PD-1 inhibitor therapy of basal cell carcinoma with pulmonary metastasis.

Basal cell carcinoma (BCC) may be challenging to differentiate from basaloid squamous cell carcinoma (bSCC), both clinically and histologically. BCC constitutes one of the most common tumours and metastatic behaviour is extremely rare. In contrast, bSCC is a rare entity with an increased propensity for distant metastasis. If these conditions develop into inoperable metastatic disease, the therapeutic alternatives are different, but the use of PD-1 inhibitors may be a valid option for both. Here, we report a case with complex histology with a component initially classified as bSCC with lung metastases and treated with the PD-1 inhibitor cemiplimab resulting in radiological and clinical responses. Re-examination of the lung biopsy using routine histomorphology in combination with immunohistochemical staining for cytokeratin 14, cytokeratin17 and BerEp4 has, however, revealed a histopathological pattern of BCC, which is in concordance with a similar analysis of the cutaneous primary tumour in the face that the patient underwent surgery for more than 5 years earlier.

Supporting clinical decision making in advanced melanoma by preclinical testing in personalized immune-humanized xenograft mouse models.

BACKGROUND: The mouse strains usually used to generate patient-derived xenografts (PDXs) are immunocompromised, rendering them unsuitable for immunotherapy studies. Here we assessed the value of immune-PDX mouse models for predicting responses to anti-PD-1 checkpoint inhibitor therapy in patients. PATIENTS AND METHODS: Melanoma biopsies contained in a retrospective biobank were transplanted into NOG mice or NOG mice expressing interleukin 2 (hIL2-NOG mice). Tumor growth was monitored, and comparisons were made with clinical data, sequencing data, and current in silico predictive tools. RESULTS: Biopsies grew readily in NOG mice but growth was heterogeneous in hIL2-NOG mice. IL2 appears to activate T-cell immunity in the biopsies to block tumor growth. Biopsy growth in hIL2-NOG mice was negatively associated with survival in patients previously treated with PD-1 checkpoint blockade. In two cases, the prospective clinical decisions of anti-PD-1 therapy or targeted BRAF/MEK inhibitors were supported by the observed responses in mice. CONCLUSIONS: Immune-PDX models represent a promising addition to future biomarker discovery studies and for clinical decision making in patients receiving immunotherapy.

BRAF mutational status as a prognostic marker for survival in malignant melanoma: a systematic review and meta-analysis.

Background: The analysis of the BRAF mutational status has been established as a standard procedure during diagnosis of advanced malignant melanoma due to the fact that BRAF inhibitors constitute a cornerstone in the treatment of metastatic disease. However, the general impact of BRAF mutational status on survival remains unclear. Our study aimed to assess the underlying prognostic significance of BRAF mutant versus wild type (WT) malignant melanoma on overall survival (OS), disease-free survival (DFS) and progression-free survival (PFS).Material and methods: A systematic literature search in EMBASE, Medline and Cochrane CENTRAL was performed. Studies were included if they reported survival outcomes for BRAF mutant versus WT patients as hazard ratios (HR) or in Kaplan-Meier (KM) curves. Random-effects meta-analysis models were used to pool HRs across the studies.Results: Data from 52 studies, representing 7519 patients, were pooled for analysis of OS. The presence of a BRAF mutation was statistically significantly associated with a reduced OS (HR [95% confidence interval (CI)]: 1.23 [1.09-1.38]), however, with substantial heterogeneity between the studies (I2: 58.0%). Meta-regression and sensitivity analyses showed that age, sex and BRAF mutation testing method did not have a significant effect on the OS HR. BRAF mutant melanoma showed comparable effect on DFS to non-BRAF mutant melanoma in stage I-III melanoma (combined HR: 1.16, 95% CI: 0.92-1.46), and on PFS in stage III-IV (HR: 0.98 (95% CI: 0.68-1.40)).Conclusion: Although there was substantial heterogeneity between the studies, the overall results demonstrated a poorer prognosis and OS in patients harbouring BRAF mutations. Future studies should take this into account when evaluating epidemiological data and treatment effects of new interventions in patients with malignant melanoma.

Downregulation of toll-like receptor 4 and IL-6 following irradiation of the rat urinary bladder.

