Concurrent absorption and secretion of airway surface liquids and bicarbonate secretion in human bronchioles.

Although small airways account for the largest fraction of the total conducting airway surfaces, the epithelial fluid and electrolyte transport in small, native airway epithelia has not been well characterized. Investigations have been limited, no doubt, by the complex tissue architecture as well as by its inaccessibility, small dimensions, and lack of applicable assays, especially in human tissues. To better understand how the critically thin layer of airway surface liquid (ASL) is maintained, we applied a "capillary"-Ussing chamber (area ≈1 mm2) to measure ion transport properties of bronchioles with diameters of ~2 mm isolated from resected specimens of excised human lungs. We found that the small human airway, constitutively and concurrently, secretes and absorbs fluid as observed in porcine small airways (50). We found that the human bronchiolar epithelium is also highly anion selective and constitutively secretes bicarbonate ( HCO3- ), which can be enhanced pharmacologically by cAMP as well as Ca2+-mediated agonists. Concurrent secretion and absorption of surface liquid along with HCO3- secretion help explain how the delicate volume of the fluid lining the human small airway is physiologically buffered and maintained in a steady state that avoids desiccating or flooding the small airway with ASL.

A young child with bilateral diaphragmatic palsy after bilateral bidirectional Glenn shunt.

A 13-months old boy was admitted in National Heart Foundation Hospital and Research Institute on 3 August 2011 with the diagnosis of Dextrocardia, A-V discordance, DORV, large perimembranous VSD, severe infundibular and valvular PS, bilateral SVC. He was operated on 10 August 2011. Bilateral bidirectional Glenn shunt was done off pump along with interruption of PDA. Antegrade pulmonary blood flow was minimized by tight PA banding. Baby was extubated 3 hours after surgery but had to reintubate immediately due to intense respiratory distress. Subsequent three trials of extubation failed. Chest x-ray revealed elevation of both the hemidiaphragm. Ultrasonogram of abdomen and Bronchogram along with fluoroscopy done and bilateral diaphragmatic palsy was diagnosed. Tracheostomy was done on 25th August 2011. Plication of left hemidiaphragm was done on 27th August and right hemidiaphragm plication was done on 10th September 2011. Though it took long period of time we managed to take him out of ventilator on 57th postoperative day. He was oxygen dependent for a period of time and finally he managed to take his own breath without tracheostomy tube from 67th postoperative day. After a long eventful postoperative hospital stay he was discharged home on 78th postoperative day. Discharge Chest x-ray revealed well expanded lung with flattened diaphragm. Echo revealed well functioning bilateral Glenn shunt. Tracheostomy wound healed nicely and there was no evidence of tracheal stenosis.

Native small airways secrete bicarbonate.

Since the discovery of Cl(-) impermeability in cystic fibrosis (CF) and the cloning of the responsible channel, CF pathology has been widely attributed to a defect in epithelial Cl(-) transport. However, loss of bicarbonate (HCO3(-)) transport also plays a major, possibly more critical role in CF pathogenesis. Even though HCO3(-) transport is severely affected in the native pancreas, liver, and intestines in CF, we know very little about HCO3(-) secretion in small airways, the principle site of morbidity in CF. We used a novel, mini-Ussing chamber system to investigate the properties of HCO3(-) transport in native porcine small airways (∼ 1 mm φ). We assayed HCO3(-) transport across small airway epithelia as reflected by the transepithelial voltage, conductance, and equivalent short-circuit current with bilateral 25-mM HCO3(-) plus 125-mM NaGlu Ringer's solution in the presence of luminal amiloride (10 µM). Under these conditions, because no major transportable anions other than HCO3(-) were present, we took the equivalent short-circuit current to be a direct measure of active HCO3(-) secretion. Applying selective agonists and inhibitors, we show constitutive HCO3(-) secretion in small airways, which can be stimulated significantly by ß-adrenergic- (cAMP) and purinergic (Ca(2+)) -mediated agonists, independently. These results indicate that two separate components for HCO3(-) secretion, likely via CFTR- and calcium-activated chloride channel-dependent processes, are physiologically regulated for likely roles in mucus clearance and antimicrobial innate defenses of small airways.

ß-adrenergic sweat secretion as a diagnostic test for cystic fibrosis.

