Bronchial Preparations Research Articles

P.2.08 Effects of the serotonin 2C receptor ligands in the nicotine-evoked conditioned hyperlocomotion and sensitization

The mode of action of nicotine was studied in a rabbit bronchial preparation. Nicotine (3 × 10−5 − 10−3 M) produced a phasic contraction. No inhibitory response to nicotine was observed. The contractile response to nicotine was inhibited by hexamethonium, pentolinium and atropine but not by tetrodotoxin. Nicotine increased the efflux of tritium from preparations which had been labelled with [3H]choline. Tetrodotoxin did not inhibit the nicotine-evoked tritium release from the bronchial preparation. The results indicate that the release of acetylcholine evoked by nicotine was not influenced by tetrodotoxin in this preparation, and that the nicotine-induced response may be produced mainly through a sodium action potential-independent process. We could not rule out a contribution by acetylcholine released from the acetylcholine store in the smooth muscle in the contractile mechanisms for nicotine.

Ion and Fluid Transport Properties of Small Airways in Cystic Fibrosis

Small airways constitute a major site of pathology in cystic fibrosis (CF) and provide most of the surface area of the conducting airways of the lung. Little is known, however, about the impact of CF on ion and fluid transport in small (bronchiolar) airways. To describe the ion and fluid transport properties of CF bronchiolar epithelium. Primary cultures of human bronchial and bronchiolar (non-CF and CF) epithelial cells were obtained. The bioelectric properties were studied in Ussing chambers and the airway surface liquid (ASL) height was measured with confocal microscopy. Primary cultures of DeltaF508 CF bronchiolar epithelial cells displayed higher transepithelial resistance than non-CF cultures, whereas baseline short circuit current and amiloride-inhibitable short circuit current were similar in both preparations. The ASL height was significantly smaller in CF compared with non-CF preparations. In the presence of amiloride, addition of forskolin increased short circuit current in non-CF but not in CF bronchiolar cultures, and the ATP-induced increase in short circuit current was lower in CF than in non-CF cultures. Non-CF bronchiolar preparations displayed larger short circuit current and fluid secretion in responses to forskolin than non-CF bronchial preparations, suggesting that CFTR-dependent Cl(-) transport may play a more important role in the regulation of fluid transport in small airways than in large airways. In CF small airways, defective Cl(-) secretion combined with unregulated (persistent) Na(+) absorption results in ASLdepletion.

Hyposecretion, Not Hyperabsorption, Is the Basic Defect of Cystic Fibrosis Airway Glands

Human airways and glands express the anion channel cystic fibrosis transmembrane conductance regulator, CFTR, and the epithelial Na(+) channel, ENaC. Cystic fibrosis (CF) airway glands fail to secrete mucus in response to vasoactive intestinal peptide or forskolin; the failure was attributed to loss of CFTR-mediated anion and fluid secretion. Alternatively, CF glands might secrete acinar fluid via CFTR-independent pathways, but the exit of mucus from the glands could be blocked by hyperabsorption of fluid in the gland ducts. This could occur because CFTR loss can disinhibit ENaC, and ENaC activity can drive absorption. To test these two hypotheses, we measured single gland mucus secretion optically and applied ENaC inhibitors to determine whether they augmented secretion. Human CF glands were pretreated with benzamil and then stimulated with forskolin in the continued presence of benzamil. Benzamil did not rescue the lack of secretion to forskolin (50 glands, 6 CF subjects) nor did it increase the rate of cholinergically mediated mucus secretion from CF glands. Finally, neither benzamil nor amiloride increased forskolin-stimulated mucus secretion from porcine submucosal glands (75 glands, 7 pigs). One possible explanation for these results is that ENaC within the gland ducts was not active in our experiments. Consistent with that possibility, we discovered that human airway glands express Kunitz-type and non-Kunitz serine protease inhibitors, which might prevent proteolytic activation of ENaC. Our results suggest that CF glands do not display excessive, ENaC-mediated fluid absorption, leaving defective, anion-mediated fluid secretion as the most likely mechanism for defective mucus secretion from CF glands.