The pathophysiology behind radiation cystitis is poorly understood. Here we investigated whether bladder irradiation affects the immune system of the rat urinary bladder. Female rats were sedated and exposed to one single radiation dose of 20 Gy or only sedated (controls) and killed 16 h to 14 days later. Rats were placed in a metabolic cage at 16 h, 3 days, 7 days and 14 days following bladder irradiation. The urinary bladders were harvested and analysed with qPCR, immunohistochemistry and/or Western blot for the expression of interferon (IFN)-γ, interleukin (IL)-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, nitric oxide synthases (eNOS, iNOS and nNOS), tumour necrosis factor (TNF)-α and toll-like receptor 4 (TLR4). Urine was collected and analysed for IL-6 and nitrite (reflecting nitric oxide activity) with ELISA and the Griess reaction, respectively. Irradiation increased bladder frequency and decreased voiding volumes 14 days following bladder irradiation. Bladder irradiation increased the expression of IL-10 and collagen in the bladder, while TLR4 and IL-6 expressions were decreased in the urothelium concomitantly with a decrease in mast cells in the submucosa and urine levels of IL-6 and nitrite. The present findings show that bladder irradiation leads to urodynamic changes in the bladder and may suppress important immunoregulatory pathways in the urinary bladder.

A phase Ib randomized multicenter trial of isolated hepatic perfusion in combination with ipilimumab and nivolumab for uveal melanoma metastases (SCANDIUM II trial).

BACKGROUND: Uveal melanoma (UM) is a rare malignancy where 50% of patients develop metastatic disease primarily affecting the liver. Approximately 40% of patients with metastatic UM respond to one-time isolated hepatic perfusion (IHP) with high-dose melphalan. This phase I trial investigates the safety and clinical efficacy of IHP combined with ipilimumab (IPI) and nivolumab (NIVO). PATIENTS AND METHODS: Immunotherapy-naïve patients were randomized in this phase I trial to receive either IHP followed by IPI 3 mg/kg and NIVO 1 mg/kg (IPI3/NIVO1) for four cycles (post-operative arm), or one cycle of preoperative IPI3/NIVO1, IHP and then three cycles of IPI3/NIVO1 (pre-post-operative arm), followed by maintenance therapy with NIVO 480 mg for 1 year. RESULTS: Eighteen patients were enrolled and randomized. Three patients did not undergo IHP as planned. In total, 11/18 patients (6 in the post-operative arm and 5 in the pre-post-operative arm) did not complete the planned four cycles of IPI3/NIVO1. Toxicity to IHP was similar in both groups, but the number of immune-related adverse events (AEs) was higher in the pre-post-operative arm. Among assessable patients, overall response rate was 57% in the post-operative arm (4/7) and 22% in the pre-post-operative arm (2/9). CONCLUSIONS: Combination therapy with IHP and IPI3/NIVO1 was associated with severe AEs. The efficacy of this combination is encouraging with high response rates. One cycle of preoperative IPI/NIVO before IHP did not show potential benefits in terms of safety or efficacy.

Understanding quality of life issues in patients with advanced melanoma: Phase 1 and 2 in the development of the EORTC advanced melanoma module.

AIMS: We aimed to develop a European Organization for Research and Treatment of Cancer (EORTC) Quality of Life (QoL) module tailored for patients with advanced (resectable or unresectable stage III/IV) melanoma receiving immune checkpoint inhibitors or targeted therapy. METHODS: Following the EORTC QoL Group module development guidelines, we conducted phases 1 and 2 of the development process. In phase 1, we generated a list of health-related (HR)QoL issues through a systematic literature review and semi-structured interviews with healthcare professionals (HCPs) and patients with advanced melanoma. In phase 2, these issues were converted into questionnaire items to create the preliminary module. RESULTS: Phase 1: we retrieved 8006 articles for the literature review, of which 35 were deemed relevant, resulting in 84 HRQoL issues being extracted to create the initial issue list. Semi-structured interviews with 18 HCPs and 28 patients with advanced melanoma resulted in 28 issues being added to the initial issue list. Following EORTC module development criteria, 26 issues were removed, and two issues were added after review by patient advocates. Phase 2: To ensure uniformity and avoid duplication, 16 issues were consolidated into eight items. Additionally, an independent expert contributed one new item, resulting in a preliminary module comprising 80 HRQoL items. CONCLUSION: We identified a range of HRQoL issues (dry skin, xerostomia, and arthralgia) relevant to patients with stage III/IV melanoma. Future module development phases will refine the questionnaire. Once completed, this module will enable standardized assessment of HRQoL in patients with (locally) advanced melanoma.

Bacterial flora of the human oral cavity, and the upper and lower esophagus.