RATIONALE: ß-Adrenergically induced sweat secretion offers an expedient method to assess native cystic fibrosis transmembrane conductance regulator (CFTR) secretory function in vivo. OBJECTIVES: To evaluate the sensitivity, specificity, and reliability of a test based on the activity and secretory function of CFTR in the sweat gland. METHODS: Primary and validation trials with prospectively ascertained healthy control subjects, obligate heterozygotes, and patients with a CFTR-related disorder and CF (pancreatic sufficient and insufficient). MEASUREMENTS AND MAIN RESULTS: Diagnostic accuracy and reliability of ß-adrenergic sweat secretory rates using an evaporimeter was assessed and compared with sweat chloride concentrations. The cholinergically stimulated mean sweat rate did not differ among groups. The mean maximal ß-adrenergically stimulated sweat rate in heterozygotes was about half the rate of healthy control subjects, and completely absent in pancreatic-insufficient patients with CF and pancreatic-sufficient patients with CF (P < 0.0001). Subjects with a CFTR-related disorder showed reduced or absent ß-adrenergic sweat secretion. The ß-adrenergic secretory response demonstrated high diagnostic accuracy (area under a characteristic receiver-operator curve = 0.99; 95% confidence interval, 0.97-1.00) and reliability (intraclass correlation, 0.90; 95% confidence interval, 0.81-0.95). The diagnostic cutoff level for CF, derived from the primary trial, correctly identified all control subjects, heterozygotes, and patients with CF in the validation cohort, whereas concurrent sweat chloride measurements misclassified one heterozygote and five subjects with CF. The cholinergic and ß-adrenergic sweat secretion rates were lower in women compared with men (P < 0.001). CONCLUSIONS: ß-Adrenergic sweat secretion rate determined by evaporimetry is an accurate and reliable technique to assess different levels of CFTR function and to identify patients with CF.

Surface fluid absorption and secretion in small airways.

Native small airways must remain wet enough to be pliable and support ciliary clearance, but dry enough to remain patent for gas flow. The airway epithelial lining must both absorb and secrete ions to maintain a critical level of fluid on its surface. Despite frequent involvement in lung diseases, the minuscule size has limited studies of peripheral airways. To meet this challenge, we used a capillary to construct an Ussing chamber (area <1 mm(2)) to measure electrolyte transport across small native airways (∼1 mm ø) from pig lung. Transepithelial potentials (V(t)) were recorded in open circuit conditions while applying constant current pulses across the luminal surface of dissected airways to calculate transepithelial electrical conductance (G(t)) and equivalent short circuit current (I(eq)(sc)) in the presence and absence of selected Na(+) and Cl(-) transport inhibitors (amiloride, GlyH-101, Niflumic acid) and agonists (Forskolin + IBMX, UTP). Considered together the responses suggest an organ composed of both secreting and absorbing epithelia that constitutively and concurrently transport fluids into and out of the airway, i.e. in opposite directions. Since the epithelial lining of small airways is arranged in long, accordion-like rows of pleats and folds that run axially down the lumen, we surmise that cells within the pleats are mainly secretory while the cells of the folds are principally absorptive. This structural arrangement could provide local fluid transport from within the pleats toward the luminal folds that may autonomously regulate the local surface fluid volume for homeostasis while permitting acute responses to maintain clearance.

Enhanced heat loss responses induced by short-term endurance training in exercising women.

We investigated the effects of short-term endurance training and detraining on sweating and cutaneous vasodilatation during exercise in young women, taking into account changes in maximal oxygen uptake (VO2max) and the phase of the menstrual cycle. Eleven untrained women participated in endurance training; cycle exercise at approximately 60% VO2max for 60 min day(-1), 4-5 days week(-1) (30 degrees C, 45% relative humidity) for three complete menstrual cycles. The standard exercise test consisted of exercise at 50% VO2max for 30 min (25 degrees C, 45% relative humidity), and was conducted before training (Pre), during training sessions (T1, T2 and T3) and after cessation of training (D1 and D2). Values of VO2max increased significantly from 32.7 +/- 1.2 to 37.8 +/- 1.2 ml min(-1) kg(-1) at the end of the training. Local sweat rate in the chest and thigh, but not in the back and forearm, were significantly greater during T1 and T2 only in women who started training from the midfollicular phase. Cutaneous blood flow did not change with training. The threshold oesophageal temperatures for heat loss responses were significantly decreased during T1 versus Pre (averaged values for each body site: sweating, 37.49 +/- 0.08 versus 37.22 +/- 0.12 degrees C; and cutaneous vasodilatation, 37.40 +/- 0.07 versus 37.17 +/- 0.10 degrees C) and maintained through T3; the sensitivities of heat loss responses were not altered. These changes returned to the Pre level by D1. Our data indicate that physical training improves heat loss responses by decreasing the threshold temperatures and that these effects occur within a month of training and disappear within a month after cessation of training. The degree of increase in sweating with training differs among body sites and might be affected by the phase of the menstrual cycle.

