Duodenal-jejunal bypass improves hypothalamic oxidative stress and inflammation in diabetic rats via glucagon-like peptide 1-mediated Nrf2/HO-1 signaling.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is often accompanied by impaired glucose utilization in the brain, leading to oxidative stress, neuronal cell injury and infla-mmation. Previous studies have shown that duodenal jejunal bypass (DJB) surgery significantly improves brain glucose metabolism in T2DM rats, the role and the metabolism of DJB in improving brain oxidative stress and inflammation condition in T2DM rats remain unclear. AIM: To investigate the role and metabolism of DJB in improving hypothalamic oxidative stress and inflammation condition in T2DM rats. METHODS: A T2DM rat model was induced via a high-glucose and high-fat diet, combined with a low-dose streptozotocin injection. T2DM rats were divided into DJB operation and Sham operation groups. DJB surgical intervention was carried out on T2DM rats. The differential expression of hypothalamic proteins was analyzed using quantitative proteomics analysis. Proteins related to oxidative stress, inflammation, and neuronal injury in the hypothalamus of T2DM rats were analyzed by flow cytometry, quantitative real-time PCR, Western blotting, and immunofluorescence. RESULTS: Quantitative proteomics analysis showed significant differences in proteins related to oxidative stress, inflammation, and neuronal injury in the hypothalamus of rats with T2DM-DJB after DJB surgery, compared to the T2DM-Sham groups of rats. Oxidative stress-related proteins (glucagon-like peptide 1 receptor, Nrf2, and HO-1) were significantly increased (P < 0.05) in the hypothalamus of rats with T2DM after DJB surgery. DJB surgery significantly reduced (P < 0.05) hypothalamic inflammation in T2DM rats by inhibiting the activation of NF-κB and decreasing the expression of interleukin (IL)-1ß and IL-6. DJB surgery significantly reduced (P < 0.05) the expression of factors related to neuronal injury (glial fibrillary acidic protein and Caspase-3) in the hypothalamus of T2DM rats and upregulated (P < 0.05) the expression of neuroprotective factors (C-fos, Ki67, Bcl-2, and BDNF), thereby reducing hypothalamic injury in T2DM rats. CONCLUSION: DJB surgery improve oxidative stress and inflammation in the hypothalamus of T2DM rats and reduce neuronal cell injury by activating the glucagon-like peptide 1 receptor-mediated Nrf2/HO-1 signaling pathway.

Epidemiological characteristics of respiratory syncytial virus infection in pediatric patients before, during the COVID-19 pandemic and after easing of COVID-19 restrictive measures in China.

We aimed to assess the epidemiological characteristics of respiratory syncytial virus (RSV) infection in Chinese children at different phases of the coronavirus disease 2019 (COVID-19) pandemic, that is, before, during the pandemic and after easing of restrictive measures. We included 123 623 patients aged 0-18 years with respiratory infection symptoms who were suspected with RSV infection from January 1, 2019 to June 30, 2023 in Hangzhou Children's Hospital. Clinical information and RSV test result were extracted from the laboratory information system. We calculated the positive rate of RSV detection by age groups, gender, seasons, types of patients and phases of COVID-19 pandemic. Nonlinear associations between age and risk of RSV infection in three phases of pandemic were assessed by restricted cubic spline regression models. Among 123 623 patients, 3875 (3.13%) were tested as positive. The highest positive rate was observed in children aged 0-28 days (i.e., 12.28%). RSV infection was most prevalent in winter (6.04%), and followed by autumn (2.52%). Although there is no statistical significance regarding the positive rate at three phases of the pandemic, we observed that the rate was lowest during the pandemic and increased after easing the measures in certain age groups (p < 0.05), which was consisted with results from the nonlinear regression analyses. In addition, regression analyses suggested that the age range of children susceptible to RSV got wider, that is, 0-3.5 years, after easing all restrictive measures compared with that before (i.e., 0-3 years) and during the pandemic (i.e., 0-1 year). Based on our findings, we called for attention from health professionals and caregivers on the new epidemiological characteristics of RSV infection in the post-pandemic era after easing the restrictive measures.