This reference study aims to survey the bacterial flora of the healthy lower human esophagus and to compare it with that of the upper esophagus and oral mucosa. The use of biopsies, in addition to brush samples, allows inclusion of not only transient bacteria present on the surface but also bacteria residing in the epithelia, and the yield of the two methods can be compared. Forty patients scheduled for surgery for reasons with no known influence on esophageal flora and with no symptoms or endoscopic signs of esophageal disease were included. Samples were collected from the oral, upper esophageal, and lower esophageal mucosa using sealed brushes and biopsy forceps. Colonies cultivated on agar plates were classified and semiquantified. Twenty-three different bacterial species were identified, with similar strains present at the three sites. The most common group of bacteria was viridans streptococci, with an occurrence rate in brush samples and biopsies of 98% and 95%, respectively. The median number of species occurring in the oral cavity, upper esophagus, and lower esophagus was between 3 and 4 (range 0-7). The total number of species in the oral cavity was significantly higher when compared with either level in the esophagus, while the yields obtained by brush and biopsy sampling were highly correlated. Hence, the normal human esophagus is colonized with a resident bacterial flora of its own, which has similarities to that of the oral mucosa. There are diverse species that make up this flora, although in relatively low amounts. The most frequent inhabitants of the esophagus are streptococci, with an occurrence rate in brush samples and biopsies of 95-98%. Comparative studies of patients with eosinophilic esophagitis and gastroesophageal reflux disease are warranted.

Nitric oxide and endothelin-1 release after one-lung ventilation during thoracoabdominal esophagectomy.

One-lung ventilation (OLV) is applied during esophagectomy to improve exposure during the thoracic part of the operation. Collapse of lung tissue, shunting of pulmonary blood flow, and changes in alveolar oxygenation during and after OLV may possibly induce an ischemia-reperfusion response in the lung, which may affect the pulmonary endothelium. Such a reaction might thereby contribute to the frequently occurring respiratory complications among these patients. In this small trial, 30 patients were randomized to either OLV (n= 16) or two-lung ventilation (TLV, n= 14) during esophagectomy. Central venous and arterial plasma samples were taken before and after OLV/TLV for analysis of nitrite and a metabolite of nitric oxide (NO), and also during the 1st, 2nd, 3rd, and 10th postoperative day for analysis of endothelin, another endothelium-derived vasoactive mediator. Lung biopsies were taken before and after OLV or TLV, and analyzed regarding immunofluorescence for isoform of NO synthase, a protein upregulated during inflammatory response and also vascular congestion. No changes in lung isoform of NO synthase immunofluorescence or vascular congestion were registered after neither OLV nor TLV. Plasma nitrite and endothelin levels were similar in the two study groups. We conclude that OLV does not seem to have any influence on key regulators of pulmonary vascular tone and inflammation, i.e. NO and endothelin. From this perspective, OLV seems to be a safe method, which defends its clinical position to facilitate surgical exposure during thoracoabdominal esophagectomy.

Validation of a clinicopathological and gene expression profile model to identify patients with cutaneous melanoma where sentinel lymph node biopsy is unnecessary.

BACKGROUND: In patients with cutaneous melanoma, sentinel lymph node biopsy (SLNB) serves as an important technique to asses disease stage and to guide adjuvant systemic therapy. A model using clinicopathologic and gene expression variables (CP-GEP; Merlin Assay) has recently been introduced to identify patients that may safely forgo SLNB. Herein we present data from an independent validation cohort of the CP-GEP model in Swedish patients. METHODS: Archival histological material (primary melanoma tissue) from a prospectively collected cohort of 421 consecutive patients with pT1-T4 melanoma undergoing SLNB between 2006 and 2014 was analyzed using the CP-GEP model. CP-GEP combines Breslow thickness and patient age with the expression levels of eight genes from the primary melanoma. Stratification is based on their risk for nodal metastasis: CP-GEP Low Risk or CP-GEP High Risk. RESULTS: The SLNB positivity rate was 13%. Of 421 primary melanomas, the CP-GEP model identified 86 patients as having a low risk for nodal metastasis. In patients with pT1-2 melanomas, the SLNB reduction rate was 35.4% (95% CI: 29.4-41.8) with a negative predictive value (NPV) of 96.5% (95% CI: 90.0-99.3). Among patients with pT1-3 melanomas, CP-GEP suggested a SLNB reduction rate of 24.0% (95% CI: 19.7-28.8) and a NPV of 96.5% (95% CI: 90.1-99.3). Only one of 118 pT3 tumors was classified as CP-GEP Low Risk, and all pT4 tumors were classified as being high risk for nodal metastasis. CONCLUSION: This study demonstrates that CP-GEP can identify patients with a low risk for nodal metastasis. Patients with pT1-2 melanomas have the highest clinical benefit from using the test, where 35% of the patients could forgo a SLNB procedure.