Iontophoretic beta-adrenergic stimulation of human sweat glands: possible assay for cystic fibrosis transmembrane conductance regulator activity in vivo.

With the advent of numerous candidate drugs for therapy in cystic fibrosis (CF), there is an urgent need for easily interpretable assays for testing their therapeutic value. Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) abolished beta-adrenergic but not cholinergic sweating in CF. Therefore, the beta-adrenergic response of the sweat gland may serve both as an in vivo diagnostic tool for CF and as a quantitative assay for testing the efficacy of new drugs designed to restore CFTR function in CF. Hence, with the objective of defining optimal conditions for stimulating beta-adrenergic sweating, we have investigated the components and pharmacology of sweat secretion using cell cultures and intact sweat glands. We studied the electrical responses and ionic mechanisms involved in beta-adrenergic and cholinergic sweating. We also tested the efficacy of different beta-adrenergic agonists. Our results indicated that in normal subjects the cholinergic secretory response is mediated by activation of Ca(2+)-dependent Cl(-) conductance as well as K(+) conductances. In contrast, the beta-adrenergic secretory response is mediated exclusively by activation of a cAMP-dependent CFTR Cl(-) conductance without a concurrent activation of a K(+) conductance. Thus, the electrochemical driving forces generated by beta-adrenergic agonists are significantly smaller compared with those generated by cholinergic agonists, which in turn reflects in smaller beta-adrenergic secretory responses compared with cholinergic secretory responses. Furthermore, the beta-adrenergic agonists, isoproprenaline and salbutamol, induced sweat secretion only when applied in combination with an adenylyl cyclase activator (forskolin) or a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine, aminophylline or theophylline). We surmise that to obtain consistent beta-adrenergic sweat responses, levels of intracellular cAMP above that achievable with a beta-adrenergic agonist alone are essential. beta-Adrenergic secretion can be stimulated in vivo by concurrent iontophoresis of these drugs in normal, but not in CF, subjects.

Changes in blood flow in a conduit artery and superficial vein of the upper arm during passive heating in humans.

The purposes of this study were (1) to evaluate changes in blood flow in the brachial artery and basilic vein of the upper arm with a rise in internal temperature during passive heating; and (2) to investigate the contributions of blood velocity and anteroposterior vessel diameter to these blood flow changes. Ten subjects rested in the supine position between a pair of tube-lined sheets. Thermoneutral water was circulated through the tubes to keep a mean skin temperature (Tsk) of 34-35 degrees C, and then hot water was circulated to maintain Tsk of 37-38 degrees C. The blood velocity and diameter in the brachial artery and basilic vein were continuously monitored by Doppler ultrasound technique and used to calculate blood flow. Blood flow in the brachial artery and basilic vein increased linearly as the oral temperature (T(or)) rose by < or =0.6 degrees C. The magnitude of the change in blood flow did not differ significantly between the two vessels. In addition, plots of DeltaT(or) versus blood flow yielded slopes that did not differ significantly between the brachial artery and the basilic vein. As T (or) increased, blood velocity, but not diameter, also increased. In conclusion, blood flow in the brachial artery and the basilic vein increased linearly as the internal temperature variable T (or) increased < or =0.6 degrees C. In both vessels, the passive heating-induced increases in blood flow resulted primarily from a change in blood velocity, rather than from a change in diameter.

Effect of skin temperature on the ion reabsorption capacity of sweat glands during exercise in humans.