Generating Kidney Organoids in Suspension from Induced Pluripotent Stem Cells.

Kidney organoids can be generated from induced pluripotent stem cells (iPSCs) through various approaches. These organoids hold great promise for disease modeling, drug screening, and potential therapeutic applications. This article presents a step-by-step procedure to create kidney organoids from iPSCs, starting from the posterior primitive streak (PS) to the intermediate mesoderm (IM). The approach relies on the APEL 2 medium, which is a defined, animal component-free medium. It is supplemented with a high concentration of WNT agonist (CHIR99021) for a duration of 4 days, followed by fibroblast growth factor 9 (FGF9)/heparin and a low concentration of CHIR99021 for an additional 3 days. During this process, emphasis is given to selecting the optimal cell density and CHIR99021 concentration at the start of iPSCs, as these factors are critical for successful kidney organoid generation. An important aspect of this protocol is the suspension culture in a low adherent plate, allowing the IM to gradually develop into nephron structures, encompassing glomerular, proximal tubular, and distal tubular structures, all presented in a visually comprehensible format. Overall, this detailed protocol offers an efficient and specific technique to produce kidney organoids from diverse iPSCs, ensuring successful and consistent results.

Characteristics of intestinal-related lymphoid hyperplasia in children and its correlation with intussusception of children.

BACKGROUND: Primary intussusception in children is a common acute abdominal disease. The cause of this disease is still not fully understood. Many articles have reported that children with intussusception are often accompanied by hyperplasia of mesenteric lymph nodes and submucosal lymphoid tissue of the terminal ileum. Therefore, hyperplasia of intestinal-associated lymphoid tissue (mesenteric lymph nodes and submucosal lymphoid tissue of the intestinal tract) may be one of the main causes of intussusception. However, the characteristics and differences of intestinal-associated lymphoid tissues in healthy children and children with intussusception at different ages have not been reported. In addition, the relationship between mesenteric lymph nodes and intestinal submucosal lymphatic tissue also needs to be further understood. METHODS: 73 patients with intussusception during the recovery phase who were treated in our hospital from October 2019 to October 2021 were collected as the observation group, while 101 children with healthy physical examination or diseases unrelated to intestinal lymphoid hyperplasia were collected as the control group. They were divided into different age groups of 1-6 months, 7-12 months, 13-18 months, 19-24 months, 25-36 months, 3-4 years, 5-6 years, and 7-8 years old. Ultrasonography was used to explore and scan mesenteric lymph nodes in fixed areas of the right lower abdomen and around the umbilicus. The size (cm3) and number (n) of detectable lymph nodes in each region were recorded and calculated, and the total mesenteric lymph node volume (cm3) of the right lower abdomen (RLTMLNV) and periumbilical region (PTMLNV) was calculated, respectively. The total mesenteric lymph node volume of each region in different ages of the two groups was analyzed. RESULTS: (1) There were significant differences between the control group and the observation group in the right lower abdominal total mesenteric lymph nodes volume (RLTMLNV) and the periumbilical total mesenteric lymph nodes volume (PTMLNV) (P = 0.001). The mesenteric lymph nodes in the observation group showed severe hyperplasia. (2) Children with intussusceptions are usually accompanied by severe mesenteric lymphoid hyperplasia. The mean volume value of RLTMLNV was greater than that of PTMLNV. Especially within 2 years of age, the mean value of RLTMLNV was significantly higher than that of PTMLNV with statistical significance (P < 0.05). (3) In normal children (control group), lymph nodes in the right lower abdomen and periumbilical area showed low hyperplasia, and there was a significant difference between age groups of < 2 years old and 2-8 years old (p = 0.001). In the children with intussusception (observation group), the hyperplasia of mesenteric lymph nodes in the right lower abdomen and around the umbilicus was severe. There was no significant difference in the proliferation of mesenteric lymphoid tissue among different age groups in the right lower abdomen (P = 0.834). There was also no significant difference in hyperplasia of periumbilical mesenteric lymphoid tissue among different age groups (P = 0.097). CONCLUSIONS: Our research shows: (1) The occurrence of primary intussusception in children is related to the hyperplasia of intestinal-associated lymphoid tissue. (2) Children with intussusceptions were usually accompanied by severe mesenteric lymphoid hyperplasia. The mesenteric lymphoid hyperplasia was more evident in the right lower abdominal ileocecal area than in the periumbilical area before 2 years of age. RLTMLNV has better predictability of intussusception than PTMLNV. The occurrence of intussusceptions was more closely related to the hyperplasia of intestinal-associated lymphoid tissue in the right lower abdomen. (3) Normal children showed a low degree of mesenteric lymphoid hyperplasia before 2 years old, moderate hyperplasia after 2 years old, and mesenteric lymphoid hyperplasia in the right lower abdominal ileocecal area was basically the same as the periumbilical area. The lymphatic tissue of the right lower abdomen and periumbilical mesentery in children with intussusceptions showed severe hyperplasia, and there were no significant differences among different age groups.