Hyperbaric oxygen treatment reverses radiation induced pro-fibrotic and oxidative stress responses in a rat model.

PURPOSE: Radiotherapy is effective in the treatment of tumors in the pelvic area but is associated with side effects such as cystitis and proctitis. Hyperbaric Oxygen Therapy (HBOT) has emerged as a treatment modality for radiation-induced side effects. In a rat model for radiation cystitis, we studied the effects of HBOT on oxidative stress and pro-fibrotic factors. MATERIALS AND METHODS: Sedated Sprague-Dawley rats underwent bladder irradiation of 20Gy with and without 20 sessions of HBOT during a fortnight. Control animals were treated with and without HBOT. All four groups of animals were euthanized 28 days later. Histopathological examinations, immunohistochemistry and quantitative polymerase chain reaction (qPCR) were used to analyze changes in oxidative stress (8-OHdG), anti-oxidative responses (SOD-1, SOD2, HO-1 and NRFα) and a panel of Th1-type and Th2-type cytokines (IL-1ß, IL-4, IL-5, IL-6, IL-10, IL-13, TNF-α, TGF-ß, IFN-γ) in the urinary bladder. RESULTS: Bladder irradiation increased the expression of 8-OHdG, SOD2, HO-1, NRFα, IL-10, TNF-α and tended to increase TGF-ß. These changes were completely reversed by HBOT while HBOT in control animals had no effects on the studied markers for oxidative stress, anti-oxidative responses and Th1-type and Th2-type cytokines. CONCLUSIONS: Radiation induced a significant elevation of oxidative stress, antioxidants and pro-fibrotic factors in our animal model for radiation cystitis that were completely reversed and normalized by HBOT. Our findings indicate that HBOT may prevent radiation-induced changes by affecting oxidative stress and inflammatory cascades induced by radiation. SUMMARY: Radiotherapy may cause the development of chronic inflammation and fibrosis, significantly impairing organ function. We hypothesized that bladder irradiation induces an oxidative stress reaction, thereby triggering the redox system and thus initiating an inflammatory and pro-fibrotic response. We aimed to assess whether these changes would be reversed by hyperbaric oxygen using an animal model for radiation cystitis. Our study show that hyperbaric oxygen therapy may reverse oxidative stress and pro-inflammatory factors induced by radiation.

Pitfalls using metalloporphyrins in carbon monoxide research.

The proposal that endogenously produced carbon monoxide (CO) may act as a biological messenger has remained controversial. Carbon monoxide is generated by haem oxygenase isoenzymes in the degradation of haem-containing molecules. Certain metalloporphyrins, which are inhibitors of haem oxygenase, have been widely used as pharmacological tools in order to establish a messenger role for CO in the brain and periphery. However, increasing evidence shows that many metalloporphyrins are also associated with a large range of undesired effects, which make the interpretation of results using such compounds very uncertain. In this article, Lars Grundemar and Lars Ny evaluate the properties and describe the nonselective effect profile of such metalloporphyrins.

Nitric oxide production in the sphenoidal sinus by the inducible and constitutive isozymes of nitric oxide synthase.

OBJECTIVE: To study the production of nitric oxide (NO), and the presence of different isoforms of the NO-synthesising enzyme, NO-synthase (NOS), in the paranasal sinus. MATERIALS AND METHODS: Ten patients, undergoing surgery for pituitary adenoma, were examined for the presence of NO gas in the sphenoidal and maxillary sinus. The distribution of different NOS isozymes in mucosal biopsies from sphenoid and maxillary sinus and ethmoidal cells was studied. RESULTS: The mean concentration of NO was 2575 ppb in the sphenoidal sinus and 6792 ppb in the maxillary sinus. Morphological analyses revealed intense NADPH-diaphorase staining throughout the epithelium. Immunoreactivity against NOS2 (inducible NOS) was observed in the apical cell layer but not of the basal layer. NOS1 (neuronal NOS)-immunoreactivity was mainly seen in the subapical part of the epithelium and NOS3 (endothelial NOS)-immunoreactivity was observed only in the most apical part of the epithelium. CONCLUSION: NO concentration in the sphenoidal sinus is about the same as in the nasal cavity and approximately half of the concentration found in the maxillary sinus. All of the three main different isozymes of NOS can be demonstrated in the mucosa of the sphenoidal and maxillary sinus and ethmoidal cells, NOS2 being the most abundant isoform.

