Nitric oxide is involved in the response of the isolated intact and epithelium-denuded rat trachea to the ß2 adrenergic receptor agonist salbutamol.

BACKGROUND: The involvement of the airway smooth muscle mediator nitric oxide (NO) in the actions of the ß2 agonist salbutamol (Sal), a well-known bronchodilator, is very poorly understood. OBJECTIVES: To determine if endogenous NO release is a major factor in the Sal-induced relaxation of the carbachol- and electrical field-stimulated rat trachea and determine the role of the tracheal epithelium as the possible source of NO involved in these effects. METHODS: Isolated carbachol- or electric field-stimulated pre-contracted in vitro male Sprague Dawley rat tracheas (with epithelium intact or denuded) were relaxed with incremental or discrete concentrations of Sal in the presence and absence of the NO synthesis inhibitor L-NAME. RESULTS: Epithelium-denuded tracheas showed a reduced relaxation response to Sal. L-NAME (1 mM) similarly decreased the sensitivity of the rat tracheas to Sal in both epithelium-intact and -denuded conditions. In the presence of L-NAME, high concentrations of Sal induced an unexpectedly large relaxation response in carbachol-stimulated rat tracheas with both intact and denuded epithelium. Sal relaxation was also affected by L-NAME in electrical field-stimulated epithelium-intact and -denuded tracheas. CONCLUSION: The results suggest that NO derived from sources other than the epithelium is an important mediator of the Sal-induced relaxation in rat tracheas.

Exercise tolerance in a hot and humid climate in heat-acclimatized girls and women.

This study compared physiological responses associated with exercise tolerance in girls (G) and women (W) of similar fitness and heat acclimatization level during exercise in a hot and humid outdoor environment (33.4 degrees C and 55.1 % RH; WBGT = 29.9 +/- 0.2 degrees C). Nine pre-menarcheal G (age = 11.3 yr) and nine W (age = 26.8 yr), matched for aerobic capacity and heat acclimatization level, performed a cycling session at 60 % VO2max until fatigue. A sports drink was provided periodically to prevent dehydration. Tolerance time was not different between the groups (G = 56.9 +/- 6.3, W = 76.5 +/- 9.9 min, p > 0.05). During exercise, sweat rate (G = 9.1 +/- 1.1, W = 12.0 +/- 1.1 ml.m(-2).min(-1)), the increase in rectal temperature [T(re)] (G = 0.9 +/- 0.1, W = 1.1 +/- 0.1 degrees C), and heat storage (G = 10.6 +/- 5.3, W = 20.5 +/- 4.5 W.m(-2)) did not differ between the groups. At fatigue, T(re) (G = 38.2 +/- 0.1, W = 38.4 +/- 0.1 degrees C), heart rate (G = 167.3 +/- 7.3, W = 171 +/- 3.3 beats.min(-1)), stroke index (G = 48.3 +/- 1.5, W = 52.4 +/- 1.8 ml.m(-2)), and forearm skin blood flow (G = 9.5 +/- 1.3, W = 11.7 +/- 1.5 ml.100 ml(- 1).min(-1)) did not differ between the groups. Similar to women, the main reasons reported by girls to stop exercising in the heat were localized leg fatigue and gluteus muscle discomfort. We conclude that heat-acclimatized girls exhibit an adequate cardiovascular and thermoregulatory adjustment while exercising in a hot and humid outdoor environment when hypohydration is prevented.

Effect of low temperature on tracheal smooth muscle contractile and relaxing responses evoked by electrical field stimulation.

OBJECTIVE: The aim of this work was to evaluate the effect of low temperature (LT) on the contractile and relaxing responses of rat tracheas (RTs) after electrical field stimulation (EFS). METHODS: Voltage-dependent (10-60 V, 40 Hz) and frequency-dependent (0.1-60 Hz, 40 V) response curves were constructed at 37 and 18 degrees C after the activation of tracheal intramural nerves with a Grass S88 stimulator. The EFS that produced half of the maximum contractile response (ES50) at 37 or 18 degrees C was determined and considered as the dependent variable. The % relaxation of pre-contracted RTs (EFS; 5 Hz, 40 V) to sodium nitroprusside (SNP; 1 x 10(-7) - 1 x 10(-3) M) isoproterenol (ISP; 1 x 10(-9) - 1 x 10(-5) M) and to 20 mM potassium chloride (KCl) after low-K+ inhibition of the Na+/K+ pump at 18 and 37 degrees C were determined. RESULTS: We found that the tracheal responses elicited by EFS at 37 and 18 degrees C were completely blocked with 1 microM atropine. LT slightly increases the voltage-dependent ES50, from 33.7 +/- 4.0 to 37.8 +/- 4.8 V, n = 5 but decreases the frequency-dependent ES50 from 19.3 +/- 4.3 to 1.0 +/- 0.28 Hz, n = 5, p < 0.05. Relaxing responses to SNP, ISP and KCl at 37 degrees C correspond to 43.5 +/- 6, 36.7 +/- 12 and 12.1 +/- 1.5% respectively. No significant tracheal relaxations were elicited at 18 degrees C. Our results indicate that in RTs, LT enhances tracheal sensitivity to EFS and decreases it in response to bronchorelaxants. The LT-dependent enhanced contractile response is observed only after a low frequency stimulation range (0.1-20 Hz), that is very close to the frequency of vagal stimuli required for inducing bronchoconstriction in vivo. Furthermore, LT abolishes the sensitivity of RTs to exogenously added bronchorelaxants (NO and ISP). In addition, LT appears to decrease the Na(+)-K+ pump activity; this effect has been associated with increased tracheal hyperreactivity in vitro. ACH appears to be the main endogenous neurotransmitter involved on neurally mediated contractile responses at 37 and 18 degrees C. CONCLUSION: Low temperature enhances the contractile response of rat tracheas in response to endogenous ACH release. The effect of LT is limited to frequencies below 20 Hz, which are within the physiological range required for bronchoconstriction. Furthermore, LT severely impairs the sensitivity of RTs to relaxant stimuli, either of endogenous of exogenous origin.

