Lynn M Iwamoto

Loop diuretics and in vitro relaxation of human fetal and newborn mouse airways.

This study was designed to test the hypotheses that furosemide directly causes relaxation in human fetal airway and that delivery of loop diuretics to either the adventitial or epithelial surface of newborn mouse airway results in equivalent relaxation. Isometric tension changes were measured in human fetal (11–16 wk) trachea and mainstem bronchus rings exposed to furosemide (300 μM) or saline after acetylcholine or leukotriene D4 constriction. Significant decreases in isometric tension to furosemide were demonstrated after constriction with acetylcholine or leukotriene D4. To examine the site of effect and mimic aerosolized and systemic administration, furosemide (3–300 μM) and bumetanide (0.3–30 μM) were applied separately to epithelial and adventitial surfaces of newborn mouse airways. No differences in airway diameter changes to epithelial or adventitial furosemide or bumetanide were observed, but a 10-fold difference in potency was found. In summary, human fetal airway relaxed to furosemide when constricted with either neurotransmitter or inflammatory mediator in vitro. Further, no differences in relaxation to equimolar epithelial and adventitial furosemide were observed in isolated newborn mouse airway. Taken together, this provides evidence that furosemide has a direct, nonepithelial-dependent effect on airway smooth muscle tone.

Methamphetamine detection from meconium and amniotic fluid in guinea pigs depends on gestational age and metabolism

Significant adverse perinatal effects of maternal methamphetamine use have been reported, but little is known about factors influencing methamphetamine screening test results during the perinatal period. We tested the hypothesis that gestational age would affect quantitative recovery of methamphetamine in meconium and amniotic fluid. Time-bred guinea pigs received an intraperitoneal (i.p.) injection of 1 mg/kg methamphetamine at either 44 days (0.65 of term, n = 5), 50 days (0.74, n = 8), 56 days (0.82, n = 9) or 63 days (0.93, n = 4) gestation. At 1 or 7 days after i.p. methamphetamine, meconium and amniotic fluid were collected for quantitative methamphetamine assay by gas chromatography-mass spectrometry. Recovery from amniotic fluid and meconium 1 day after injection was influenced by gestational age. Greater values in amniotic fluid and meconium and a higher percentage of positive samples were seen in older fetuses. Collectively at all gestational ages, combined testing of amniotic fluid and meconium yielded detectable methamphetamine or its metabolites in 87% of guinea pigs 1 day after injection. However, methamphetamine was not detectable 1 week after injection in any sample (n = 63) at either 0.74 or 0.82 of term except for one positive amniotic fluid sample. Finally, demethylation of methamphetamine to amphetamine was higher in older fetuses.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of environmental conditions on emergency department use by wheezing children.

STUDY OBJECTIVE To examine in children the relationship of wheezing to measurable environmental factors. STUDY DESIGN Multiple regression analysis was used to measure correlation with air quality, weather, and seasonal and infection-related variables. RESULTS Daily wheezing census was significantly correlated with weather and seasonal variables and the daily infection census. We are not certain which weather variable is the dominant factor in the weather association because all of the weather variables have some degree of colinearity. Air quality as measured by carbon monoxide and airborne particles was not shown to be associated with wheezing. CONCLUSION A high incidence of pediatric emergency department presentations for wheezing are associated with weather, infections, and months of the year.

Acute hyperoxic injury attenuates the relaxing effects of "loop" diuretics and salbutamol on large airways of newborn guinea pigs.

ABSTRACT: We have previously found an age-dependent relaxing effect of furosemide in normal fetal, newborn, and adult guinea pig airways with fetal trachea exhibiting the greatest relaxation and adult tissue the least. This study was designed to expand upon this finding by determining if in vivo hyperoxic exposure would influence in vitro airway relaxation mediated by the loop diuretics, furosemide and ethacrynic acid, and the β2-adrenoceptor agonist, salbutamol. Newborn guinea pigs were raised in >95% Fi02 until ill; controls in room air. Isometric relaxation to 3 × 10−5 M furosemide, 3 × 10−6 M ethacrynic acid, or 10−8−10−6 M salbutamol was recorded in 3 × 10−6 M histamine-constricted airway rings. Ethacrynic acid, like furosemide, relaxed newborn guinea pig airways. Hyperoxia did not alter the contractile effect of 3 × 10−6 M histamine but did significantly decrease the relaxing effect of furosemide, ethacrynic acid, and salbutamol. Loop diuretic mediated airway relaxation was accentuated in HEPES buffer when compared with Krebs, whereas salbutamol-mediated relaxation was unaffected. These results suggest that hyperoxia nonspecifically decreases airway responsiveness to the relaxing agents studied.

Amiloride-induced contraction of isolated guinea pig, mouse, and human fetal airways.

