Pertuzumab plus trastuzumab and chemotherapy for HER2-positive metastatic gastric or gastro-oesophageal junction cancer (JACOB): final analysis of a double-blind, randomised, placebo-controlled phase 3 study.

BACKGROUND: Adding pertuzumab to trastuzumab and chemotherapy improves survival in HER2-positive early breast cancer and metastatic breast cancer. We assessed the efficacy and safety of pertuzumab versus placebo in combination with trastuzumab and chemotherapy in first-line HER2-positive metastatic gastric or gastro-oesophageal junction cancer. METHODS: JACOB was a double-blind, placebo-controlled, randomised, multicentre, phase 3 trial in patients aged 18 years or older with HER2-positive metastatic gastric or gastro-oesophageal junction cancer. Eligible patients had measurable or evaluable non-measurable disease at baseline, Eastern Cooperative Oncology Group performance status of 0 or 1, and baseline left ventricular ejection fraction of 55% or more. Patients at 197 oncology clinics (in 30 countries) were randomly assigned (1:1) to receive either pertuzumab (840 mg intravenously) or placebo every 3 weeks, with trastuzumab (8 mg/kg loading dose, then 6 mg/kg every 3 weeks intravenously), plus chemotherapy (cisplatin 80 mg/m2 every 3 weeks intravenously, oral capecitabine 1000 mg/m2 twice a day [2000 mg/m2 every 24 h] for 28 doses every 3 weeks, or 5-fluorouracil 800 mg/m2 every 24 h intravenously [120 h continuous infusion] every 3 weeks). Randomisation was by a central permuted block randomisation scheme (block size of 4) with an interactive voice or web response system, stratified by geographical region, previous gastrectomy, and HER2 positivity. The primary endpoint was overall survival in the intention-to-treat population. This trial is registered with Clinicaltrials.gov, number NCT01774786 (ongoing, but closed to enrolment). FINDINGS: Between June 10, 2013, and Jan 12, 2016, of 3287 patients assessed, 780 eligible patients were randomly assigned to receive either pertuzumab plus trastuzumab and chemotherapy (pertuzumab group, n=388) or placebo plus trastuzumab and chemotherapy (control group, n=392). Median duration of follow-up was 24·4 months (95% CI 22·3-26·1) in the pertuzumab group and 25·0 months (22·3-28·9) in the control group. After 242 deaths in the pertuzumab group and 262 deaths in the control group (the study was not stopped at this point), overall survival was not significantly different between treatment groups (median overall survival 17·5 months [95% CI 16·2-19·3] in the pertuzumab group and 14·2 months [12·9-15·5] in the control group; hazard ratio 0·84 [95% CI 0·71-1·00]; p=0·057). Serious adverse events occurred in 175 (45%) of 385 patients in the pertuzumab group and 152 (39%) of 388 patients in the control group. Diarrhoea was the most common serious adverse event in both groups (17 [4%] patients in the pertuzumab group vs 20 [5%] patients in the control group). The most common grade 3-5 adverse events were neutropenia (116 [30%] patients in the pertuzumab group vs 108 [28%] patients in the control group), anaemia (56 [15%] vs 65 [17%]), and diarrhoea (51 [13%] vs 25 [6%]). Treatment-related deaths occurred in seven (2%) patients in the control group; no treatment-related deaths occurred in the pertuzumab group. INTERPRETATION: Adding pertuzumab to trastuzumab and chemotherapy did not significantly improve overall survival in patients with HER2-positive metastatic gastric or gastro-oesophageal junction cancer compared with placebo. Further studies are needed to identify improved first-line treatment options in these types of cancer and to identify patients with HER2-driven tumours who might benefit from dual HER2-targeted therapy. FUNDING: F. Hoffmann-La Roche Ltd.

A Phase II Study of the c-Met Inhibitor Tivantinib in Combination with FOLFOX for the Treatment of Patients with Previously Untreated Metastatic Adenocarcinoma of the Distal Esophagus, Gastroesophageal Junction, or Stomach.