Surgical treatment of recurrent intussusception induced by intestinal lymphoid hyperplasia in a child: is bowel resection always necessary? A case report.

BACKGROUND: Intussusception recurrence (IR) induced by intestinal lymphoid hyperplasia (ILH) in children is rare, and surgical treatment is the final resort if IR is refractory to medications and non-surgical interventions. To date, only a few case reports have described surgical management of ILH-induced IR in children, all involving bowel resection regardless of whether there are bowel necrosis and perforation. CASE PRESENTATION: A 2-year-old boy was transferred to our department due to IR. His main complaint was abdominal pain. Color Doppler ultrasound confirmed ileocecal intussusception while no other abnormalities were found. A final diagnosis of IR with unknown causes was made. Repeated saline enema reductions and dexamethasone failed to cure the IR. Laparotomy was eventually performed after almost 10 episodes of IR. Intraoperatively, distal ileum thickening with palpable masses without bowel necrosis and perforation was noted. ILH was suspected and a biopsy of the affected intestine was performed. Histopathological analysis confirmed ILH. The intussusception was manually reduced, the terminal ileum and the ileocecal junction were fixed to the paralleled ascending colon and the posterior peritoneum respectively, and no bowel resection was performed. The postoperative recovery was uneventful and no IR was observed during over 5 years of follow-up. CONCLUSIONS: As far as we are aware, this is the first report of successful surgical treatment of ILH-induced pediatric IR without bowel resection in a child. Our experience suggests bowel resection may be unnecessary if bowel necrosis and perforation are absent.

Role of Proximal Intestinal Glucose Sensing and Metabolism in the Blood Glucose Control in Type 2 Diabetic Rats After Duodenal Jejunal Bypass Surgery.

BACKGROUND: Although gastric surgery can significantly improve blood glucose homeostasis in type 2 diabetes mellitus (T2DM), its mechanism remains unclear. This study evaluated the role of intestinal glucose sensing, glucose transport, and metabolism in the alimentary limb (A limb) of T2DM rats after duodenal jejunal bypass (DJB) surgery. METHODS: A T2DM rat model was induced via a high-glucose high-fat diet and low-dose streptozotocin injection. The diabetic rats were divided into two groups: the DJB surgery (T2DM-DJB) group and the sham surgery (T2DM-Sham) group. Wistar rats were used as wild-type control (Control). Small animal PET was used to assess the change in glucose metabolic status in the intestine. The intestinal villi height and the number of EECs after DJB were evaluated. The expressions of sweet taste receptors (T1R2/T1R3), glucose transporters (SGLT1/GLUT2), and key enzymes involved in glucose metabolism (HK2, PFK2, PKM2, G6Pase, and PCK1) in the A limb after DJB was detected by Western blot and qRT-PCR. RESULTS: Small animal PET analysis showed the intestinal glucose metabolism increased significantly 6 weeks after DJB surgery. The intestinal villi height and the number of EECs in the A limb 6 weeks after surgery increased significantly in T2DM-DJB rats comparing to T2DM-Sham rats. The mRNA and protein expression of T1R1/T1R3 and SGLT1/GLUT2 were downregulated in DJB-T2DM rats, while enzymes involved in glucose metabolism was upregulated in the A limb in T2DM-DJB rats. CONCLUSION: Proximal intestinal glucose sensing and metabolism play an important role in blood glucose homeostasis by DJB.