Effect of the airway epithelium on the contraction of rat isolated trachea under conditions of low temperature.

BACKGROUND: The exposure of the airways to low temperature (LT) is a well-known trigger of bronchoconstriction and asthma. However, the mechanisms involved in this response are not well understood. In particular, the specific role of the airway epithelium in the response to LT has not been well established. OBJECTIVE: The purpose of this work is to study the regulatory role of the airway epithelium in the contractile response of the rat trachea under conditions of LT. METHODS: Isolated rat tracheas were stimulated with acetylcholine (ACH) at 37 and 18 degrees C, and dose-response curves were generated in epithelium-intact or epithelium-denuded conditions. EC(50) and E(max) were determined under these conditions. RESULTS: Epithelium denudation at 37 degrees C, increases the tracheal sensitivity to ACH, (EC(50) from 5.9 to 2.6 microM, p = 0. 003). The effects of denudation were even more pronounced at 18 degrees C. E(max) was significantly decreased under these conditions. Exposure of intact tracheas to 18 degrees C also produced a significant increase in tracheal sensitivity (EC(50) from 5.9 to 1.5 microM, p = 0.0004), although no changes in E(max) were noted. CONCLUSIONS: These results strongly suggest that the airway epithelium diminishes the sensitivity of the airway smooth muscle to ACH at 37 and 18 degrees C, and that it is also needed to observe the maximal contractile response under LT conditions. Therefore, the spasmogenic effect of low temperature is significantly potentiated by the absence of airway epithelium.

Chronic imipramine treatment induces downregulation of alpha-2 receptors in rat's locus coeruleus and A2 region of the tractus solitarius.

Imipramine is an effective antidepressant agent that blocks the reuptake of monoamines. In order to understand some of its basic mechanisms of action, we investigated the effects of chronic imipramine administration (10 mg/kg, i.p.; 21 days) on the alpha-2 receptor population of several brain sites. Alpha-2 receptor density was estimated by in vitro autoradiography using [3H]Idazoxan. The densitometric analysis revealed a decreased receptor density in the A2 region of the tractus solitarius (20%) and locus coeruleus (16%). No changes were observed in the amygdala, pyriform cortex, periacueductal gray and the bed nucleus of the stria terminalis. These results suggest that chronic imipramine treatment selectively modulates the alpha-2 receptor population localized in the brain stem norepinephrine-rich nuclei and not in the population present on limbic structures innervated by noradrenergic terminal projections. The possible physiological consequences of this selective modulation of alpha-2 receptors are discussed.

Differential antagonism by amiloride and pirenzepine of the muscarinic receptors of rat tracheal smooth muscle.

Amiloride (AM) is a well known potassium sparing diuretic. The effects of AM at the cellular level include blockade of Na+/H+ exchange in several tissues and inhibition of passive sodium flux in epithelial cells. In this study we have explored the interactions of amiloride with muscarinic receptors, using isolated rat tracheal rings and compared its effects to those of the muscarinic receptor subtype-selective antagonist pirenzepine (PZ). The results obtained demonstrate the ability of AM (100 microM to 1mM) to inhibit the ACh induced rat tracheal contractions. The inhibition resulted in the reduction of the Emax values of ACh in this preparation, and the apparent Ki for AM was of 478 microM. This effect was also observed in a sodium-free choline medium, indicating that it is independent from sodium transport mechanisms sensitive to AM. In contrast to AM, PZ displayed a surmountable type of antagonism with a pA2 value of 6.52. The results demonstrate a differential antagonism by AM and PZ of the muscarinic receptors present in the smooth muscle of the rat trachea.

Role of Na+ and Ca++ in guinea pig trachealis contraction induced by cooling.