BACKGROUND: This phase I/II study was designed to determine the maximum tolerated dose of tivantinib in combination with standard dose FOLFOX for the treatment of patients with advanced solid tumors and to evaluate the safety and efficacy of this combination for patients with previously untreated metastatic adenocarcinoma of the distal esophagus, gastroesophageal (GE) junction, or stomach. METHODS: Patients with advanced solid tumors for which FOLFOX would be appropriate chemotherapy received escalating doses of tivantinib BID (days 1-14) in a standard 3 + 3 design in phase I. In phase II, patients with advanced GE cancer received standard FOLFOX day 1 and tivantinib (360 mg PO BID) days 1-14 of each 2-week cycle. Restaging occurred every four cycles. The primary phase II endpoint was response rate (RR). RESULTS: Forty-nine patients were enrolled (15 on phase I and 34 on phase II). The expansion dose was established as tivantinib 360 mg BID in combination with FOLFOX. Thirty-two phase II patients were treated for a median of eight cycles (range, 1-38), with an overall RR of 38%. Treatment-related toxicities included neutropenia, fatigue, diarrhea, nausea, and peripheral neuropathy. Median progression-free survival (PFS) was 6.1 hmonths with a median time to progression of 7.0 months. Median overall survival was 9.6 months. Two patients remain on study at the time of this analysis. CONCLUSIONS: The combination treatment of tivantinib plus FOLFOX in patients with advanced GE cancer showed a response and PFS in the range of historical controls for first-line FOLFOX therapy. However, two patients had extended time on study treatment (36 and 45 cycles) at the time of data cutoff.

Central nervous system relapse in patients with untreated HER2-positive esophageal or gastroesophageal junction adenocarcinoma.

Although HER2-positive breast cancers demonstrate a propensity for central nervous system (CNS) metastasis, it is unknown whether other HER2-positive tumors, including adenocarcinomas of the esophagus/gastroesophageal junction (EAC), share this characteristic. Insight into this association may inform the development of HER2-targeted therapies that penetrate the blood-brain barrier. We examined HER2 overexpression and gene amplification in 708 patients with EAC who underwent curative-intent surgery during a time period (1980-1997) when no patient received HER2-targeted therapy. We identified patients whose site of first cancer recurrence was CNS and those who had a CNS relapse at any time. After a median follow-up of 61.2 months, 3.4% (24/708) of patients developed CNS relapse (all involved the brain). Patients with HER2-positive (vs -negative) primary tumors showed a higher 5-year cumulative incidence of CNS relapse as first recurrence (5.8% vs. 1.2%; p = 0.0058) and at any time (8.3% vs. 2.4%; p = 0.0062). In a multivariable model that included covariates previously associated with HER2 or with CNS relapse in breast cancer, HER2 positivity was the only variable that was statistically significantly associated with shorter time to CNS relapse as first recurrence (p = 0.0026) or at any time (hazard ratio 4.3 [95% confidence interval 1.8 to 10.3]; p = 0.001). These are the first data in a non-breast cancer to demonstrate an association between HER2 positivity and higher CNS relapse risk after surgery, and suggest that HER2-positive EACs have a predilection for CNS metastases.

The prognostic impact of epidermal growth factor receptor in patients with metastatic gastric cancer.

BACKGROUND: The epidermal growth factor receptor (EGFR) is a potential target of anticancer therapy in gastric cancer. However, its prognostic role in metastatic gastric or gastroesophageal junction (GE) cancer has not been established yet. METHODS: EGFR status was analyzed by immunohistochemistry (IHC) in paraffin-embedded samples from 357 patients who received chemotherapy in 4 first-line trials. Automated RNA extraction from paraffin and RT-quantitative PCR were additionally used to evaluate EGFR mRNA expression in 130 patients. RESULTS: EGFR protein expression (any grade) and overexpression (3+) were observed in 43% and 11% of patients, respectively. EGFR positivity correlated with intestinal type histology (p = 0.05), but not with other clinicopathologic characteristics. Median follow-up was 18.2 months. Median overall survival (OS) was similar in patients with EGFR positive vs. those with EGFR negative tumors, regardless whether positivity was defined as ≥1+ (10.6 vs. 10.9 months, p = 0.463) or as 3+ (8.6 vs. 10.8 months, p = 0.377). The multivariate analysis indicated that EGFR status is not an independent prognostic factor (hazard ratio 0.85, 0.56 to 1.12, p = 0.247). There were also no significant differences in overall survival when patients were categorized according to median (p = 0.116) or quartile (p = 0.767) distribution of EGFR mRNA gene expression. Similar distributions of progression-free survival according to EGFR status were observed. CONCLUSIONS: Unlike different cancer types where EGFR-positive disease is associated with an adverse prognostic value, EGFR positivity is not prognostic of patient outcome in metastatic gastric or GE cancer.