Nebulized amiloride has been proposed as therapy in cystic fibrosis to block Na+ hyperabsorption in airway epithelium and prevent dehydration of secretions. Patients with cystic fibrosis often have reactive airways. Bovine and canine trachea relax to amiloride in vitro, suggesting another benefit as a bronchodilator, whereas guinea pig trachea, a useful model of human airways, does not. We hypothesized that human airways would respond like guinea pig airways. Airway ring segments from guinea pigs, mice, and human fetuses were constricted with the concentration of acetylcholine producing 50-75% maximum contraction. Subsequent changes in isometric tension to cumulative additions of amiloride (10-8-10-4M) were measured. Guinea pig airways contracted 29 ± 5%, mouse airways contracted 23 ± 6%, and human fetal airways contracted 30 ± 8%. Contraction to amiloride was mimicked by dimethylamiloride, a more selective inhibitor of the Na+/H+antiporter, and was attenuated by protein kinase C (PKC) inhibition with GF109203X and staurosporine. The present study indicates that amiloride-induced airway contraction in guinea pigs and mice closely parallels the response in isolated human airways and that the mechanism may involve the Na+/H+antiporter and PKC.Nebulized amiloride has been proposed as therapy in cystic fibrosis to block Na+ hyperabsorption in airway epithelium and prevent dehydration of secretions. Patients with cystic fibrosis often have reaction airways. Bovine and canine trachea relax to amiloride in vitro, suggesting another benefit as a bronchodilator, whereas guinea pig trachea, a useful model of human airways, does not. We hypothesized that human airways would respond like guinea pig airways. Airway ring segments from guinea pigs, mice, and human fetuses were constricted with the concentration of acetylcholine producing 50-75% maximum contraction. Subsequent changes in isometric tension to cumulative additions of amiloride (10(-8)-10(-4) M) were measured. Guinea pig airways contracted 29 +/- 5%, mouse airways contracted 23 +/- 6%, and human fetal airways contracted 30 +/- 8%. Contraction to amiloride was mimicked by dimethylamiloride, a more selective inhibitor of the Na+/H+ antiporter, and was attenuated by protein kinase C (PKC) inhibition with GF109203X and staurosporine. The present study indicates that amiloride-induced airway contraction in guinea pigs and mice closely parallels the response in isolated human airways and that the mechanism may involve the Na+/H+ antiporter and PKC.

RELAXATION TO INTRALUMINAL AND EXTRALUMINAL FUROSEMIDE IN NEWBORN MOUSE TRACHEA. |[bull]| 1519

Furosemide has been given as a bronchodilator by nebulization for direct application to the airways in an attempt to localize its actions and minimize systemic effects. It is also given by systemic administration in the treatment of infants with chronic lung disease. Although clinical studies demonstrate variable results, furosemide relaxes airway smooth muscle in vitro, using isometric tension measurement. A disadvantage of this technique is that in this system, tissues are stretched in a two dimensional fashion. Therefore, we used a cannulated, perfused video micrometry system to determine if the way in which furosemide reaches the airway, by epithelial deposition or by systemic absorption, affects the amount of relaxation. Trachea (600-800 μ diameter) from newborn mice (2-4 d), C57BL/6J and AJ strains, were cannulated and mounted onto a video micrometry system (Living Systems, Burlington, VT). Tissues were bathed in and perfused with HEPES buffer, pH 7.4 at 37°C, bubbled with 100% O2. After an equilibration period, the airways were preconstricted with extraluminal 10 μM Acetylcholine. When stable tension was achieved, 300 μM furosemide was added either extraluminally or intraluminally. Effects of extraluminal and intraluminal furosemide were compared (% change in airway diameter relative to Acetylcholine preconstriction). Relaxation to furosemide given intraluminally (65 ± 9%), with direct epithelial exposure, was not different from relaxation of extraluminally (52 ± 11%) exposed airway (p = 0.4, n=7). Thus the route of furosemide administration did not change the magnitude of relaxation when the same concentration was present either intra- or extraluminally, indicating that furosemide mediated airway relaxation is non-epithelial dependent. Further, systemically administered furosemide may produce airway relaxation if adequate concentrations reach the airway. Preliminary data herein suggest that this cannulated, perfused system may allow for a more physiologic response of the intact airway and may allow for the differentiation of epithelially mediated responses. Funding: NIH-HL45220, Leahi Trust, & US Army HSC.

