Incidence and risk factors for hospital-acquired cholecystitis.

BACKGROUND: Acute cholecystitis can occur both inside and outside hospital settings. However, little is known about the clinical characteristics of hospital-acquired cholecystitis (HAC). AIM: To investigate the clinical characteristics of HAC in a tertiary academic hospital. METHODS: This retrospective cohort study included hospitalized patients who were found to have gallstones without cholecystitis or cholangitis on admission between January 2018 and December 2021. Multi-variate logistic regression analysis was used to make comparisons between patients with and without HAC. FINDINGS: In total, 890 patients met the inclusion criteria and were evaluated in this study. Forty-one patients (4.6%) developed HAC during the study period. Multi-variate logistic regression analysis showed that a history of cholecystitis or cholangitis, fasting for ≥1 day, and gallstones in the gallbladder neck were independently associated with increased risk of HAC. HAC occurred most frequently after several weeks of admission, and only four patients (9.8%) had bacteraemia. CONCLUSIONS: HAC was relatively common among hospitalized patients. Physicians should be aware of the possibility of HAC in symptomatic hospitalized patients with certain risk factors.

The impact of heptavalent pneumococcal conjugate vaccine on the incidence of childhood community-acquired pneumonia and bacteriologically confirmed pneumococcal pneumonia in Japan.

Heptavalent pneumococcal conjugate vaccine (PCV7) was introduced to Japan in 2010. We investigated the impact of PCV7 on childhood community-acquired pneumonia (CAP) and pneumococcal pneumonia (PP). Children aged <5 years living in Chiba city, Japan, who were admitted to hospitals were enrolled to estimate the incidence of CAP based on the mid-year population. PP was determined by the presence of Streptococcus pneumoniae in cultured blood and/or sputum samples of CAP patients. The incidence of CAP and S. pneumoniae isolated from PP patients was compared before (April 2008-March 2009) and after (April 2012-March 2013) the introduction of PCV7 immunization. The annual incidence of CAP was reduced [incidence rate ratio 0·81, 95% confidence interval (CI) 0·73-0·90]. When comparing post-vaccine with pre-vaccine periods, the odds ratio for PP incidence was 0·60 (95% CI 0·39-0·93, P = 0·024). PCV7-covered serotypes markedly decreased (66·6% in pre-vaccine vs. 15·6% in post-vaccine, P < 0·01), and serotypes 6C, 15A, 15C and 19A increased. Multidrug-resistant international clones in the pre-vaccine period (Spain6B-2/ST90, Taiwan19F-14/ST236) decreased, while Sweden15A-25/ST63 was the dominant clone in the post-vaccine period. A significant reduction in the incidence of both CAP hospitalizations and culture-confirmed PP of vaccine serotypes was observed at 2 years after PCV7 vaccination.

Effect of aerobic exercise training followed by a low-calorie diet on metabolic syndrome risk factors in men.

BACKGROUND AND AIMS: Whether low-volume, high-intensity, interval training (HIIT) is an adequate exercise method for improving metabolic risk factors is controversial. Moreover, it is not known if performing a short-term, low-calorie diet intervention (LCDi) after a HIIT program affects risk factors. This study investigated how an 8-week, 3 times/week exercise intervention (EXi) incorporating either HIIT or moderate-intensity continuous training (MICT) followed by a 4-week LCDi affects risk factors. METHODS AND RESULTS: Twenty-six male workers with metabolic risk factors (47.4 ± 7.1 years; cardiorespiratory capacity (VO2peak) of 28.5 ± 3.9 ml/kg/min) were randomly assigned to either the HIIT (3 sets of 3-min cycling with a 2-min active rest between sets, 180 kcal) or MICT (45 min, 360 kcal) group. After the EXi, all subjects participated in a 4-week LCDi (4 counseling sessions). During the EXi, VO2peak improved more (P < 0.05) through HIIT (25.4 ± 14.6%) than through MICT (14.9 ± 12.8%), whereas improvements in body fat and HDL cholesterol were similar. During the LCDi, some risk factors improved further (P < 0.05) without any group differences, while VO2peak in the HIIT group decreased (P < 0.05) to the same level as in the MICT group. CONCLUSION: VO2peak increased more with HIIT than with MICT during the EXi despite HIIT having a lower exercise volume than MICT, but this advantage of HIIT promptly disappeared through detraining. An intervention strategy consisting of 8 weeks of either HIIT or MICT followed by a 4-week LCDi has a positive effect on metabolic risk factors. CLINICAL TRIAL REGISTRATION: UMIN11352.