SMARCA4/SMARCA2-deficient Carcinoma of the Esophagus and Gastroesophageal Junction.

Undifferentiated carcinoma of the esophagus and gastroesophageal junction is a recently recognized entity in the fifth edition of the World Health Organization Classification of Digestive Tumors and is diagnostically challenging, particularly on small biopsies. SMARCA4 and SMARCA2 are chromatin remodeling genes with key roles in oncogenesis. We retrieved 14 cases of SMARCA4/SMARCA2-deficient undifferentiated carcinoma of the gastroesophageal junction and esophagus from the authors' institutions. The tumors showed similar histologic findings: the sheet-like proliferation of tumor cells characterized by discohesion, large nuclei, and prominent macronucleoli with many tumor cells exhibiting a rhabdoid appearance. In 8 cases, adjacent specialized intestinal metaplasia was noted and 3 cases exhibited adjacent high-grade dysplasia. Immunohistochemically, tumors variably expressed keratins and disclosed loss of expression of SMARCA4 in 12 and SMARCA2 in 7 cases. In 2 cases SMARCA2 alone was lost without SMARCA4 loss. A mutant p53 immunohistochemical pattern was seen in 4 of 4 cases, 3 of which showed diffuse, strong nuclear expression, and 1 case displayed a complete loss of nuclear expression of p53, including invasive carcinoma and associated dysplasia, when present. Limited clinical follow-up was available, but 3 patients died of disease within 0.6, 2, and 7 months of diagnosis. We present the first series of undifferentiated carcinoma of the esophagus and gastroesophageal junction with this characteristic morphology associated with loss of SMARCA4 and/or SMARCA2 expression. This tumor type likely arises from dedifferentiation of a lower grade carcinoma in some cases, and Barrett esophagus and appears to be associated with an aggressive clinical course.

Randomized, Phase II Study of Trastuzumab Beyond Progression in Patients With HER2-Positive Advanced Gastric or Gastroesophageal Junction Cancer: WJOG7112G (T-ACT Study).

PURPOSE: This study evaluated the continuous use of trastuzumab beyond progression (TBP) in human epidermal growth factor receptor 2 (HER2)-positive advanced gastric or gastroesophageal junction (G/GEJ) cancer. PATIENTS AND METHODS: Patients with HER2-positive advanced G/GEJ cancer refractory to first-line chemotherapy with trastuzumab in combination with fluoropyrimidine and platinum were eligible. Patients were randomly assigned to the paclitaxel (80 mg/m2, days 1, 8, and 15, every 4 weeks) or paclitaxel with trastuzumab (PT; initially 8 mg/kg followed by 6 mg/kg, every 3 weeks) arms. The primary endpoint was progression-free survival (PFS). Secondary endpoints included overall survival (OS), response rate, and safety. Biomarkers such as HER2 expression status in tumor tissue after first-line treatment, HER2 amplification evaluated in serum cell-free DNA, and soluble HER2 levels were analyzed. RESULTS: Overall, 91 patients were allocated to the paclitaxel (n = 46) and PT (n = 45) arms. The median PFS in the paclitaxel and PT arms was 3.2 and 3.7 months, respectively (hazard ratio [HR], 0.91; 80% CI, 0.67 to 1.22; P = .33), and the median OS in both arms was 10 months (HR, 1.2; 95% CI, 0.75 to 2.0; P = .20). The overall response rates in the paclitaxel and PT arms were 32% and 33%, respectively (P = 1.00), and safety was comparable between the 2 arms. On exploratory analyses, HER2 positivity of tumor tissues was lost after first-line chemotherapy in 11 (69%) of 16 patients whose tumor tissues were available, and circulating HER2 DNA amplification was detected in 41 (60%) of 68 patients. However, no biomarkers associated with efficacy of TBP were found. CONCLUSION: The TBP strategy failed to improve PFS in patients with HER2-positive advanced G/GEJ cancer, and no beneficial biomarkers were found.