Localization and activity of nitric oxide synthases in the gastrointestinal tract of Trypanosoma cruzi-infected mice.

Chagas' disease, caused by the protozoan parasite, Trypanosoma cruzi, is associated with gastrointestinal abnormalities. Since nitric oxide (NO) has been shown to be a factor influencing intestinal function we evaluated the distributions and activities of the NO synthase (NOS) isoforms, in the gut of mice infected with T. cruzi. Ca2+-dependent (NOS1/NOS3) activity was decreased, whereas Ca2+-independent (NOS2) activity was increased in infected mice. NOS2-immunoreactivity was demonstrated in cells within the muscle layers and epithelium in infected mice and NOS1 immunoreactivity was seen in nerve structures. These data indicate that alterations in the NO-system may be important in the pathogenesis of the gastrointestinal manifestations in Chagas' disease.

Heme oxygenase-1, heme oxygenase-2 and biliverdin reductase in peripheral ganglia from rat, expression and plasticity.

The expression of inducible and constitutive heme oxygenase and biliverdin reductase was studied in normal and cultured peripheral ganglia from adult rats, using immunocytochemistry and in situ hybridization. Dramatic changes were induced by one to two days' culturing of dorsal root ganglia, nodose ganglia, otic ganglia, sphenopalatine ganglia and superior cervical ganglia. An up-regulation of inducible heme oxygenase was found in satellite cells of the cultured nodose ganglia, dorsal root ganglia, sphenopalatine ganglia and otic ganglia, whereas only a few satellite cells in the superior cervical ganglia responded with an increase in inducible heme oxygenase immunoreactivity. In the superior cervical ganglia inducible heme oxygenase also appeared in a subpopulation of macrophages. During culturing, expression of inducible heme oxygenase immunoreactivity also increased in axons and in nerve cell bodies. In situ hybridization corroborated the immunocytochemical findings, revealing a strong up-regulation of inducible heme oxygenase messenger RNA in satellite cells, and less pronounced up-regulation in nerve cell bodies. Constitutive heme oxygenase immunoreactivity was found in most neurons in all of the ganglia studied. No significant changes in constitutive heme oxygenase immunoreactivity could be observed in cultured ganglia. Biliverdin reductase immunoreactivity was barely detectable in any of the normal ganglia; however, after culturing it appeared in axons, single nerve cell bodies and nerve cell nuclei. The results show that inducible heme oxygenase is up-regulated in peripheral ganglia after axonal injury, and suggest a role for carbon monoxide in cellular signaling and a requirement for the antioxidant (bilirubin) during the regeneration process.

Inhibition by zinc protoporphyrin-IX of receptor-mediated relaxation of the rat aorta in a manner distinct from inhibition of haem oxygenase.

1. Carbon monoxide (CO), produced by haem oxygenase through degradation of haem, has been claimed to be a neuromessenger and a possible regulator of vascular tone. We examined whether the haem oxygenase inhibitor, zinc protoporphyrin-IX (ZnPP) and other porphyrins affect the relaxation evoked by various agents in the rat isolated aorta. 2. Pretreatment with ZnPP (0.1 mM) virtually abolished the relaxation evoked by vasoactive intestinal peptide (VIP) and atrial natriuretic peptide (ANP). ZnPP also evoked a rightward shift of the concentration-response curve for the relaxation induced by acetylcholine. 3. In contrast, ZnPP did not affect the relaxation evoked by forskolin and 3-morpholino-sydnonimine, agents which directly activate adenylate and guanylate cyclase, respectively. 4. Although, less effective than ZnPP, tin protoporphyrin-IX (SnPP; 0.1 mM) and protoporphyrin-IX (PP; 0.1 mM) also attenuated the VIP-evoked relaxation. 5. The elevation of cyclic AMP and cyclic GMP levels evoked by VIP and ANP, respectively, were abolished by pretreatment with ZnPP (0.1 mM). 6. ZnPP, SnPP and PP did not affect the contraction evoked by phenylephrine. 7. The results show that ZnPP inhibits relaxation induced by VIP, ANP and acetylcholine, probably by interfering with membrane receptor-coupled signal transduction pathways. This inhibition does not seem to be dependent upon inhibition of haem oxygenase. The lack of specificity of the haem oxygenase inhibiting metalloporphyrins makes them less suitable as pharmacological tools in the investigation of a messenger role for CO.