Serum matrix metalloproteinase-13 as a diagnostic biomarker for cutaneous squamous cell carcinoma.

BACKGROUND: A significant proportion of newly diagnosed patients with cutaneous squamous cell carcinoma (cSCC) have metastasis and eventually die of the disease, necessitating the exploration of novel biomarkers for early detection of cSCC aggressiveness, risk assessment and monitoring. Matrix metalloproteinase-13 (MMP-13) has been implicated in cSCC pathogenesis. Serum MMP-13 levels have been shown to predict survival in patients with esophageal SCC, but their diagnostic value for cSCC has not been explored. METHODS: We conducted a case-control study to examine serum MMP-13 as a biomarker for cSCC. Patients with cSCC undergoing surgical resection and health controls undergoing plastic surgery were recruited. ELISA for measurement of serum MMP-13 and immunohistochemistry for detection of tissue MMP-13 were performed, and the results were compared between the case and the control group, and among different patient groups. ROC curve analysis was performed to determine the diagnostic value of serum MMP-13 levels. RESULTS: The ratio of male to female, and the age between the case (n = 77) and the control group (n = 50) were not significantly different. Patients had significantly higher serum MMP-13 levels than healthy controls. Subjects with stage 3 cSCC had markedly higher serum MMP-13 levels than those with stage 1 and stage 2 cSCC. Patients with invasive cSCC had remarkably higher serum MMP-13 than those with cSCC in situ. Post-surgery serum MMP-13 measurement was done in 12 patients, and a significant MMP-13 decrease was observed after removal of cSCC. Tumor tissues had a remarkably higher level of MMP-13 than control tissues. Serum MMP-13 predicted the presence of invasive cSCC with an AUC of 0.87 (95% CI [0.78 to 0.95]) for sensitivity and specificity of 81.7 and 82.4%, respectively for a cut-off value of 290 pg/mL. Serum MMP-13 predicted lymph node involvement with an AUC of 0.94 (95% CI [0.88 to 0.99]) for sensitivity and specificity of 93.8 and 88.5%, respectively for a cut-off value of 430 pg/mL. CONCLUSION: Serum MMP-13 might serve as a valuable biomarker for early detection of cSCC invasiveness and monitoring of cSCC progression.

DNA methylation in Hepatoblastoma-a literature review.

Hepatoblastoma (HB) is the most common malignant liver tumor in children. Abnormal activation of the Wnt/ß-catenin signaling pathway plays an important role in the formation and development of HB. Genes in HB show a global hypomethylation change, accompanied by hypermethylation of specific tumor suppressor genes (TSGs). This article reviews the hypermethylation changes in several TSGs, such as RASSF1A, SOCS1, APC, HHIP, and P16, and analyzes the pathways and mechanisms of TSGs regulating gene expression. The role of the methylation-regulating enzymes DNA methyltransferases (DNMTs) and ten-eleven translocation (TET) family members enzymes in the methylation changes of HB was analyzed, and it was speculated that the occurrence of HB is partly due to the obstruction of liver differentiation in the early stage of differentiation. The origin cells may be incompletely differentiated hepatocytes remaining in the liver of children after birth. Therefore, further studying the role of methylation regulating enzymes in methylation changes in HB is a promising future research direction.

Duodenal-Jejunal Bypass Ameliorates Type 2 Diabetes Mellitus by Activating Insulin Signaling and Improving Glucose Utilization in the Brain.