INTESTINAL ISCHEMIA/REPERFUSION INDUCES BRONCHIAL HYPERREACTIVITY AND INCREASES SERUM TNF-α IN RATS

Intestinal or hepatic ischemia/reperfusion induces acute lung injury in animal models of multiple organ failure. Tumor necrosis factor (TNF)- alpha is involved in the underlying inflammatory mechanism of acute respiratory distress syndrome. Although the inflammatory cascade leading to acute respiratory distress syndrome has been extensively investigated, the mechanical components of acute respiratory distress syndrome are not fully understood. Our hypothesis is that splanchnic ischemia/reperfusion increases airway reactivity and serum TNF-alpha levels. To assess bronchial smooth muscle reactivity under methacholine stimulation, and to measure serum TNF-alpha levels following intestinal and/or hepatic ischemia/reperfusion in rats. Rats were subjected to 45 minutes of intestinal ischemia, or 20 minutes of hepatic ischemia, or to both (double ischemia), or sham procedures (control), followed by 120 minutes of reperfusion. The animals were then sacrificed, and the bronchial response to increasing methacholine molar concentrations (10(-7) to 3 x 10(-4)) was evaluated in an ex-vivo bronchial muscle preparation. Serum TNF-alpha was determined by the L929-cell bioassay. Bronchial response (g/100 mg tissue) showed increased reactivity to increasing methacholine concentrations in the intestinal ischemia and double ischemia groups, but not in the hepatic ischemia group. Similarly, serum TNF-alpha (pg/mL) concentration was increased in the intestinal ischemia and double ischemia groups, but not in the hepatic ischemia group. Intestinal ischemia, either isolated or associated with hepatic ischemia, increased bronchial smooth muscle reactivity, suggesting a possible role for bronchial constriction in respiratory dysfunction following splanchnic ischemia/reperfusion. This increase occurred in concomitance with serum TNF-alpha increase, but whether the increase in TNF-alpha caused this bronchial contractility remains to be determined.

Augmentation of Endothelin-1-Induced Phosphorylation of CPI-17 and Myosin Light Chain in Bronchial Smooth Muscle from Airway Hyperresponsive Rats

Airway hyperresponsiveness (AHR) associated with heightened airway resistance and inflammation is a characteristic feature of bronchial asthma. It has been demonstrated that contraclile responsiveness to endothelin-1 (ET-1) in repeated antigen challenge-induced airway hyperresponsive bronchial preparation was significantly increased. ET-1 is a potent contracting substance for various smooth muscles including airways. In addition to the classical Ca(2+)-mediated contraction, ET-1 also induced Ca(2+) sensitization of contraction. However, it is not clear whether ET-1 stimulation also activates the CPI-17 (PKC-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa) pathway in airway smooth muscles. Therefore, the changes in ET-1-induced activation/phosphorylation of CPI-17 and myosin light chain (MLC) in bronchial smooth muscle of repeatedly antigen-challenged rats were examined. The levels of ET-1-induced phosphorylation of CPI-17 and MLC were increased much more markedly in the AHR group than in the sensitized control animals. It might be suggested that the augmented activation of CPI-17 observed in the hyperresponsive bronchial smooth muscle is responsible for the enhanced agonists-induced contraction of bronchial smooth muscle in AHR rats.

Effect of cold storage on cholinergic responses induced by electrical field stimulation in human bronchi

The aim of the present study was to examine the effects of cold storage on the responses induced by electrical field stimulation (EFS) in human bronchial preparations. Responses induced by EFS and acetylcholine were studied in human bronchial rings mounted in organ baths, either on the day of surgery or after storage at 4 °C in Krebs–Henseleit solution for 24 and 48 h, respectively. The responses induced by EFS were studied at different voltages (20, 40 and 60 V) and at a range of frequencies (2, 4, 8, 10, 30 and 60 Hz). EFS induced a triphasic response, consisting of a cholinergic contraction, followed by a relaxation and subsequently a slow sustained contraction. The amplitude of the EFS-induced response was enhanced with increasing voltages and increasing frequencies. None of the three EFS-induced phases were significantly altered by cold storage at 24 h, whereas storage for 48 h significantly decreased the reactivity of the preparations. Likewise, the contractions induced by acetylcholine were unaltered after 24 h, but significantly depressed after 48 h. These results suggest that the reactivity of human bronchial preparations to EFS is not altered when tissues are conserved for 24 h, whereas prolonged storage should be avoided.

Possible Involvement of CPI-17 in Augmented Bronchial Smooth Muscle Contraction in Antigen-Induced Airway Hyper-Responsive Rats

Airway hyper-responsiveness (AHR) associated with heightened airway resistance and inflammation is a characteristic feature of asthma. It has been demonstrated that contractile responsiveness and Ca(2+) sensitization to acetylcholine (ACh) in repeated antigen challenge-induced airway hyper-responsive bronchial preparation were significantly increased. The CPI-17 (PKC-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa) is activated by protein kinase C and acts on a myosin light-chain phosphatase-specific target. The aim of the present study was to explore the role of CPI-17 in hyper-responsiveness of bronchial smooth muscle in antigen-induced AHR rats. In immunoblotting, the levels of expression of CPI-17 mRNA and protein were significantly increased in bronchus from rats that were repeatedly challenged with antigen. ACh-induced CPI-17 phosphorylation and translocation to membrane fraction were also significantly increased in bronchus from antigen-challenged rats. In conclusion, we suggest that augmented expression and activation of CPI-17 observed in the hyper-responsive bronchial smooth muscle might be responsible for the enhanced ACh-induced Ca(2+) sensitization of bronchial smooth muscle contraction associated with AHR.

