenance of the integrity of the oesophageal mucosa. The significant contribution of salivary EGF to the quality of the oesophageal mucosal barrier has been demonstrated in an experimental setting and in a clinical scenario. Patients with low salivary EGF levels are predisposed to severe oesophageal damage if they develop gastro-oesophageal reflux and are a high-risk group for development of Barrett's oesophagus. Not only the salivary glands but also the human oesophagus has a profound ability to elaborate and release EGF. Some changes in luminal release of EGF during oesophageal mucosal exposure to intraluminal damaging factors imply its role in the oesophageal protective mechanisms. To exert biological effects within the oesophageal mucosal compartment, EGF requires binding to the ligand-binding domain of its receptor. This process results in receptor dimerisation, autophosphorylation and activation of intracellular signal transduction pathways. EGF receptors are localised on the basolateral and luminal aspect of the mucosal cells playing an important role in fast regeneration of oesophageal epithelium through the high mitotic activity of its proliferative zone. An increase in the rate of salivary EGF secretion during masticatory stimulation suggests its potential therapeutic benefit in the treatment of patients with damaged oesophageal mucosa.

The pro-protein convertase PC1 is induced in the transected sciatic nerve and is present in cultured Schwann cells: comparison with PC5, furin and PC7, implication in pro-BDNF processing.

Injury of peripheral nerves induces expression of several pro-protein convertases (PCs) involved in processing of precursor proteins into their diverse active end-products. In this study, the focus was on convertase PC1 which, although undetectable in control nerves, is strongly induced in injured nerves. High concentrations of PC1 mRNA of 9.0, 5.5, 3.0, 2.5 and 1.6 kb were observed on day 4 post-lesion in proximal and distal segments. By in situ hybridization PC1 mRNA was detected in most of endoneurial cells, which were further identified by immunocytochemistry as myelin 2', 3'-cyclic nucleotide 3'-phosphodiesterase containing Schwann cells. PC1 mRNA and protein were also present in cultured Schwann cells also containing convertases PC5, furin and PC7 as well as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Mostly unprocessed pro-NGF of 35 kDa and pro-BDNF of 35 kDa were found on Western blotting of Schwann cells. Expression of exogenous neurotrophins by infection with vaccinia virus vector showed that mouse pro-NGF and rat pro-BDNF are cleaved intracellularly on smaller forms of 13.5 kDa NGF and 14 kDa BDNF. Infection experiments demonstrated that Schwann cells contain active processing enzymes. In conclusion, this work provides in vivo evidence of the presence of several PCs in the injured rat sciatic nerve and ex vivo in cultured Schwann cells.

Pex mRNA is localized in developing mouse osteoblasts and odontoblasts.

Mutations in PEX, a phosphate-regulating gene with homology to endopeptidase on the X chromosome, were recently identified in patients with X-linked hypophosphatemia (XLH), an inherited disorder of phosphate homeostasis characterized by growth retardation and rachitic and osteomalacic bone disease. To understand the mechanism by which loss of PEX function elicits the mutant phenotype, a study of its mRNA localization and ontogenesis was undertaken. Using the reverse transcriptase-nested polymerase chain reaction (RT-nested PCR) with polyA+ RNA purified from mouse testis, a 337-bp Pex cDNA fragment was generated and cloned in the pCRII plasmid. The cDNA was used to generate sense and anti-sense Pex riboprobes for in situ hybridization (ISH) and Northern analysis. To survey a large number of different tissues, sagittal sections of embryos and newborn mice were examined. ISH showed the presence of Pex mRNA in osteoblasts and odontoblasts. Pex gene expression was detectable on Day 15 of embryonic development, which coincides with the beginning of intercellular matrix deposition in bones. Finally, Northern analysis of total RNA from calvariae and teeth of 3-day-old and adult mice showed that the abundance of the 7-kb Pex transcript is decreased in adult bones and in nongrowing teeth. The present study demonstrates that Pex mRNA is expressed in bones and teeth and suggests that this putative endopeptidase plays an important role in the development of these tissues.

Role of platelet-activating factor in Chinese hamster ovary cell responses to cholera toxin.