The effect of skin temperature on the ion reabsorption capacity of sweat glands during exercise in humans is unknown. In this study, eight healthy subjects performed a 60-min cycling exercise at a constant intensity (60% VO(2max)) under moderate (25 degrees C) and cool (15 degrees C) ambient temperatures at a constant relative humidity of 40%. The sweating rate (SR), index of sweat ion concentration (ISIC) by using sweat conductivity, esophageal temperature (Tes), mean skin temperature, and heart rate (HR) were measured continuously under both ambient temperatures. The SR and ISIC were significantly lower at the cool ambient temperature versus the moderate temperature. There were no significant differences in the changes in HR and esophageal temperature between these ambient temperature conditions, while the mean skin temperature was significantly lower at the cool ambient temperature by almost 3 degrees C (P < 0.05). The slopes of the relationships between Tes and the SR and ISIC were significantly lower and the thresholds of these relationships were significantly higher at the cool ambient temperature (P < 0.05). The ion reabsorption capacity of the sweat glands was significantly lower (P < 0.05) in a cool environment (0.21 +/- 0.04 vs. 0.52 +/- 0.06 mg/cm(2)/min at 15 and 25 degrees C, respectively) as evaluated using the relationships for SR and ISIC. The results suggest that the ion reabsorption capacity of the sweat glands is influenced by skin temperature during exercise in humans.

Changes in the index of sweat ion concentration with increasing sweat during passive heat stress in humans.

To investigate the pattern changes in the index of sweat ion concentration at skin surface with increasing sweat during passive heat stress in humans, we measured conductivity of the perfused water with sweat as the index of sweat ion concentration and sweat rate, continuously at the chest skin surface. Eight healthy subjects (22.4 +/-1.0 years) were passively heated by lower-leg immersion in a hot water bath of 42 degrees C for 50 min in an ambient temperature of 28 degrees C and relative humidity of 50%. The internal temperature (Tor) thresholds of sweat rate and index of sweat ion concentration were almost similar. Concomitant onset for the index of sweat ion concentration and sweat rate occurred but two types of linear regression lines were identified in the relationship between the index of sweat ion concentration and sweat rate at a boundary sweat rate value of 0.30 +/- 0.08 mg cm(-2) min(-1). The slope of the regression line at low levels of sweat (slope 0.02 +/- 0.01 V mg(-1) cm(-2) min(-1)) was significantly gradual compared with that at moderate levels of sweat (slope 0.30 +/- 0.08 V mg(-1) cm(-2) min(-1)) (P<0.05). These results suggest that at low levels of sweat the index of sweat ion concentration responds gradually with respect to sweat rate, which may be due to the ion reabsorption capacity of the sweat duct, and then the index of sweat ion concentration increased steeply with sweat rate.

Continuous monitoring of single-sweat-gland activity.

A conductivity measurement system using a small ion-free-solution perfusion chamber has been developed to monitor single sweat-gland activity (SSGA) continuously at the skin surface. The chamber has a small open space of 0.2 mm2 at the bottom and has a transparent window. Single sweat pores were visualized by the starch/iodine method and the chamber was attached onto a single sweat pore using a magnifying lens affixed at the window. Silver electrodes were installed inside the chamber, and, by perfusing ion-free solution through the chamber at a constant flow rate, the conductivity of the solution was measured at the inlet and the outlet of the chamber. Continuous SSGA was monitored at the palm, finger tip and chest skin surface when the subjects were seated in a resting position and under stresses such as hand grasping with a dynamometer and performing mental arithmetic. Different types of response were observed from different sweat pores. The response time of this system was less than 0.15 s. The present results reveal that continuous sweat activity can be monitored even from a single sweat gland.

Continuous monitoring of sweating by electrical conductivity measurement.

A method of continuous monitoring of sweating was developed in which sweat was detected by changes in the conductivity of perfusing water. An ion-free solution was perfused at a constant flow rate through a chamber attached to the skin surface. The chamber was designed so that the electrodes were installed inside at the inlet and outlet, and a 14 mm3 channel was constructed at the bottom to wash out sweat. The 90% response time was 0.12 s. Attaching the chamber to the palm allowed measurements to be made with the subject seated in a comfortable environment. The sweat rate and heart rate were measured simultaneously with an air-ventilation chamber and a heart rate counter, respectively, with the subjects at rest, and under stresses such as grasping hands and doing mental arithmetic. This method yields sweat responses similar to those obtained with an air-ventilation chamber and simultaneous heart rate measurements. The main advantages of this method are faster response time and smaller observation area.

Immunocytochemical localization of benzo(a)pyrene-DNA adducts in human tissue.