BACKGROUND: Duodenal-jejunal bypass (DJB) can dramatically improve type 2 diabetes independent of weight loss and food restriction. Increasing evidence has demonstrated that brain insulin signaling plays an important role in the pathophysiology of type 2 diabetes. This study explores whether the antidiabetic effect of DJB is involved in brain insulin signaling activation and brain glucose utilization. METHODS: A diabetic rat model was established by high-fat and high-glucose diet. DJB or sham surgery was performed in diabetic rats. 18F-FDG PET scanning was used to detect glucose uptake in different organs, particularly in the brain. The levels of glucose transporters, glucose utilization-related proteins (HK1 and PFK2), insulin, and insulin signaling pathway-related proteins (InsR, IRS1/2, PI3K, and p-Akt) in the brain tissues were evaluated and analyzed. RESULTS: The results showed that DJB significantly improved basal glycemic parameters and reversed the decreasing glucose uptake in the brains of type 2 diabetic rats. DJB significantly increased not only the expression levels of brain insulin, IRS1/2, PI3K, and p-Akt but also the levels of the glucose utilization enzymes HK1 and PFK2 in the brain. CONCLUSION: These results indicate that enhanced brain insulin signaling transduction and brain glucose utilization play important roles in the antidiabetic effect of DJB.

Germline stem cell gene PIWIL2 mediates DNA repair through relaxation of chromatin.

DNA damage response (DDR) is an intrinsic barrier of cell to tumorigenesis initiated by genotoxic agents. However, the mechanisms underlying the DDR are not completely understood despite of extensive investigation. Recently, we have reported that ectopic expression of germline stem cell gene PIWIL2 is associated with tumor stem cell development, although the underlying mechanisms are largely unknown. Here we show that PIWIL2 is required for the repair of DNA-damage induced by various types of genotoxic agents. Upon ultraviolet (UV) irradiation, silenced PIWIL2 gene in normal human fibroblasts was transiently activated after treatment with UV light. This activation was associated with DNA repair, because Piwil2-deficienct mouse embryonic fibroblasts (mili(-/-) MEFs) were defective in cyclobutane pyrimidine dimers (CPD) repair after UV treatment. As a result, the UV-treated mili(-/-) MEFs were more susceptible to apoptosis, as characterized by increased levels of DNA damage-associated apoptotic proteins, such as active caspase-3, cleaved Poly (ADP-ribose) polymerase (PARP) and Bik. The impaired DNA repair in the mili(-/-) MEFs was associated with the reductions of histone H3 acetylation and chromatin relaxation, although the DDR pathway downstream chromatin relaxation appeared not to be directly affected by Piwil2. Moreover, guanine-guanine (Pt-[GG]) and double strand break (DSB) repair were also defective in the mili(-/-) MEFs treated by genotoxic chemicals Cisplatin and ionizing radiation (IR), respectively. The results indicate that Piwil2 can mediate DNA repair through an axis of Piwil2 → histone acetylation → chromatin relaxation upstream DDR pathways. The findings reveal a new role for Piwil2 in DNA repair and suggest that Piwil2 may act as a gatekeeper against DNA damage-mediated tumorigenesis.

Identification of Piwil2-like (PL2L) proteins that promote tumorigenesis.

PIWIL2, a member of PIWI/AGO gene family, is expressed in the germline stem cells (GSCs) of testis for gametogenesis but not in adult somatic and stem cells. It has been implicated to play an important role in tumor development. We have previously reported that precancerous stem cells (pCSCs) constitutively express Piwil2 transcripts to promote their proliferation. Here we show that these transcripts de facto represent Piwil2-like (PL2L) proteins. We have identified several PL2L proteins including PL2L80, PL2L60, PL2L50 and PL2L40, using combined methods of Gene-Exon-Mapping Reverse Transcription Polymerase Chain Reaction (GEM RT-PCR), bioinformatics and a group of novel monoclonal antibodies. Among them, PL2L60 rather than Piwil2 and other PL2L proteins is predominantly expressed in various types of human and mouse tumor cells. It promotes tumor cell survival and proliferation in vitro through up-regulation of Stat3 and Bcl2 gene expressions, the cell cycle entry from G(0/1) into S-phase, and the nuclear expression of NF-κB, which contribute to the tumorigenicity of tumor cells in vivo. Consistently, PL2L proteins rather than Piwil2 are predominantly expressed in the cytoplasm or cytoplasm and nucleus of euchromatin-enriched tumor cells in human primary and metastatic cancers, such as breast and cervical cancers. Moreover, nuclear PL2L proteins are always co-expressed with nuclear NF-κB. These results reveal that PL2L60 can coordinate with NF-κB to promote tumorigenesis and might mediate a common pathway for tumor development without tissue restriction. The identification of PL2L proteins provides a novel insight into the mechanisms of cancer development as well as a novel bridge linking cancer diagnostics and anticancer drug development.