AMILORIDE DIFFERENTIALLY AFFECTS INFLAMMATORY- AND NEUROLOGIC-INDUCED AIRWAY TONE. 1788

Nebulized amiloride (AMIL) is used in cystic fibrosis (CF) to block Na+ hyperabsorption and dehydration of mucus. CF patients often have inflammation and reactive airways. Bovine and canine airways relax to AMIL in vitro, suggesting a benefit as a bronchodilator, but we found (Pediatr Res 1996;39:387A) that guinea pig (GP), mouse, and human fetal airways contract to AMIL when tone is induced by acetylcholine (ACh, neurotransmitter), independent of airway epithelium. However, the effect of AMIL on airway tone induced by inflammatory mediators, such as leukotriene D4 (LTD4), is not known. Thus, we further defined the mechanism of AMIL-mediated airway contraction, and compared this to the response on airway tone induced by other agonists. Methods. GP (n=6-16), mouse (n=5-9), and human fetal (6 fetuses, 11-16 wks gestation) airway ring segments were constricted with an EC50-75 of ACh, KCl, or LTD4. Changes in isometric tension to cumulative additions of AMIL (0.01-100 μM) were measured. Results. Developmental regulation was not detected. AMIL-mediated contraction was attenuated by inhibition of protein kinase C (PKC) with GF109203X (p<0.01) and staurosporine (p<0.01). However, after constriction with KCl, AMIL relaxed GP (108±15%), mouse (57±32%), and human fetal airway tone (137±58%), and after constriction with LTD4, AMIL relaxed GP(82±10%) and human fetal airway tone (62±23%). Mice did not respond to LTD4. Conclusions. After constriction with ACh, airways from all three species contracted to AMIL in vitro, and this contraction may be mediated by PKC. However, after constriction with KCl and LTD4, AMIL caused relaxation. AMIL may be useful clinically as a bronchodilator when airway hyperresponsiveness is mediated by leukotrienes. Funded by: US Army HSC, NIH RR/AI11091, and NIH HL-45220.

META-HYDROXY BENZOYLECGONINE RECOVERY IN FETAL GUINEA PIGS. † 919

Cocaine (COC) is rapidly hydrolyzed to benzoylecgonine (BE), a major vasoactive metabolite measured in screening infants for prenatal exposure to COC. Recently, detection of meta-hydroxy benzoylecgonine (m-OH BE) has been suggested to improve identification of these infants. However the pathway of m-OH BE formation is unknown. This study was designed to determine if m-OH BE is a late metabolite derived directly from COC or via BE. Pregnant Dunkin-Hartley guinea pigs at 63 d gestation were injected i.p. with 10 mg/kg COC or BE. Amniotic fluid (AF) and meconium (MEC) from each fetus were collected upon sacrifice at 24 and 48 h post-cocaine; and 24, 48, and 72 h post BE injection. COC metabolites were quantitated by gas chromatography-mass spectroscopy. After maternal COC injection, recovery of COC in AF (75%) and MEC (88%) decreased from 24 (n=12) to 48 h (n=9). While BE levels post-COC injection declined in both AF (58%) and MEC (69%) from 24 (n=11) to 48 h(n=7), m-OH BE recovery in MEC increased (1600%). Similarly, after maternal BE injection, mean recovery of BE decreased from 24 (n=11) to 48 h (n=7) in both MEC (69%) and AF (64%), while m-OH BE recovery increased (633%) in MEC. By 72 h (n=11), BE recovery decreased another 85% in both AF and MEC, however m-OH BE was still present in MEC and unchanged from 48 h. There was no significant recovery of m-OH BE in AF after COC or BE injection. Colon from BE exposed fetuses were independently analyzed to determine if m-OH BE is formed in MEC or released into MEC. BE was present at 24 h and levels were undetectable by 72 h, however no m-OH was found. These results indicate that 1) m-OH BE is a metabolite of BE rather than of COC; and 2) levels of m-OH BE may be detectable longer in MEC than BE. Finally, failure to test for m-OH BE in MEC may result in underreporting of cocaine exposure. Funded by: Kapiolani MC& Childrens Miracle Network, US Army HSC.No financial support received from US Drug Testing Labs.

FUROSEMIDE-INDUCED DESENSITIZATION TO SALBUTAMOL IN ISOLATED NEONATAL GUINEA PIG AIRWAYS. ▴ 2300

FUROSEMIDE-INDUCED DESENSITIZATION TO SALBUTAMOL IN ISOLATED NEONATAL GUINEA PIG AIRWAYS. ▴ 2300

EFFECT OF AMILORIDE ON AIRWAY TONE. |[utrif]| 2306

Amiloride, an inhibitor of the Na+-H+ antiporter in airway epithelium, has been proposed as nebulized therapy to loosen secretions in patients with cystic fibrosis. Studies with bovine and canine trachea have determined that amiloride produces airway relaxation in vitro, suggesting that it may also have potential benefit as a bronchodilator. However, amiloride has not been shown to relax guinea pig trachea. Since guinea pig trachea is felt to be a useful model of human airway, we hypothesized that human airway response to amiloride would be similar to that of guinea pigs. Newborn guinea pig along with mouse tracheal responses to amiloride were compared to those of human fetal airway. Airway ring segments were constricted with an EC50-75 dose of acetylcholine and changes in isometric force to cumulative additions of amiloride (10-8 to 10-4 M) were recorded. In guinea pig airway with intact epithelium, amiloride produced contraction, increasing tension at maximum by 26±4%(n=16). Removal of epithelium did not alter this response (41±10%, n=8, p=0.12). Mouse trachea also responded with an increase in tension(10±6%, n=4). Likewise, human fetal airway tone increased by 14±9% [n=6 from 2 fetuses (16 wk gestation)]. In summary, unlike adult bovine and canine trachea, human fetal airway contracted upon exposure to amiloride. Guinea pig and mouse airways also contracted, and this contraction was independent of epithelium. These results, along with previous work employing furosemide, support the notion that guinea pigs and mice are useful paradigms in the study of ion transporters in the regulation of human airway tone. Funded by: US Army HSC, CRC, and NIH HL-45220.