Salivary contribution to exhaled nitric oxide.
Dietary and metabolic nitrate is distributed from the blood to the saliva by active uptake in the salivary glands, and is reduced to nitrite in the oral cavity by the action of certain bacteria. Since it has been reported that nitric oxide may be formed nonenzymatically from nitrite this study aimed to determine whether salivary nitrite could influence measurements of exhaled NO. Ten healthy subjects fasted overnight and ingested 400 mg potassium nitrate, equivalent to approximately 200 g spinach. Exhaled NO and nasal NO were regularly measured with a chemiluminescence technique up to 3 h after the ingestion. Measurements of exhaled NO were performed with a single-breath procedure, standardized to a 20-s exhalation, at a flow of 0.15 L x s(-1), and oral pressure of 8-10 cmH2O. Values of NO were registered as NO release rate (pmol x s(-1)) during the plateau of exhalation. Exhaled NO increased steadily over time after nitrate load and a maximum was seen at 120 min (77.0+/-15.2 versus 31.2+/-3.0 pmol x s(-1), p<0.01), whereas no increase was detected in nasal NO levels. Salivary nitrite concentrations increased in parallel; at 120 min there was a four-fold increase compared with baseline (1.56+/-0.44 versus 0.37+/-0.09 mM, p<0.05). The nitrite-reducing conditions in the oral cavity were also manipulated by the use of different mouthwash procedures. The antibacterial agent chlorhexidine acetate (0.2%) decreased NO release by almost 50% (p<0.01) 90 min after nitrate loading and reduced the preload control levels by close to 30% (p<0.05). Sodium bicarbonate (10%) also reduced exhaled NO levels, but to a somewhat lesser extent than chlorhexidine acetate. In conclusion, salivary nitric oxide formation contributes to nitric oxide in exhaled air and a large intake of nitrate-rich foods before the investigation might be misinterpreted as an elevated inflammatory activity in the airways. This potential source of error and the means for avoiding it should be considered in the development of a future standardized method for measurements of exhaled nitric oxide. (+info)
Regulation of thick ascending limb ion transporter abundance in response to altered acid/base intake.
Changes in ammonium excretion with acid/base perturbations are dependent on changes in medullary ammonium accumulation mediated by active NH4+ absorption by the medullary thick ascending limb. To investigate whether alterations in the abundance of medullary thick ascending limb ion transporters, namely the apical Na+/K+(NH4+)/2Cl- -cotransporter (BSC-1), the apical Na+/H+ -exchanger (NHE3), and the Na+/K+ -ATPase alpha1-subunit, may be responsible in part for altered medullary ammonium accumulation, semiquantitative immunoblotting studies were performed using homogenates from the inner stripe of the rat renal outer medulla. After 7 d of NH4Cl (7.2 mmol/220 g body wt per d) loading (associated with increased medullary ammonium accumulation), neither BSC-1 nor Na+/K+ -ATPase protein expression was altered, but NHE3 protein abundance was significantly increased. On the other hand, both BSC-1 and Na+/K+ -ATPase protein abundance was increased significantly in rats fed NaHCO3 (7.2 mmol/220 g body wt per d) for 7 d. Rats fed a high-NaCl diet (7.7 mEq Na+/220 g body wt per d) for 5 d also showed marked increases in both BSC-1 and Na+/K+ -ATPase expression. The expression level of NHE3 protein did not change with either NaHCO3 or high NaCl intake. None of these three transporters showed a significant difference in abundance between the groups fed equimolar (7.2 mmol/220 g body wt per d for 7 d) NaHCO3 or NaCl. It is concluded that outer medullary BSC-1 and Na+/K+ -ATPase alpha1-subunit protein abundance is increased by chronic Na+ loading but not by acid/base perturbations and that outer medullary NHE3 protein abundance is increased by chronic NH4Cl loading. (+info)
Thiorhodospira sibirica gen. nov., sp. nov., a new alkaliphilic purple sulfur bacterium from a Siberian soda lake.