Homozygous deletion but not mutation of exons 5 and 8 of the fragile histidine triad (FHIT) gene is associated with features of differentiated thyroid carcinoma.

The fragile histidine triad (FHIT) gene encompasses the most common human fragile site, FRA3B at 3p14.2, a region that is involved in homozygous deletions in a variety of human tumors. FHIT is considered to be a tumor suppressor gene that is frequently inactivated in various types of cancer. To study the role of the FHIT gene in thyroid tumorigenesis, we looked for homozygous deletions or mutations of exons 5 and 8 of the FHIT gene in 65 cases of differentiated thyroid carcinoma (DTC) and their matched non-cancerous epithelium (NCE), using exon-specific PCR amplification and PCR single strand conformation polymorphism (PCR-SSCP) techniques. In DTC, the incidence of homozygous deletion of exon 5 was 30.8% (20/65), and it was associated with tumor metastasis to lymph nodes (p <0.05). The incidence of homozygous deletion of exon 8 was 29.2% (19/65), and it was associated with the tumor pathological grade, TNM stage, and lymph node metastasis (p <0.05). There was strong correlation between homozygous deletions of exon 5 and exon 8 (p <0.01). No point mutations were observed in either exon 5 or exon 8. These findings suggest that: (a) exons 5 and 8 of FHIT are key target regions of deletion, (b) homozygous deletions of exon 5 and exon 8 may be good biomarkers for the biological behavior of DTC, and (c) point mutation of these exons may not be involved in the inactivation of the FHIT gene in DTC.

Association of the promoter methylation and protein expression of Fragile Histidine Triad (FHIT) gene with the progression of differentiated thyroid carcinoma.

The role of aberrant methylation of fragile histidine triad (FHIT) promoters in the differentiated thyroid carcinoma (DTC) is not yet clear. Therefore, we investigated the association of the status of FHIT promoter methylation and FHIT protein expression with the clinicopathological progression of DTC, using PCR-based methylation assay and immunohistochemical technique. While no FHIT gene promoter methylation was observed in the matched non-cancerous epithelium (NCE) specimens, 24.6% of DTC samples demonstrated methylation in the FHIT promoter region. The protein expression of FHIT in NCE and DTC was 100.0% and 41.5% (P<0.01), respectively. There was a negative correlation between promoter methylation and protein expression of FHIT gene (P<0.05). Additionally, the methylation status appeared to be significantly associated with the pathological grade, tumor TNM stage, and lymph node metastasis (P<0.05), and FHIT proteins were weakly expressed in only about 20% of DTC with grade II pathological changes, TNM stage III/IV, or lymph node metastasis. Finally, the gender and tumor classification but not age marginally affected the promoter methylation and protein expression of FHIT. Our results suggest that methylation of the promoter region may play a key role in inactivation of FHIT - possibly leading to subsequent carcinogenesis and progression of DTC.

Precancerous stem cells can serve as tumor vasculogenic progenitors.