Binding of αvß1 and αvß6 integrins to tenascin-C induces epithelial-mesenchymal transition-like change of breast cancer cells.

Tenascin-C (TNC), a large hexameric extracellular glycoprotein, is a pleiotropic molecule with multiple domains binding to a variety of receptors mediating a wide range of cellular functions. We earlier reported that TNC induces epithelial-mesenchymal transition (EMT)-like change in breast cancer cells. In the present study, we clarified TNC receptor involvement in this process. Among integrins previously reported as TNC receptors, substantial expression of αv, α2, ß1 and ß6 subunits was detected by quantitative PCR and immunoblotting in MCF-7 cells. Integrin ß6 mRNA was remarkably upregulated by transforming growth factor (TGF)-ß1 treatment, and protein expression was prominently increased by additional exposure to TNC. Immunofluorescent labeling demonstrated integrin αvß6 accumulation in focal adhesions after TNC treatment, especially in combination with TGF-ß1. The α2 and ß1 subunits were mainly localized at cell-cell contacts, αv being found near cell cluster surfaces. Immunoprecipitation showed increase in αvß1 heterodimers, but not α2ß1, after TNC treatment. Activated ß1 subunits detected by an antibody against the Ca(2+)-dependent epitope colocalized with αv in focal adhesion complexes, associated with FAK phosphorylation at tyrosine 925. Neutralizing antibodies against αv and ß1 blocked EMT-like change caused by TNC alone. In addition, anti-αv and combined treatment with anti-ß1 and anti-αvß6 inhibited TGF-ß1/TNC-induced EMT, whereas either of these alone did not. Integrin subunits αv, ß1 and ß6, but not α2, bound to TNC immobilized on agarose beads in a divalent cation-dependent manner. Treatments with neutralizing antibodies against ß1 and αvß6 reduced αv subunit bound to the beads. Immunohistochemistry of these receptors in human breast cancer tissues demonstrated frequent expression of ß6 subunits in cancer cells forming scattered nests localized in TNC-rich stroma. These findings provide direct evidence that binding of αvß6 and αvß1 integrins to TNC as their essential ligand induces EMT-like change in breast cancer cells.

Functional variants in the thromboxane A2 receptor gene are associated with lung function in childhood-onset asthma.

BACKGROUND: The thromboxane A2 receptor (TBXA2R) gene is associated with asthma, but no functional genetic variations are known to associate with the disease or its related phenotypes. OBJECTIVE: To investigate the association of TBXA2R polymorphisms with asthma susceptibility and related phenotypes and to identify functionally relevant polymorphisms. METHODS: We performed comprehensive sequencing of the TBXA2R gene in 48 Japanese control subjects and found a set of variants (SNP1 G>T rs2238634, SNP2 T>G rs2238633, SNP3 C>T rs2238632 and SNP4 G>A rs2238631) in intron 1 in linkage disequilibrium with c.795 T>C rs1131882, which was previously reported to be associated with asthma and related phenotypes. To investigate the effect of four common haplotypes (H1, H2, H3 and H4) on transcriptional activity, we performed a luciferase assay in primary bronchial smooth muscle cells (BSMCs) and human airway epithelial cells (BEAS-2B). We also studied the haplotype association with lung function, TBXA2R mRNA levels, and eosinophil fraction/count in peripheral blood in childhood-onset asthma patients and/or controls. RESULTS: H2 and H4, containing minor alleles of SNP2 and SNP3, had significantly higher transcriptional activities than H1 consisting of major alleles (P < 0.001 in BSMCs and BEAS-2B). Homozygotes for redefined haplotype h2 corresponding to minor alleles of SNP2 and SNP3 were associated with lower lung function in childhood-onset asthma patients compared to other zygotes (baseline Forced expiratory volume in one second (FEV1)/ Forced vital capacity (FVC) and Forced expiratory flow between 25% and 75% of the FVC (%FEF(25-75%)): P = 0.00201 and 0.0128, respectively, and post-bronchodilator FEV1/FVC and %FEF(25-75%): P = 0.00224 and 0.0393 respectively). Haplotype h2 was also associated with higher mRNA levels in control peripheral blood cells and higher blood eosinophil fractions and counts in female controls. CONCLUSIONS AND CLINICAL RELEVANCE: Genetic variants were identified in the TBXA2R gene that influenced transcriptional activity and were associated with asthma-related phenotypes. Thromboxane pathways may therefore play important roles in airway inflammation and remodelling in asthma patients.