[The indices of pro- and antioxidant state of the gastro-esophageal junction tissues in experimental gastroesophageal reflux disease].

Experimental investigation on studying of pro- and antioxidant state in esophageal tissues was performed. It was established, that accumulation of products of the proteins and lipids oxidation during inflammatory process course coincides with significant lowering of the antioxidant enzymes activity. The data obtained trusts the various diagnostic significance of diienic conjugates and malonic dialdehyde (MDA) levels. The increase of MDA concentration as well as of products of the oxidational modifications proteins, owing basic character, was revealed as a typical biochemical marker of acid-peptic injury of esophageal tissues.

HER2 testing of gastro-oesophageal adenocarcinoma: a commentary and guidance document from the Association of Clinical Pathologists Molecular Pathology and Diagnostics Committee.

The use of biologics targeted to the human epidermal growth factor receptor 2 (HER2) protein is the latest addition to the armamentarium used to fight advanced gastric or gastro-oesophageal junction adenocarcinoma. The decision to treat with the biologic trastuzumab is completely dependent on HER2 testing of tumour tissue. In 2017, the College of American Pathologists, American Society for Clinical Pathology and the American Society of Clinical Oncology jointly published guidelines for HER2 testing and clinical decision making in gastro-oesophageal adenocarcinoma. The Association of Clinical Pathologists Molecular Pathology and Diagnostics Committee has issued the following document as a commentary of these guidelines and, in parallel, to provide guidance on HER2 testing in National Health Service pathology departments within the UK. This guidance covers issues related to case selection, preanalytical aspects, analysis and interpretation of such HER2 testing.

Reflux esophagitis or Helicobacter infection? - diagnostic value of the inflammatory pattern in metaplastic mucosa at the squamocolumnar junction.

OBJECTIVES: Metaplastic glandular mucosa with goblet cells at the squamocolumnar junction is induced either by reflux or by Helicobacter infection. We investigated whether the accompanying inflammation may give information about the etiology of these metaplastic changes and whether there are further criteria which are helpful in differentiating Helicobacter-induced vs. reflux-caused metaplasia. METHODS: One hundred and nine patients with intestinal metaplasia diagnosed in biopsies obtained immediately below the Z-line were evaluated. Further biopsies were taken from the gastric body and antrum. Patients were diagnosed as having a normal Z-line, or as showing short tongues or segments of Barrett's esophagus endoscopically. Inflammation was graded according to the updated Sydney-system. Metaplasia was typed using Gomori's-aldehyde-fuchsin-Alcianblue staining. RESULTS: Compared to patients with Barrett's esophagus, the active (p=0.0002) and chronic inflammation (p=0.0004) at the squamocolumnar junction was higher in patients with a normal Z-line and frequently accompanied by lymphoid aggregates (p<0.0001) and regular cardia- (p=0.0044) and/or corpus-type glands (p=0.0004). Pseudogoblet cells were more frequent in Barrett's esophagus (p=0.0159). CONCLUSIONS: The endoscopic aspect of the Z-line, the inflammatory pattern, and the type of glands in biopsies from the squamocolumnar junction, as well as the presence of pseudogoblet cells are helpful tools in distinguishing Barrett's mucosa from Helicobacter-associated intestinal metaplasia.

HER2 Testing and Clinical Decision Making in Gastroesophageal Adenocarcinoma: Guideline From the College of American Pathologists, American Society for Clinical Pathology, and the American Society of Clinical Oncology.