Specimens of human lung, uterine cervix, ovary, and placenta were studied for the presence of benzo(a)pyrene 7,8-diol 9,10 epoxide (BPDE)-DNA adducts by using rabbit anti-BPDE-DNA antibody and light microscopic immunocytochemistry. BPDE-DNA antigenicity was detected in the bronchial epithelial cells, cervical epithelium, oocytes, luteal cells, corpora albicans, and hyalinized media of arteries within the ovaries and trophoblastic cells of the placental villi. In conjunction with immunoassay detection of BPDE-DNA adducts in human peripheral blood lymphocytes, this study demonstrates that a variety of human tissues can metabolize and bind the ubiquitous carcinogen benzo(a)pyrene. The identification and localization of this carcinogen-DNA antigenicity in various tissues and cells may not only help in monitoring exposed persons but also give insight to organ site carcinogenesis, transplacental carcinogenesis, and teratogenesis.

Is the corneal posterior cell layer truly endothelial?

The posterior cell layer of the normal human cornea or "endothelium" was investigated by electron microscopy and immunocytochemistry. Ultrastructurally, the cells lacked the characteristic marker for endothelial cells (Weibel-Palade body). Immunoperoxidase studies demonstrated these cells to be negative for factor VIII antigen, but strongly positive for keratine, vimentin, S-100 protein, and neuron-specific enolase. The anterior epithelial cell layer showed identical immunoreactivity. These studies strongly suggest that the posterior cell layer of the cornea lacks ultrastructural and immunocytochemical markers of endothelial cells and both the anterior and posterior cell layers share similar cell markers. The authors propose that the posterior cell layer of the cornea should, therefore, not be misnamed as "endothelium."

Ultrastructural features of normal mouse colon epithelium. Unique characteristics of a species.

Ultrastructural studies of the colon epithelium of normal C57Bl/Ha and ICR/Ha mice revealed some unique characteristics not seen in human or rat colon. The most striking feature was the presence of a unique type of intracytoplasmic organelle with crystalline internal structure. These organelles measured 1-2 micron in diameter, usually rounded or oval showing marked variation in size and shape. They were surrounded by a single layer of trilaminar plasma membrane and their core structure was mildly electron dense with markedly dense crystalline substructure. Grimelius silver stain done at ultrastructural level revealed these to have staining properties identical to neuroendocrine granules. Approximately 1-5% of the epithelial cells of mouse colon were usually filled with these organelles. But occasionally they were also present in the endocrine cells of colon. The other striking feature of the mouse colon epithelium is the presence of an inordinate number of bacteria. These rod shaped bacteria were present deep inside crypts in large numbers. They were also present within the goblet type of mucous cells and the so-called columnar cells of the surface epithelium. A third unique feature of mouse colon was the presence of mitotic figures in cells with conspicuous mucous vacuoles. This contrasts with human and rat colon where mitosis occurs in cells with little or no mucus. Since the ultrastructural morphology of the normal mouse colon is distinctly different from the human and rat, caution must be exercised in extrapolating colon carcinogenesis data from mouse to human.

Malignant conversion and metastasis of mouse skin tumors: a comparison of SENCAR and CD-1 mice.

The progression of papillomas to squamous cell carcinomas (malignant conversion) was studied in the skin of SENCAR and Charles River CD-1 mice, using a three-stage treatment protocol. After initiation with 7,12-dimethylbenz(a)anthracene (DMBA) (stage 1) and limited promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) (stage II), papilloma-bearing mice were treated (stage III) with either tumor initiators, such as urethane, N-methyl-N'nitro-N-nitrosoguanidine (MNNG) or 4-nitroquinoline-n-oxide (R-NQO), the promoter TPA, or solvent (acetone). Similar final carcinoma yields were found in the mice treated in stage III with TPA or acetone, although carcinomas developed earlier in the TPA-treated mice. In contrast, treatment with tumor initiators in stage III increased both the rate of appearance and the final yield of carcinomas. Similar results were obtained in both SENCAR and CD-1 mice. A papilloma stage appears to be necessary for carcinoma development since elimination of TPA treatment in stage II greatly reduced the incidence of both papillomas and carcinomas in both stocks of mice. The heterogeneity of papillomas with regard to progression to carcinomas is demonstrated by the low rate of conversion of TPA-dependent papillomas and the high rate of conversion of persistent papillomas in CD-1 mice. The carcinomas that develop using the three-stage regimen vary in metastatic potential. In CD-1 mice, the frequency of metastases to lymph nodes were similar in groups treated in stage III with MNNG, urethane, 4-NQO, TPA, or acetone, but treatment with urethane substantially increased metastases to the lung.(ABSTRACT TRUNCATED AT 250 WORDS)