Cholera toxin (CT)-induced intestinal secretion and Chinese hamster ovary cell (CHO) elongation involves cyclic adenosine monophosphate and protein synthesis-dependent prostaglandin formation. We previously reported inhibition of CT-induced intestinal secretion and CHO elongation by platelet-activating factor (PAF) receptor antagonists and secretion of PAF by human intestinal epithelial cells exposed to CT. Herein, we show that PAF is involved after cAMP and that PAF, like CT, mediates prostaglandin E2 synthesis in CHO cells. CT-induced CHO elongation was blocked by specific PAF receptor antagonists, BN52021 and SR27417. SR27417 blocked dibutyryl cAMP-induced CHO elongation, but did not alter CHO elongation caused by PGE2. Neither CT-stimulated cAMP accumulation nor PGE2 production was inhibited by SR27417. Both PGE2 and PAF caused significant CHO elongation, but the latter did not stimulate significant cAMP production. In addition, PAF, like CT and dibutyryl cAMP, stimulated significant PGE2 production. Finally, the protein synthesis inhibitor cycloheximide, which completely blocks the effect of CT on prostaglandin synthesis, also blocked that of PAF, suggesting that PAF also mediates protein synthesis-dependent prostaglandin formation. We conclude that PAF is involved in CHO cytoskeletal responses to CT after the accumulation of cAMP and, like CT, PAF stimulates protein synthesis-dependent prostaglandin accumulation.

The isoforms of proprotein convertase PC5 are sorted to different subcellular compartments.

The proprotein convertase PC5 is encoded by multiple mRNAs, two of which give rise to the COOH-terminal variant isoforms PC5-A (915 amino acids [aa]) and PC5-B (1877 aa). To investigate the differences in biosynthesis and sorting between these two proteins, we generated stably transfected AtT-20 cell lines expressing each enzyme individually and examined their respective processing pattern and subcellular localization. Biosynthetic analyses coupled to immunofluorescence studies demonstrated that the shorter and soluble PC5-A is sorted to regulated secretory granules. In contrast, the COOH-terminally extended and membrane-bound PC5-B is located in the Golgi. The presence of a sorting signal in the COOH-terminal 38 amino acids unique to PC5-A was demonstrated by the inefficient entry into the regulated secretory pathway of a mutant lacking this segment. EM of pancreatic cells established the presence of immunoreactive PC5 in glucagon-containing granules, demonstrating the sorting of this protein to dense core secretory granules in endocrine cells. Thus, a single PC5 gene generates COOH-terminally modified isoforms with different sorting signals directing these proteins to distinct subcellular localization, thereby allowing them to process their appropriate substrates.

Impact of Helicobacter pylori colonization on immunoreactive epidermal growth factor and transforming growth factor-alpha in gastric juice. Its potential pathogenetic implications.

Epidermal growth factor (EGF), pivotal in mucosal protection, is partly degraded proteolytically at low pH in the gastric milieu; gastric acid secretion, on the other hand, remains influenced by H. pylori colonization. The aim of this study, therefore, was to evaluate the impact of low pH and H. pylori colonization status on immunoreactive EGF and the other member of EGF-family, immunoreactive transforming growth factor-alpha (TGF-alpha). Eighteen patients with nonulcer dyspepsia (NUD) colonized by H. pylori and 55 NUD patients without H. pylori colonization were investigated. Gastric juice samples were aspirated at the beginning of the endoscopy procedure and immediately placed on ice, and their pH was recorded. The measurement of immunoreactive EGF and TGF-alpha was performed using commercially available radioimmunoassays (RIAs) after adjustment of pH to neutral using an assay buffer. Statistical analysis was performed using sigma-Stat for Windows. The concentration of immunoreactive EGF in patients with NUD colonized by H. pylori was 80% lower (P < 0.02) than in those without H. pylori and in both groups immunoreactive EGF was significantly lower when the pH of gastric juice was below 4.0. The concentration of immunoreactive EGF in H. pylori(+) and H. pylori(-) patients was similar when the pH of aspirated gastric juice was above 4.0. However, with gastric juice pH < 4.0, the EGF concentration was 64% lower in H. pylori(+) patients than H. pylori(-) patients (P < 0.05). In general, the concentration of immunoreactive TGF-alpha in gastric juice was unaffected by H. pylori colonization or pH of gastric juice. It is concluded that: (1) significantly lower immunoreactive EGF concentrations in patients with pH below 4.0 indicate that immunoreactive EGF but not immunoreactive TGF-alpha is affected by an acidic gastric milieu; (2) the further reduction of gastric juice immunoreactive EGF at pH below 4.0 in patients colonized by H. pylori suggests that this microorganism may elaborate factors that accelerate its proteolytic degradation or inhibit its rate of synthesis and/or secretion; and (3) this diminished content of immunoreactive EGF at low pH, especially in patients colonized by H. pylori, may facilitate the development and/or progression of mucosal damage.