The role of extracellular Na+ and Ca++ in cooling-induced contraction (CIC) was studied in guinea pig trachealis muscle. Tracheal preparations were tested in Krebs-Ringer bicarbonate (KRB) solution and then in either: (1) a low Na+ sucrose KRB, (2) KRB plus amiloride, a sodium channel blocker, or (3) KRB plus nifedipine, a calcium channel blocker. When the trachealis muscle was cooled from 37 to 20 degrees C in KRB, the value of the isometric tension increased on average by 25% of the maximal tension induced by acetylcholine; however, when the tissue was tested in the low Na+ sucrose KRB or the KRB containing amiloride, CIC was totally prevented. In the presence of nifedipine the mean value of CIC was only 10% of the acetylcholine-induced contraction, which was also observed not to be affected by extracellular Na+ or Ca++; whereas the KCl-induced contraction was affected by Ca++ but not by Na+. The results suggest that rapid cooling of the smooth muscle cells causes an increase in sodium conductance and a decrease in the activity of the Na+-K+ pump, resulting in membrane depolarization. During depolarization a significant quantity of Ca++ enters the cell, contributing to the magnitude of CIC.

Effects of ethanol on calcium transport across the liver cell plasma membrane.

The effect of ethanol on calcium transport by the liver cell was studied by using a rat liver slice preparation. Ethanol was shown to decrease by about 30% the rate constant for 45Ca efflux from the intracellular compartment. This inhibitory effect of ethanol was not observed in the absence of Ca2+ or Na+ from the incubation medium. Ethanol was also shown to greatly increase non-insulin calcium uptake by liver slices. This effect of ethanol appeared to be dose dependent and was not observed in the absence of Na+ from the incubation medium. The ability of ethanol to increase calcium uptake by the hepatocyte was completely blocked by 1 mM Amiloride. Amiloride, however, did not affect the increased entry of either Na+ or Ca2+ produced by 10 mM Ouabain, a specific inhibitor of the sodium pump. Carbon tetrachloride (CCl4), a well known hepatotoxin, also increased calcium uptake by the hepatocyte. Amiloride, however, was not able to block the CCl4-induced calcium uptake. These results suggest that ethanol activates a Na+ entry pathway, probably represented by a Na+/H+ exchanger, which in turn stimulates an entry of Ca2+ through a Na+/Ca2+ exchange mechanism located in the plasma membrane of the hepatocyte.

Mixed actions of hemicholinium at autonomic receptor sites of the rat vas deferens.

1. The effects of hemicholinium (HC-3) on several autonomic agents in the isolated rat vas deferens were investigated. 2. HC-3 reduced slightly the effects of NE and DA. 3. The responses by cholinergic agents on the M1-ACh receptors were not modified, however HC-3 reduced, significantly, the responses on the M2-ACh receptors. 4. These results suggest that HC-3 besides its anticholinergic properties possesses ability to interact with adrenoceptors in the isolated rat vas deferens.

The Na+/Ca2+ exchange system of the liver cell.

The efflux of 45Ca (Caeff) from rat liver slices incubated in a Krebs Ringer bicarbonate buffer showed two components with first order rate constants (kCa) of 0.0625 min-1 and 0.0101 min-1 respectively. The slow component corresponds to Caeff from the intracellular medium. Ouabain decreased kCa from the slow component (ksCa) by about 30%. External Na+ or Ca2+ did not affect ksCa. However, the inhibitory effect of ouabain was not observed in the absence of either Na+ or Ca2+ from the incubation medium. Concomitant to a decrease in ksCa, ouabain increased 45Ca uptake (Caup) by about 30%. Sodium ionophores also increased Caup, an effect which was not observed in the absence of external Na+. 22Na efflux was inhibited 50% by ouabain. In the presence of ouabain, external Ca2+ was able to significantly increase ksNa from 0.0475 min-1 to 0.0506 min-1. External Na+ dit not affect ksCa from preloaded slices containing high calcium and low sodium when incubated in a Ca2+-free medium. These results would indicate that a Na+/Ca2+ exchange system in operative in the plasma membrane of the hepatocyte. It is estimated that about 60% of the calcium entering the hepatocyte moves in through this exchanger. The evidence obtained also suggests that the Na+/Ca2+ exchanger operates unidirectionally, extruding internal Na+ in exchange for external Ca2+.

The pulmonary ethanol metabolizing system (PET).

Rat lung slices incubated in a Krebs ringer bicarbonate buffer respired at a rate of 0.62 mumoles/g lung X min. Respiration was inhibited by cyanide and stimulated by dinitrophenol. PET was insensitive to the action of pyrazole, cyanide or azide. Lung microsomes metabolized ethanol at rates which were less than 1% of PET. They showed an apparent Km for ethanol of the order of 10 mM and a Vmax of about 7 nmoles acetaldehyde/mg prot X min. Oxidation of ethanol by lung slices to acetaldehyde and CO2 was less than 0.3% and 1.15% than PET, respectively. Chromatographic determination of the products of ethanol metabolism by lung slices showed that a metabolite similar to glucuronic acid was formed. It is concluded that PET is a nonoxidative process which from the evidence presented in this work, would seem to involve the formation of ethyl glucuronide.