Context ERBB2 (erb-b2 receptor tyrosine kinase 2 or HER2) is currently the only biomarker established for selection of a specific therapy for patients with advanced gastroesophageal adenocarcinoma (GEA). However, there are no comprehensive guidelines for the assessment of HER2 in patients with GEA. Objectives To establish an evidence-based guideline for HER2 testing in patients with GEA, formalize the algorithms for methods to improve the accuracy of HER2 testing while addressing which patients and tumor specimens are appropriate, and to provide guidance on clinical decision making. Design The College of American Pathologists (CAP), American Society for Clinical Pathology (ASCP), and the American Society of Clinical Oncology (ASCO) convened an Expert Panel to conduct a systematic review of the literature to develop an evidence-based guideline with recommendations for optimal HER2 testing in patients with GEA. Results The Panel is proposing 11 recommendations with strong agreement from the open comment participants. Recommendations The Panel recommends that tumor specimen(s) from all patients with advanced GEA, who are candidates for HER2-targeted therapy, should be assessed for HER2 status before the initiation of HER2-targeted therapy. Clinicians should offer combination chemotherapy and an HER2-targeted agent as initial therapy for all patients with HER2-positive advanced GEA. For pathologists, guidance is provided for morphologic selection of neoplastic tissue, testing algorithms, scoring methods, interpretation and reporting of results, and laboratory quality assurance. Conclusion This guideline provides specific recommendations for assessment of HER2 in patients with advanced GEA while addressing pertinent technical issues and clinical implications of the results.

Novel Targeted Therapies for Esophagogastric Cancer.

Gastroesophageal cancer (GEC) remains a major cause of cancer-related mortality worldwide. Although the incidence of distal gastric adenocarcinoma (GC) is declining in the United States, proximal esophagogastric junction adenocarcinoma (EGJ) is increasing in incidence. GEC, including GC and EGJ, is treated uniformly in the metastatic setting. Overall survival in the metastatic setting remains poor. Molecular characterization of GEC has identified mutations and copy number variations, along with other oncogenes, biomarkers, and immuno-oncologic checkpoints that may serve as actionable therapeutic targets. This article reviews these key aberrations, their impact on protein expression, therapeutic implications, and clinical directions within each pathway.

Update on Gastroesophageal Adenocarcinoma Targeted Therapies.

Gastroesophageal cancer (GEC) remains a major cause of cancer-related mortality worldwide. Although the incidence of distal gastric adenocarcinoma (GC) is declining in the United States, proximal esophagogastric junction adenocarcinoma (EGJ) incidence is rising. GC and EGJ, together, are treated uniformly in the metastatic setting as GEC. Overall survival in the metastatic setting remains poor, with few molecular targeted approaches having been successfully incorporated into routine care to date-only first-line anti-HER2 therapy for ERBB2 amplification and second-line anti-VEGFR2 therapy. This article reviews aberrations in epidermal growth factor receptor, MET, and ERBB2, their therapeutic implications, and future directions in targeting these pathways.

Anti-reflux anastomosis following esophagectomy for adenocarcinoma of the esophagogastric junction: impact of duodenogastroesophageal reflux and expression of cyclooxygenase-2 in the remnant esophagus.

OBJECTIVE: Reflux is the principal complication for patients after esophagectomy with gastric reconstruction. The aim of this study was to investigate the effect of the modified Nissen fundoplication after resection of adenocarcinoma from the esophagogastric junction (AEG) on controlling the reflux and the role of duodenogastroesophageal reflux (DGER) and cyclooxygenase-2 (COX-2) expression level in the remnant esophagus. PATIENTS AND METHODS: Sixty patients with AEG were randomly divided into two groups: (i) the conventional anastomosis group and (ii) the anti-reflux anastomosis group. Fifty esophagectomized patients were invited to participate in postoperative follow-up after 6 to 12 months. Among those we had 29 cases in the conventional anastomosis group and 21 in the anti-reflux anastomosis group. We used endoscopy, simultaneous 24 hours esophageal pH and bilirubin monitoring in this study. The COX-2 expression level in the remnant esophagus was detected using real-time PCR. RESULTS: The reflux esophagitis prevalence in anti-reflux anastomosis group was comparable to that in the conventional group (p = 0.154). DeMeester score and fraction time of bilirubin abs >0.14 decreased more intensely in the anti-reflux anastomosis group (p < 0.05). The COX-2 expression level in of anti-reflux anastomosis group was evidently lower than that in the conventional anastomosis group (p = 0.022) while it was meaningfully higher compared to the normal control group (p = 0.046). COX-2 up-regulation as well as high prevalence of esophagitis were observed in simultaneous acid reflux and DGER (p < 0.05). CONCLUSIONS: Although modified fundoplication following resection of AEG did not achieve an optimal effect on controlling reflux, it was very effective in decreasing the reflux. COX-2 expression monitoring can be considered as a possible new way to evaluate the impact of anti-reflux surgery. DGER occurring in acidic environment could develop severe reflux esophagitis and up-regulate the COX-2 expression.