A new purple sulfur bacterium was isolated from microbial films on decaying plant mass in the near-shore area of the soda lake Malyi Kasytui (pH 9.5, 0.2% salinity) located in the steppe of the Chita region of south-east Siberia. Single cells were vibrioid- or spiral-shaped (3-4 microns wide and 7-20 microns long) and motile by means of a polar tuft of flagella. Internal photosynthetic membranes were of the lamellar type. Lamellae almost filled the whole cell, forming strands and coils. Photosynthetic pigments were bacteriochlorophyll a and carotenoids of the spirilloxanthin group. The new bacterium was strictly anaerobic. Under anoxic conditions, hydrogen sulfide and elemental sulfur were used as photosynthetic electron donors. During growth on sulfide, sulfur globules were formed as intermediate oxidation products. They were deposited outside the cytoplasm of the cells, in the peripheral periplasmic space and extracellularly. Thiosulfate was not used. Carbon dioxide, acetate, pyruvate, propionate, succinate, fumarate and malate were utilized as carbon sources. Optimum growth rates were obtained at pH 9.0 and optimum temperature was 30 degrees C. Good growth was observed in a mineral salts medium containing 5 g sodium bicarbonate l-1 without sodium chloride. The new bacterium tolerated up to 60 g sodium chloride l-1 and up to 80 g sodium carbonates l-1. Growth factors were not required. The DNA G + C composition was 56.0-57.4 mol%. Based on physiological, biochemical and genetic characteristics, the newly isolated bacterium is recognized as a new species of a new genus with the proposed name Thiorhodospira sibirica. (+info)
Evaluation of signals activating ubiquitin-proteasome proteolysis in a model of muscle wasting.
The ubiquitin-proteasome proteolytic system is stimulated in conditions causing muscle atrophy. Signals initiating this response in these conditions are unknown, although glucocorticoids are required but insufficient to stimulate muscle proteolysis in starvation, acidosis, and sepsis. To identify signals that activate this system, we studied acutely diabetic rats that had metabolic acidosis and increased corticosterone production. Protein degradation was increased 52% (P < 0.05), and mRNA levels encoding ubiquitin-proteasome system components, including the ubiquitin-conjugating enzyme E214k, were higher (transcription of the ubiquitin and proteasome subunit C3 genes in muscle was increased by nuclear run-off assay). In diabetic rats, prevention of acidemia by oral NaHCO3 did not eliminate muscle proteolysis. Adrenalectomy blocked accelerated proteolysis and the rise in pathway mRNAs; both responses were restored by administration of a physiological dose of glucocorticoids to adrenalectomized, diabetic rats. Finally, treating diabetic rats with insulin for >/=24 h reversed muscle proteolysis and returned pathway mRNAs to control levels. Thus acidification is not necessary for these responses, but glucocorticoids and a low insulin level in tandem activate the ubiquitin-proteasome proteolytic system. (+info)
Enhancement of chemotherapy by manipulation of tumour pH.
The extracellular (interstitial) pH (pHe) of solid tumours is significantly more acidic compared to normal tissues. In-vitro, low pH reduces the uptake of weakly basic chemotherapeutic drugs and, hence, reduces their cytotoxicity. This phenomenon has been postulated to contribute to a 'physiological' resistance to weakly basic drugs in vivo. Doxorubicin is a weak base chemotherapeutic agent that is commonly used in combination chemotherapy to clinically treat breast cancers. This report demonstrates that MCF-7 human breast cancer cells in vitro are more susceptible to doxorubicin toxicity at pH 7.4, compared to pH 6.8. Furthermore 31P-magnetic resonance spectroscopy (MRS) has shown that the pHe of MCF-7 human breast cancer xenografts can be effectively and significantly raised with sodium bicarbonate in drinking water. The bicarbonate-induced extracellular alkalinization leads to significant improvements in the therapeutic effectiveness of doxorubicin against MCF-7 xenografts in vivo. Although physiological resistance to weakly basic chemotherapeutics is well-documented in vitro and in theory, these data represent the first in vivo demonstration of this important phenomenon. (+info)
Roles of bicarbonate, cAMP, and protein tyrosine phosphorylation on capacitation and the spontaneous acrosome reaction of hamster sperm.