Mediators and mechanisms of relaxation in rabbit urethral smooth muscle.

1. Electrophysiological and mechanical experiments were performed to investigate whether the nitric oxide (NO)-mediated relaxation of rabbit urethral smooth muscle is associated with a hyperpolarization of the membrane potential. In addition, a possible role for vasoactive intestinal peptide (VIP) and carbon monoxide (CO) as relaxant agents in rabbit urethra was investigated. 2. Immunohistochemical experiments were performed to characterize the NO-synthase (NOS) and VIP innervation. Possible target cells for NO were studied by using antisera against cyclic GMP. The cyclic GMP-immunoreactivity was investigated on tissues pretreated with 1 mM IBMX, 0.1 mM zaprinast and 1 mM sodium nitroprusside. 3. Intracellular recordings of the membrane potential in the circular smooth muscle layer revealed two types of spontaneous depolarizations, slow waves with a duration of 3-4 s and an amplitude of 30-40 mV, and faster (0.5-1 s), more irregular depolarizations with an amplitude of 5-15 mV. The resting membrane potential was 39 +/- 1 mV (n = 12). Application of NO (30 microM), CO (30 microM) or VIP (1 microM) did not change the resting membrane potential. 4. Both NO (1-100 microM) and VIP (1 nM-1 microM) produced concentration-dependent relaxations amounting to 87 +/- 4% and 97 +/- 2% (n = 6), respectively. The relaxant effect of CO (1-30 microM) amounted to 27 +/- 4% (n = 5) at the highest concentration used. 5. Immunohistochemical experiments revealed a rich supply of NOS-immunoreactive nerve fibres in the smooth muscle layers. Numerous spinous cyclic GMP-immunoreactive cells were found interspersed between the smooth muscle bundles, mainly localized in the outer layer. These cells had long processes forming a network surrounding the smooth muscle bundles. VIP-immunoreactivity was sparse in comparison to NOS-immunoreactive nerves. 6. The rich supply of NOS-immunoreactive nerve fibres supports the view that NO is an important NANC-mediator in the rabbit urethra. In contrast to several other tissues, the relaxant effect of NO in the rabbit urethra does not seem to be mediated by hyperpolarization. The network of cyclic GMP-immunoreactive cells may constitute target cells for NO, but their function remains to be established.

Alpha-latrotoxin-induced transmitter release in feline oesophageal smooth muscle: focus on nitric oxide and vasoactive intestinal peptide.

1. The effects of alpha-latrotoxin (alpha LTX) on muscle tone, resting membrane potential, cyclic nucleotide content, and ultrastructure were examined in feline oesophageal smooth muscle, including the lower oesophageal sphincter (LOS). 2. In circular smooth muscle strips from LOS developing active tone alpha LTX (1 nM) induced a 94 +/- 3% (n = 16) relaxation. Intermittent treatment with alpha LTX for 4 h abolished the response. Pretreatment with NG-nitro-L-arginine (L-NOARG; 0.1 mM) attenuated the relaxation. 3. In carbachol-contracted circular smooth muscle strips from the LOS and oesophageal body (OB), alpha LTX induced a 95 +/- 5% (n = 6) and 73 +/- 9% (n = 8) relaxation, respectively. The relaxations were attenuated by L-NOARG, and in LOS strips, the relaxation was abolished by the combination of L-NOARG and vasoactive intestinal peptide (VIP)-antiserum (1:25). At resting tension in circular smooth muscle strips from the OB, alpha LTX induced a scopolamine sensitive contraction in the presence of L-NOARG. 4. In circular LOS and OB preparations, alpha LTX changed the resting membrane potential from -49 +/- 2mV to -59 +/- 3 mV (n = 4), and -62 +/- 2 mV to -71 +/- 3 mV (n = 4), respectively. 5. The alpha LTX-induced relaxation of LOS and OB muscle was associated with a 138% and 72% increase in cyclic GMP levels, respectively. No changes in cyclic AMP levels were observed. 6. Ultrastructural analysis of LOS and OB revealed a rich supply of nerve profiles containing small synaptic and large dense core vesicles. alpha LTX treatment resulted in a loss of both types of vesicle. 7. These results suggest that alpha LTX induces relaxation of oesophageal circular smooth muscle associated with NO-generation and transmitter release from synaptic vesicles. Beside NO, VIP seems to be involved in the relaxant effects of alpha LTX on the LOS. In addition, alpha LTX may have contractile effects by release of acetylcholine.

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.