HER2 testing in gastric cancer: An update.

Human epidermal growth factor receptor 2 (HER2) overexpression is increasingly recognized as a frequent molecular abnormality in gastric and gastroesophageal cancer. With the recent introduction of HER2 molecular targeted therapy for patients with advanced gastric cancer, determination of HER2 status is crucial in order to select patients who may benefit from this treatment. This paper provides an update on our knowledge of HER2 in gastric and gastroesophageal cancer, including the prognostic relevance of HER2, the key differences between HER2 protein expression interpretation in breast and gastric cancer, the detection methods and the immunohistochemistry scoring system.

Hypermethylation and loss of expression of glutathione peroxidase-3 in Barrett's tumorigenesis.

Chronic gastroesophageal reflux disease is a known risk factor for Barrett's esophagus (BE), which induces oxidative mucosal damage. Glutathione peroxidase-3 (GPx3) is a secretory protein with potent extracellular antioxidant activity. In this study, we have investigated the mRNA and protein expression of GPx3, and explored promoter hypermethylation as an epigenetic mechanism for GPx3 gene inactivation during Barrett's carcinogenesis. Quantitative real-time reverse transcription polymerase chain reaction on 42 Barrett's adenocarcinomas (BAs) revealed consistently reduced levels of GPx3 mRNA in 91% of tumor samples. GPx3 promoter hypermethylation was detected in 62% of Barrett's metaplasia, 82% of dysplasia, and 88% of BA samples. Hypermethylation of both alleles of GPx3 was most frequently seen in BAs (P = .001). Immunohistochemical staining of GPx3 in matching tissue sections (normal, BE, Barrett's dysplasia, and BA) revealed strong immunostaining for GPx3 in normal esophageal and gastric tissues. However, weak to absent GPx3 staining was observed in Barrett's dysplasia and adenocarcinoma samples where the promoter was hypermethylated. The degree of loss of immunohistochemistry correlated with the hypermethylation pattern (monoallelic versus biallelic). The observed high frequency of promoter hypermethylation and progressive loss of GPx3 expression in BA and its associated lesions, together with its known function as a potent antioxidant, suggest that epigenetic inactivation and regulation of glutathione pathway may be critical in the development and progression of BE.

Innervation of the esophagus in mice that lack MASH1.

The striated muscle of the esophagus differs from other striated muscle, because it develops by the transdifferentiation of smooth muscle, and the motor end plates receive a dual innervation from vagal (cholinergic) motor neurons and nitric oxide synthase (NOS)-containing enteric neurons. Mash1-/- mice have no enteric neurons in their esophagus and die within 48 hours of birth without milk in their stomachs (Guillemot et al. [1993] Cell 75:463-476). In this study, the innervation of the esophagus of newborn Mash1-/-, Mash1+/- and wild type mice was examined. There was no difference between Mash1-/-, Mash1+/-, and wild type mice in the transdifferentiation of the muscle and the development of nicotinic receptor clusters. However, there were significantly more cholinergic nerve terminals per motor end plate in Mash1-/- mice than Mash1+/- or wild type mice. Each of the Mash1-/- mice had fewer than 50 NOS neurons per esophagus, compared with approximately 3,000 in wild type mice. Newborn Mash1+/- mice also contained significantly fewer NOS neurons than wild type mice. In Mash1-/- mice, NOS nerve fibers were virtually absent from the external muscle but were present at the myenteric plexus. Unlike that of newborn wild type mice, the lower esophageal sphincter of Mash 1-/- mice lacked NOS nerve fibers; this may explain the absence of milk in the stomach. We conclude that 1) the transdifferentiation of the esophageal muscle and the development of the extrinsic innervation do not require enteric neurons or MASH1, 2) extrinsic NOS neurons only innervate the myenteric plexus.