Involvement of endothelin-1 in habitual exercise-induced increase in arterial compliance.

AIM: Habitual aerobic exercise results in a significant increase in central arterial compliance. Endothelin-1 (ET-1) is a potent endothelium-derived vasoconstrictor peptide and could play a role in mediating the habitual aerobic exercise-induced increase in central arterial compliance. The aim of the present study was to examine whether ET-1 is involved in the mechanisms underlying the increase in central arterial compliance with aerobic exercise training. METHODS: Seven apparently healthy middle-aged and older (60 +/- 3 years) adults underwent systemic endothelin-A/B (ET(A/B))-receptor blockade (500 mg of Tracleer) before and after 12 weeks of aerobic exercise training (70 +/- 1% of maximal heart rate, 44 +/- 2 min day(-1), 4.4 +/- 0.1 days week(-1)). RESULTS: Basal carotid arterial compliance (via simultaneous B-mode ultrasound and arterial applanation tonometry on the common carotid artery) increased significantly after exercise training. Resting plasma ET-1 concentration decreased significantly after exercise training. Before exercise intervention, carotid arterial compliance increased significantly with the administration of the ET(A/B)-receptor blockade. After training, however, increases in carotid arterial compliance previously observed with the ET(A/B)-receptor blockade before training were abolished. CONCLUSIONS: Regular aerobic exercise training enhances central arterial compliance in middle-aged and older humans. The increase in arterial compliance was associated with the corresponding reduction in plasma ET-1 concentration as well as the elimination of ET-1-mediated vascular tone. These results suggest that reductions in ET-1 may be an important mechanism underlying the beneficial effect of exercise training on central artery compliance.

Differences in the composition of intestinal Bifidobacterium species and the development of allergic diseases in infants in rural Japan.

BACKGROUND: Bifidobacterium is a dominant genus in the intestinal microbiota of infants and comprises many different species. A series of studies performed in northern Europe showed differences in the composition of Bifidobacterium species between allergic infants and healthy controls. Additional studies are needed to confirm this observation. OBJECTIVE: To investigate the composition of fecal Bifidobacterium species in allergic infants and healthy controls in Japan, using molecular methods. METHODS: Full-term born babies were followed up to 6 months of age at a local hospital in rural Japan. The presence or absence of allergy was determined based on allergic symptoms and skin prick tests. Fecal Bifidobacterium species in allergic infants (n=10), and healthy controls (n=16) were evaluated using nine Bifidobacterium species-specific or group-specific primers based on 16S rDNA sequences at 1, 3, and 6 months of age. RESULTS: The number of the infants in whom no Bifidobacterium species could be found was four (15.4%) at 1 month, two (7.7%) at 3 months, and one (3.3%) at 6 months of age, all of whom were healthy controls. At 1 month of age, allergic infants had a higher prevalence f the Bifidobacterium catenulatum group than healthy controls (60.0% vs. 6.3%, P<0.01). At 6 months of age, allergic infants had a higher prevalence of B. bifidum than healthy controls (70.0% vs. 12.5%, P<0.01). These differences were not related to feeding method. CONCLUSIONS: Our results in infants in rural Japan support the hypothesis that a compositional difference in intestinal Bifidobacterium species may be associated with the development of allergy in early infancy, although the responsible species might vary among countries or races.

Usefulness of magnetic resonance sialography in patients with juvenile Sjögren's syndrome.

OBJECTIVE: Sialography is an important means for evaluating parotid gland damage in patients with Sjögren's syndrome (SS). However, 'conventional' X-ray sialography is invasive and sometimes difficult to perform and repeat, especially for young patients. Recently, magnetic resonance (MR) sialography has been used in adult SS patients. In this study, we investigated the usefulness of MR sialography for evaluating parotid gland damage in juvenile SS. METHODS: Eight young patients suffering from SS were studied. MR sialography and X-ray sialography were performed simultaneously in the same patients. The images obtained by both methods were assessed with Rubin-Holt staging. RESULTS: MR sialography detected ductal dilatation in 5 of 8 patients, while it was detected in 7 of 8 patients by X-ray sialography. The stages were the same in 4 patients by both methods. In 3 patients, the stages on X-ray sialography were higher than those on MR sialography; in 1 patient, the stage on MR sialography was higher. The correlation between the stages determined by the 2 methods was 0.85. There were no side effects in MR sialography, whereas 3 patients complained of pain during X-ray sialography. CONCLUSION: MR sialography can evaluate Stage II approximately III parotid gland damage in juvenile SS. Although MR sialography cannot detect subtle changes in the duct, it has no side effects and can be performed repeatedly in young patients. We propose that MR sialography be chosen as the first tool for diagnosing and during follow-up of the status of the glands in juvenile SS.