Capacitation is a prerequisite for successful fertilization by mammalian spermatozoa. This process is generally observed in vitro in defined NaHCO3-buffered media and has been shown to be associated with changes in cAMP metabolism and protein tyrosine phosphorylation. In this study, we observed that when NaHCO3 was replaced by 4-(2-hydroxyethyl)1-piperazine ethanesulfonic acid (HEPES), hamster sperm capacitation, measured as the ability of the sperm to undergo a spontaneous acrosome reaction, did not take place. Addition of 25 mM NaHCO3 to NaHCO3-free medium in which spermatozoa had been preincubated for 3.5 h, increased the percentage of spontaneous acrosome reactions from 0% to 80% in the following 4 h. Addition of anion transport blockers such as 4,4'-diiso thiocyano-2, 2'-stilbenedisulfonate (DIDS) or 4-acetomido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) to the NaHCO3-containing medium inhibited the acrosome reaction, with maximal inhibition at 600 microM, and with an EC50 of 100 microM. Increasing either extracellular or intracellular pH did not induce the acrosome reaction in NaHCO3-free medium. In contrast, addition of 500 microM dibutyryl cAMP (dbcAMP), alone or together with 100 microM 1-methyl-3-isobutylxanthine (IBMX), induced the acrosome reaction in spermatozoa incubated in NaHCO3-free medium. These compounds also partially reversed the inhibition of the acrosome reaction caused by the DIDS or SITS in complete medium. In contrast to these results, IBMX or dbcAMP did not induce acrosome reactions in cells incubated in Ca2+-free medium. When hamster sperm were incubated in the absence of NaHCO3 or in the presence of NaHCO3 and DIDS, cAMP concentrations were significantly lower than the values obtained from sperm incubated in complete medium. Protein tyrosine phosphorylation has also been shown to be highly correlated with the onset of capacitation in many species. During the first hour of capacitation, an increase in protein tyrosine phosphorylation was observed in complete medium. In the absence of NaHCO3, the increase in protein tyrosine phosphorylation was delayed for 45 min, and this delay was overcome by the addition of dbcAMP and IBMX. The induction of the acrosome reaction by calcium ionophore A23187 in NaHCO3-free medium was delayed 2 h, as compared with control medium. This delay was not observed in the presence of dbcAMP and IBMX. Taken together, these results suggest that a cAMP pathway may mediate the role of NaHCO3 in the capacitation of hamster spermatozoa and that protein tyrosine phosphorylation is necessary but not sufficient for complete capacitation. (+info)
A minimally invasive tracer protocol is effective for assessing the response of leucine kinetics and oxidation to vaccination in chronically energy-deficient adult males and children.
In disadvantaged populations, recurrent infections lead to a loss of body nitrogen and worsen nutritional status. The resulting malnutrition, in its turn, produces a greater susceptibility to infection. This study aimed to examine the ability of a new minimally invasive tracer protocol to measure leucine oxidation, and then to use it to quantify the effect of vaccination on leucine kinetics and oxidation. Undernourished men (n = 5; body mass index 16.3 +/- 0.9 kg/m(2)) and children (n = 9; age 4.1 +/- 0.6 y; weight-for-age Z-score -2.3 +/- 0.7) underwent metabolic studies 6 d before and 1 d after vaccination with diphtheria, pertussis and tetanus (DPT). The tracer protocol was performed in the fed state and involved two 3-h sequential periods of frequent (20 min) oral doses of NaH(13)CO(3) or [1-(13)C] leucine. Frequent breath samples and urine collections were made. Blood samples were obtained from the men and used for the determination of the isotopic enrichment of alpha-ketoisocaproic acid. The prevaccination oxidation of leucine (percentage of dose +/- SD) was 18.1 +/- 2.3 (men) and 16.7 +/- 3.8 (children). One day after vaccination, these values had risen to 19. 9 +/- 1.9 (P < 0.05) in the men and to 19.5 +/- 4.6 (P < 0.01) in the children. In the adults, vaccination was associated with a rise in whole-body protein breakdown [mg protein/(kg.h)] from 200 +/- 40 to 240 +/- 10 (P < 0.05). A minor simulated infection increases leucine catabolism in undernourished humans and this new, minimally invasive protocol is sufficiently sensitive to measure these changes. (+info)
Combined effects of buffer and adrenergic agents on postresuscitation myocardial function.
Although buffer agents alone have failed to improve the success of resuscitation, we now examine the widely held concept that it is the combined effect of alkaline buffer and adrenergic agents that improves outcomes of cardiopulmonary resuscitation. In the present report, the effects of both CO(2)-consuming and CO(2)-generating buffer agents in combination with adrenergic vasopressor drugs were investigated. Ventricular fibrillation was electrically induced in Sprague-Dawley rats weighing between 450 and 550 g. Precordial compression and mechanical ventilation were initiated after 8 min of untreated ventricular fibrillation. Animals were then randomized to receive bolus injections of either inorganic sodium bicarbonate buffer, organic tromethamine buffer, or saline placebo. The beta(1) adrenergic effects of epinephrine were blocked with esmolol. The vasopressor amine was injected 2 min after injection of the buffer agent. Electrical defibrillation was attempted at the end of 8 min of precordial compression. In 15 additional animals, the sequence of administration of the adrenergic vasopressor and buffer agents was reversed such that the adrenergic vasopressor was injected before the buffer agents. All animals were restored to spontaneous circulation. Both bicarbonate and tromethamine significantly decreased coronary perfusion pressure from 26 to 15 mm Hg and reduced the magnitude of the vasopressor effect of the adrenergic vasopressor. When the vasopressor preceded the buffer, declines in coronary perfusion pressure after administration of buffer agents were prevented. In each instance, however, greater impairment of postresuscitation myocardial function and decreased postresuscitation survival were observed after treatment with buffer agents. (+info)