Tumor neo-vascularization is critical for tumor growth, invasion and metastasis, which has been considered to be mediated by a mechanism of angiogenesis. However, histopathological studies have suggested that tumor cells might be the progenitor for tumor vasculature. Recently, we have reported that the precancerous stem cells (pCSCs) representing the early stage of developing cancer stem cells (CSCs), have the potential for both benign and malignant differentiation. Therefore, we investigated whether pCSCs serve as progenitors for tumor vasculogenesis. Herein, we report that in the pCSC-derived tumors, most blood vessels were derived from pCSCs. Some pCSCs constitutively expressed vasculogenic receptor VEGFR-2, which can be up-regulated by hypoxia and angiogenesis-promoting cytokines, such as GM-CSF, Flt3 ligand, and IL-13. The pCSCs are much more potent in tumor vasculogenesis than the differentiated tumor monocytic cells (TMCs) from the same tumor, which had comparable or even higher capacity to produce some vascular growth factors, suggesting that the potent tumor vasculogenesis of pCSCs is associated with their intrinsic stem-like property. Consistently tumor vasculogenesis was also observed in human cancers such as cervical cancer and breast cancer and xenograft lymphoma. Our studies indicate that pCSCs can serve as tumor vasculogenic stem/progenitor cells (TVPCs), and may explain why anti-angiogenic cancer therapy trials are facing challenge.

Potency of authentic nitric oxide in inducing aortic relaxation.

The reported EC(50) of vasorelaxation for authentic nitric oxide (NO) is from the nM to muM range. The cause of this large difference is undetermined. In this study, NO electrodes were used to monitor the actual NO concentration in the organ chamber during the recording of the relaxation of rat aortic rings. It was demonstrated that both the O(2) concentration in the solution and the rate of stirring the solution markedly affected the actual NO concentration, while the vasorelaxation response to NO was also changed with the composition of the buffer solution. It was observed that the apparent EC(50) of aortic relaxation for authentic NO is 340+/-40nM and 81+/-4nM in the conventional organ chamber containing Krebs-Ringer buffer bubbled with a high content (95% O(2)+5% CO(2)) and a low content (20% O(2)+5% CO(2)+75% N(2)) of O(2) gas mixture, respectively. The apparent EC(50) was further reduced to 45+/-2nM after the Krebs-Ringer buffer, a bicarbonate buffer, was replaced by a phosphate buffer solution bubbled with a low content of O(2) gas mixture (20% O(2)+80% N(2)) or with air. By using an organ chamber, which makes low concentrations of NO more stable in the solution, it was determined that the apparent EC(50) was 9.7+/-0.4nM and the threshold of NO concentration in dilating the aortas was approximately 0.3nM. This modified organ chamber will be useful for quantitatively measuring EC(50) of vasorelaxation for authentic NO under physiological and pathological conditions.

Estimation of nitric oxide concentration in blood for different rates of generation. Evidence that intravascular nitric oxide levels are too low to exert physiological effects.

Endothelium-derived nitric oxide (NO) is a potent vasodilator in the cardiovascular system. Several lines of experimental evidence suggest that NO or NO equivalents may also be generated in the blood. However, blood contains a large amount of hemoglobin (Hb) in red blood cells (RBCs). The RBC-encapsulated Hb can react very quickly with NO, which is only limited by the rate of NO diffusion into the RBCs. It is unclear what the possible NO concentration levels in blood are and how the NO diffusion coefficient (D) and the permeability (Pm) of RBC membrane to NO affect the level of NO concentration. In this study, a steady-state concentration experimental method combined with a spherical diffusion model are presented for determining D and Pm and examining the effect of NO generation rate (V0) and hematocrit (Hct) on NO concentration. It was determined that Pm is 4.5 +/- 1.5 cm/s and D is 3410 +/- 50 microm2/s at 37 degrees C. Simulations based on experimental parameters show that, when the rate of NO formation is as high as 100 nm/s, the maximal NO concentration in blood is below 0.012 nM at Pm = 4.5 cm/s and Hct = 45%. Thus, it is unlikely that NO is directly exported or generated from the RBC as an intravascular signaling molecule, because its concentration would be too low to exert a physiological role. Furthermore, our results suggest that, if RBCs export NO bioactivity, this would be through NO-derived species that can release or form NO rather than NO itself.