Respiratory syncytial virus infection suppresses IFN-gamma production of gammadelta T cells.

The immunological mechanisms by which respiratory syncytial virus (RSV) contributes to the development of asthma are poorly understood. gammadelta T cells are important in mucosal defence, and may contribute to the establishment of primary immune responses by producing cytokines early during respiratory infections. Thus, we used flow cytometry and intracellular cytokine staining to investigate the expression of interferon (IFN)-gamma and interleukin (IL)-4 by mitogen-stimulated gammadelta T cells from the peripheral blood of 15 hospitalized infants with RSV bronchiolitis, seven rotavirus-infected infants and eight normal controls. gammadelta T cells from RSV-infected infants had a lower proportion of IFN-gamma-producing cells (median, 4.00%; range, 0.58-6.60%) and a slightly but significantly higher proportion of IL-4-producing cells (median, 0.40%; range, 0.13-2.76%) than rotavirus-infected infants (median, 32.10%; range, 14.43-61.21%; P < 0.01, median, 0.00%; range, 0.00-0.00%; P < 0.05) in the acute phase. By contrast, differences in cytokine production by total CD3+ T cells did not differ significantly between patient groups. Thus, reduced IFN-gamma-production by gammadelta T cells in the peripheral blood of RSV-infected infants is accompanied by increased Th2 cytokine production during the acute phase of disease. At follow-up, eight children had recurrent episodes of wheezing. The frequencies of IFN-gamma-producing gammadelta T cells were significantly lower in patients who developed recurrent wheezing (median, 0.65%; range, 0.02-1.75%) than in patients without recurrent wheezing (median, 6.90%; range, 5.25-10.98%; P < 0.005). Cytokine production by gammadelta T cells may therefore be important in the pathogenesis of acute RSV disease, and play a part in the development of recurrent childhood wheezing after bronchilolitis.

Catecholamines and opioid peptides increase in plasma in humans during possession trances.

Naturally induced possession trances have been observed in healthy people of many societies. The neurophysiological basis of this phenomenon remains unknown, however, because of the difficulty in accessing subjects in trances due to their sacred context. In the present study, we measured the plasma levels of several neuroactive substances from subjects exhibiting or lacking possession trance characteristics during Balinese dedicatory dramas under natural conditions. The trance group exhibited significant increases in plasma concentrations of noradrenaline, dopamine and beta-endorphin, compared with controls who performed the same actions as the trance group. The present finding suggests that catecholamines and opioid peptides are involved in possession trances.

Impairment of spermatogenesis in rats by methylmercury: involvement of stage- and cell- specific germ cell apoptosis.

Methylmercury has been shown to affect the male reproductive organs. However, the specific mode of impairment of spermatogenesis during methylmercury exposure remains unknown. In this study, we characterized the induction of germ cell apoptosis and reproductive toxicity in Wistar male rats that had been exposed to methylmercuric chloride (MMC). Subcutaneous injection of MMC at a dose of 10 mg/kg per day for 8 days resulted in a 28% testicular weight loss at 14 days after the first injection. In addition, the ventral and dorso-lateral prostatic lobes showed a 65 and 52% decrease, respectively, at 14 days, although no effects were observed in the epididymis. Sperm production also was suppressed by the administration of MMC. After exposure to MMC, fragmentation of testicular DNA was found to be increased at 3 days after the first injection, with a 20-fold increase over control levels at 14 days. In situ detection of apoptosis by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) staining revealed that spermatocytes and spermatids at stages VII-VIII and IX-XI, respectively, steps which are considered to be highly sensitive to testosterone, were the major cell types affected. Consequently, a marked cell loss in elongated spermatids at stages XII-XIV and I was observed at 14 days. In addition, plasma testosterone levels were reduced at 6 days after exposure to MMC, and remained at approximately 20% of control levels during the 14-day observation period. Our results suggest that methylmercury impairs spermatogenesis by germ cell deletion via cell- and stage- specific apoptosis.