Myogenic nitric oxide synthase activity in canine lower oesophageal sphincter: morphological and functional evidence.

1. Studies on canine lower oesophageal sphincter (LOS) evaluated the existence and function of a myogenic, nitric oxide synthase (NOS) by use of immunocytochemistry for NOS isozymes, NADPH-d histochemistry, [3H]-L-arginine to [3H]-L-citrulline transformation. In addition, functional studies in the muscle bath were performed. 2. Smooth muscle bundles or freshly isolated smooth muscle cells of LOS were NADPH-d reactive but did not recognize some antibodies against neural, endothelial or inducible NOS. NADPH-d reactivity and immunoreactivity to a neural NOS antibody were colocalized in LOS enteric nerves. Muscle plasma membrane-enriched fractions from fresh and cultured LOS cells converted [3H]-L-arginine to [3H]-L-citrulline; activity was mostly Ca2+/calmodulin-dependent. 3. N-Nitro-L-arginine (L-NOARG) persistently increased tone (blocked by L-arginine) in muscle strips despite blockade of nerve function. Nifedipine prevented or abolished L-NOARG-induced, but not carbachol-induced, contraction showing that tone increase by L-NOARG required functional L-Ca channels. 4. Membrane-bound, myogenic NOS in canine LOS may release NO continuously when Ca2+ entry through L-Ca channels occurs under physiological conditions and thereby modulate tone in LOS.

Localization and activity of haem oxygenase and functional effects of carbon monoxide in the feline lower oesophageal sphincter.

1. In the feline lower oesophageal sphincter (LOS), the distribution of the carbon monoxide (CO) producing enzymes haem oxygenase (HO)-1 and -2 was studied by immunohistochemistry and confocal microscopy, the HO activity was measured and the possible role for CO as a mediator of relaxation was investigated. 2. HO-2 immunoreactivity was abundant in nerve cell bodies of the submucosal and myenteric plexus. Approximately 50% of the HO-2-containing myenteric cell bodies were also nitric oxide synthase- and vasoactive intestinal peptide (VIP)-immunoreactive. In addition, HO-2 immunoreactivity was seen in nerve fibres, in non-neuronal cells dispersed in the smooth muscle and in arterial endothelium. HO-1 immunoreactivity was confined to non-neuronal cells in the smooth muscle, similar to those positive for HO-2. 3. Activity of HO, measured as CO production, was observed in LOS homogenates at a rate of 1.00 +/- 0.05 nmol mg-1 protein h-1. This production was inhibited by the HO inhibitor, zinc protoporphyrin-IX (ZnPP). 4. In isolated circular smooth muscle strips of LOS, developing spontaneous tone, exogenously administered CO evoked a concentration-dependent relaxation reaching a maximum of 93 +/- 3%. This relaxation was accompanied by an increase in cyclic GMP, but not cyclic AMP levels. The relaxant response was attenuated by methylene blue, but unaffected by tetrodotoxin. Repeated exposure to CO resulted in a progressive reduction of the relaxant response. 5. ZnPP caused a rightward-shift of the concentration-response curves for the relaxant responses to VIP, peptide histidine isoleucine, and pituitary adenylate cyclase activating peptide 27. 6. ZnPP and tin protoporphyrin-IX (another inhibitor of HO) did not affect nonadrenergic, noncholinergic relaxations induced by electrical field stimulation. Nor did ZnPP affect relaxations induced by 3-morpholino-sydnonimine or forskolin. 7. The present findings, showing localization of HO immunoreactivity to both neuronal and nonneuronal cells of the feline LOS, ability of LOS to produce CO and a relaxant effect of CO in circular LOS muscle, suggest a role for CO as a peripheral messenger.

Carbon monoxide-induced relaxation and distribution of haem oxygenase isoenzymes in the pig urethra and lower oesophagogastric junction.

1. The distribution of the carbon monoxide (CO) producing enzymes haem oxygenase (HO)-1 and -2 was studied by immunohistochemistry in the pig's lower urinary tract, including bladder extramural arteries, and the oesophagogastric junction (OGJ). In isolated smooth muscle from the urethra and the OGJ, the mechanisms for CO-induced relaxations were characterized by measurement of cyclic nucleotide levels and by responses to the guanylate cyclase inhibitor methylene blue and some K+ channel inhibitors. 2. HO-2 immunoreactivity was observed in coarse nerve trunks within the smooth muscle of the urethra and OGJ, and in nerve cell bodies of the enteric plexuses of the OGJ. Furthermore, the vascular endothelium of the intramural vessels of the urethra, bladder and OGJ, and the extramural vessels of the bladder, displayed HO-2 immunoreactivity. Two different antisera against HO-1 were used, but only one displayed immunoreactivity in neuronal structures. HO-1 immunoreactivity, as displayed by this antiserum, was seen in nerve cells, coarse nerve trunks and varicose nerve fibres in the smooth muscle of the urethra and OGJ. Some HO-2 and/or HO-1 (as displayed by both HO-1 antisera) immunoreactive cells with a non-neuronal appearance were observed within the smooth muscle of the OGJ, bladder and urethra. 3. In the urethral preparations, exogenously applied CO (72 microM) evoked a relaxation amounting to 76 +/- 6%. The relaxation was associated with an increase in cyclic GMP, but not cyclic AMP, content. CO-evoked relaxations were not significantly reduced by treatment with methylene blue, or by inhibitors of voltage-dependent (4-aminopyridine), high (iberiotoxin, charybdotoxin) and low (apamin) conductance Ca(2+)-activated, and ATP-sensitive (glibenclamide) K+ channels. Bladder strips, and ring preparations from the extramural arteries of the bladder, did not respond to exogenously administered CO (12-72 microM). 4. In the OGJ, exogenously applied CO evoked a relaxation of 86 +/- 6%, which was associated with an increase in cyclic GMP, but not cyclic AMP, content. Treatment with 30 microM methylene blue raised the spontaneously developed muscle tone, and reduced the maximum relaxation evoked by CO to 33 +/- 9%. Addition of 4-aminopyridine, apamin, glibenclamide, iberiotoxin, charybdotoxin or glibenclamide had no effect on the relaxations. 4-aminopyridine (0.1-1 mM), iberiotoxin (0.1 microM) and charybdotoxin (0.1 microM) increased the spontaneously developed tone, and a combination of charybdotoxin and apamin reduced CO-induced (24 microM CO) relaxations. 5. The present findings demonstrate the presence of HO in both neuronal and non-neuronal cells in the pig OGJ and lower urinary tract. CO produces relaxation of the smooth muscle in the OGJ and urethra, associated with a small increase in cyclic GMP concentration in both regions. Relaxations evoked by CO in the urethra do not seem to involve voltage-dependent, low and high conductance, or ATP-dependent K+ channels. However, in the OGJ relaxations evoked by CO can be attenuated by methylene blue and a combination of charybdotoxin and apamin.

Carbon monoxide as a putative messenger molecule in the feline lower oesophageal sphincter of the cat.

The distribution of the carbon monoxide (CO) producing enzymes haem oxygenase (HO) type 1 and 2 were studied in the feline lower oesophageal sphincter (LOS), as were HO activity and functional effects of CO. HO-2 immunoreactivity was observed in nerve cell bodies in the submucosal and myenteric plexus, nerve fibres, non-neuronal cells surrounding smooth muscle bundles, and in arterial endothelium, HO-1 immunoreactivity was confined to non-neuronal cells in the smooth muscle layer. CO production, indicating HO activity, was demonstrated in tissue homogenates. CO relaxed the LOS, and activated the cyclic GMP system. These results show that HO is present in the LOS, and suggest that CO can be generated by neuronal and non-neuronal structures and may have a role as a peripheral messenger.