Exposure to ozone aggravates nasal allergy-like symptoms in guinea pigs.

The ability of O3 exposure to aggravate ovalbumin (OVA)-induced nasal allergy-like symptoms was studied in guinea pigs. Guinea pigs were exposed to filtered air or to 0.4 ppm O3 for 5 weeks. During the exposure, 1% OVA or saline was administered into the nasal cavities once a week. Sneezes and nasal secretions were measured for a 20-min period following OVA administration. The number of eosinophils infiltrating both nasal epithelium and subepithelium and titers of specific anti-OVA-IgG were measured 24 h after the last administration. Ozone increased OVA-induced sneezing and nasal secretion, as well as induced nasal hyper-responsiveness to physical stimuli. The number of eosinophils infiltrating the nasal subepithelium was increased by O3, and the titer of anti-OVA-IgG tended to increase in the O3-exposed animals. Thus, exposure to O3 aggravated nasal allergy-like symptoms by inducing nasal hyper-responsiveness, the infiltration of eosinophils, and by tending to increase the production of anti-OVA-IgG.

Ozone exposure enhances expression of cell-surface molecules associated with antigen-presenting activity on bronchoalveolar lavage cells in rats.

In this study, we investigated the effects of ozone (O3) exposure on expression of cell-surface molecules associated with antigen presentation and on accessory activity of bronchoalveolar lavage cells (BAL cells). Rats were exposed to 1 ppm O3 for 3 days. Expression of cell-surface molecules was measured by flow cytometry. Accessory activity of BAL cells was assessed by the allogeneic mixed lymphocyte reaction (MLR) and specific antigen-presenting activity. O3 exposure increased the expression of Ia, B7.1, B7.2, and CD11b/c on BAL cells. Morphological and immunological studies showed the Ia-positive cells to have monocyte-like features. Peripheral blood monocytes expressed Ia, B7.1, B7.2, and CD11b/c. The Ia expression on the monocytes was further increased by treatment of them with BAL fluid from O3-exposed rats (O3-BALF). Resident alveolar macrophages, however, did not express Ia antigen, and the Ia expression was not increased by O3-BALF. Neutrophils, which also infiltrated in response to O3 exposure, did not express Ia, B7.1, and B7.2. Therefore, infiltrating monocytes may have caused the increased expression of Ia and B7 molecules on BAL cells exposed to O3. The accessory activity of BAL cells in terms of MLR and specific antigen-presenting activity was also enhanced by O3 exposure. The present study suggests that monocytes infiltrating in response to O3 exposure caused enhancements of the expression of Ia and costimulatory molecules on and the accessory activity of BAL cells.

Phenanthraquinone inhibits eNOS activity and suppresses vasorelaxation.

Diesel exhaust particles cause an impairment of endothelium-dependent vasorelaxation and are associated with cardiopulmonary-related diseases and mortality, but the mechanistic details are poorly understood. Since we reported previously that phenanthraquinone, an environmental chemical contained in diesel exhaust particles, suppresses neuronal nitric oxide synthase (nNOS) activity by shunting electrons away from the normal catalytic pathway, it was hypothesized that phenanthraquinone inhibits endothelial NOS (eNOS) activity and affects vascular tone. Therefore, the effects of phenanthraquinone on eNOS activity, endothelium-dependent relaxation, and blood pressure were examined in the present study. Phenanthraquinone inhibited NO formation evaluated by citrulline formed by total membrane fraction of bovine aortic endothelial cells with an IC(50) value of 0.6 microM. A kinetic study revealed that phenanthraquinone is a competitive inhibitor with respect to NADPH and a noncompetitive inhibitor with respect to L-arginine. Endothelium-dependent relaxation of rat aortic rings by ACh was significantly inhibited by phenanthraquinone (5 microM), whereas the endothelium-independent relaxation by nitroglycerin was not. Furthermore, an intraperitoneal injection of phenanthraquinone (0.36 mmol/kg) to rats resulted in an elevation of blood pressure (1.4-fold, P < 0.01); under this condition, plasma levels of stable NO metabolites, nitrite/nitrate, in phenanthraquinone-treated rats was reduced to 68% of control levels. The present findings suggest that phenanthraquinone has a potent inhibitory action on eNOS activity via a similar mechanism reported for nNOS, thereby causing the suppression of NO-mediated vasorelaxation and elevation of blood pressure.

Homing receptor expression on cord blood T lymphocytes and the development of atopic eczema in infants.

Expression of the gut-homing receptor integrin alphaEbeta7, but not cutaneous lymphocyte-associated antigen (CLA), on milk allergen-stimulated cord blood T lymphocytes precedes the development of milk-induced eczema in early infancy. The data indicate the involvement of integrin alphaEbeta7 in the development of infantile allergic eczema and provide a clue to the avoidance of specific allergens and novel therapy targeting homing receptors in food allergy.

A novel mouse model of Graves' disease: implications for a role of aberrant MHC class II expression in its pathogenesis.

Mice immunized with fibroblasts expressing an MHC class II molecule and human thyrotropin receptor (TSHR), but not either alone, develop major features characteristic of Graves' disease (GD), such as thyroid-stimulating autoantibodies directed against TSHR, increased serum thyroid hormone levels, and enlarged thyroid glands. The results indicate the need for the simultaneous expression of a class II molecule and the TSHR on the surface of the fibroblasts to develop stimulating anti-TSHR antibodies and full-blown GD in our model. A T cell line established from a mouse with hyperthyroidism proliferates in response to fibroblasts expressing a class II molecule and TSHR, but not to the fibroblasts expressing only TSHR, indicating that the class II molecules on the fibroblasts present TSHR-derived peptide(s) to T cells. These results strongly suggest that the acquisition of antigen-presenting ability by thyrocytes can lead to the induction or progression of GD. We identified a T cell epitope of TSHR by the proliferative response of spleen cells from mice immunized with fibroblasts expressing a class II molecule and TSHR to 80 overlapping peptides spanning the extracellular domain of human TSHR. The identification of a major T cell epitope provides an important clue to a novel therapy of GD.

Graves' disease: a host defense mechanism gone awry.

In this report we summarize evidence to support a model for the development of Graves' disease. The model suggests that Graves' disease is initiated by an insult to the thyrocyte in an individual with a normal immune system. The insult, infectious or otherwise, causes double strand DNA or RNA to enter the cytoplasm of the cell. This causes abnormal expression of major histocompatibility (MHC) class I as a dominant feature, but also aberrant expression of MHC class II, as well as changes in genes or gene products needed for the thyrocyte to become an antigen presenting cell (APC). These include increased expression of proteasome processing proteins (LMP2), transporters of antigen peptides (TAP), invariant chain (Ii), HLA-DM, and the co-stimulatory molecule, B7, as well as STAT and NF-kappaB activation. A critical factor in these changes is the loss of normal negative regulation of MHC class I, class II, and thyrotropin receptor (TSHR) gene expression, which is necessary to maintain self-tolerance during the normal changes in gene expression involved in hormonally-increased growth and function of the cell. Self-tolerance to the TSHR is maintained in normals because there is a population of CD8- cells which normally suppresses a population of CD4+ cells that can interact with the TSHR if thyrocytes become APCs. This is a host self-defense mechanism that we hypothesize leads to autoimmune disease in persons, for example, with a specific viral infection, a genetic predisposition, or even, possibly, a TSHR polymorphism. The model is suggested to be important to explain the development of other autoimmune diseases including systemic lupus or diabetes.

Intense exercise causes decrease in expression of both endothelial NO synthase and tissue NOx level in hearts.

Cardiac myocytes produce nitric oxide (NO). We studied the effects of intense exercise on the expression of NO synthase (NOS) and the tissue level of nitrite (NO(2)(-))/nitrate (NO(3)(-)) (i.e., NOx), which are stable end products of NO in the heart. Rats ran on a treadmill for 45 min. Immediately after this exercise, the heart was quickly removed. Control rats remained at rest during the same 45-min period. The mRNA level of endothelial NOS (eNOS) in the heart was markedly lower in the exercised rats than in the control rats. Western blot analysis confirmed downregulation of eNOS protein in the heart after exercise. Tissue NOx level in the heart was significantly lower in the exercised rats than in the control rats. The present study revealed for the first time that production of NO in the heart is decreased by intense exercise. Because NO attenuates positive inotropic and chronotropic responses to beta(1)-adrenergic stimulation in the heart, the decrease in cardiac production of NO by intense exercise may contribute to the acceleration of increase in myocardial contractility and heart rate during intense exercise.