Acetylcholine-induced phosphorylation of CPI-17 in rat bronchial smooth muscle: the roles of Rho-kinase and protein kinase C

It has been demonstrated that CPI-17 provokes an inhibition of myosin light chain phosphatase to increase myosin light chain phosphorylaton and Ca(2+) sensitivity during contraction of vascular smooth muscle. However, expression and agonist-mediated regulation of CPI-17 in bronchial smooth muscle have not been documented. Thus, expression and phosphorylation of CPI-17 mediated by PKC and ROCK were investigated using rat bronchial preparations. Acetylcholine (ACh)-induced contraction and Ca(2+) sensitization were both attenuated by 10(-6) mol Y-27632 /L, a ROCK inhibitor, 10(-6) mol calphostin C/L, a PKC inhibitor, and their combination. A PKC activator, PDBu, induced a Ca(2+) sensitization in alpha-toxin-permeabilized bronchial smooth muscle. In this case, the Ca(2+) sensitizing effect was significantly inhibited by caphostin C but not by Y-27632. An immunoblot study demonstrated CPI-17 expression in the rat bronchial smooth muscle. Acetylcholine induced a phosphorylation of CPI-17 in a concentration-dependent manner, which was significantly inhibited by Y-27632 and calphostin C. In conclusion, these data suggest that both PKC and ROCK are involved in force development, Ca(2+) sensitization, and CPI-17 phosphorylation induced by ACh stimulation in rat bronchial smooth muscle. As such, RhoA/ROCK, PKC/CPI-17, and RhoA/ROCK/CPI pathways may play important roles in the ACh-induced Ca(2+) sensitization of bronchial smooth muscle contraction.

IL-4 and IL-13 induce fibroblast-myofibroblast differentiation

RATIONALE: Chronic asthmatic inflammation is accompanied by airway remodeling. Increasing number of studies suggest an important role in this process for the fibroblast-myofibroblast differentiation. Our aim was to compare the effects of IL-4 and IL-13 on human lung fibroblasts (HLF) with tracheal (TSM) and bronchial (BSM) smooth muscle preparations.

Cholinesterase activity in human pulmonary arteries and veins: correlation with mRNA levels

Isolated intact human pulmonary arteries and veins were used to determine the acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) activities in the absence or presence of two selective cholinesterase (ChE) inhibitors, iso-OMPA or BW284c51, respectively. These results were compared with the mRNA levels for each enzyme in human pulmonary vessels. Total ChE activities measured in presence of acetylthiocholine (ACTI, 1 mM) in intact vascular preparations were 45 ± 04 and 114 ± 07 mU/g tissue in human pulmonary arteries (n = 14) and veins (n = 14), respectively. These activities were completely abolished in presence of 10 μM neostigmine. In both types of vessels AChE and BChE activities were observed. These activities were at least 2-fold higher in human pulmonary veins when compared with arteries and were correlated with the accumulation of the corresponding transcripts (n = 8). In each type of vessel, similar total ChE activities were detected in homogenized and intact preparations, while in human bronchial preparations this activity was 5-fold higher in homogenates than in intact preparations. Together these results provide evidence that the ChE activities in human pulmonary vessels may be extracellular and that the higher activity measured in veins as compared to arteries was associated with the differential accumulation of the corresponding transcripts.

Epithelium-dependent and -independent inhibitory effects of sivelestat, a neutrophil elastase inhibitor, on substance P-induced contraction of airway smooth muscle in lipopolysaccharide-treated guinea-pigs

The underlying mechanism involved in the interaction between neutrophil elastase inhibitors and tachykinins has not been elucidated. In this study we have examined the effects of sivelestat, a neutrophil elastase inhibitor, on the in vitro responses of airways from lipopolysaccharide (LPS)-untreated or -treated guinea-pigs to substance P. Substance P (0.01-30 micromol/l) produced concentration-dependent contractions of both tracheal and bronchial ring preparations of LPS-untreated or -treated guinea-pigs. Responsiveness to substance P in these isolated airway preparations was augmented by either epithelium removal or LPS treatment. In epithelium-intact tracheal ring preparations isolated from LPS-untreated guinea-pigs, sivelestat (100 micromol/l) significantly inhibited substance P-induced contractions. The inhibitory action was markedly attenuated by pretreatment with L-NAME (100 micromol/l) or indomethacin (2 micromol/l), and was almost undetected following removal of the epithelium. On the other hand, in bronchial ring preparations isolated from LPS-untreated guinea-pigs, sivelestat had only a very slight effect on substance P-induced contraction of the epithelium-intact preparation, whereas sivelestat greatly inhibited contraction in epithelium-removed bronchial ring preparations. In LPS-treated guinea-pigs, whether the epithelium was intact or not, sivelestat significantly inhibited the substance P-induced contraction of bronchial ring preparations. Pretreatment with L-NAME (100 micromol/l) or indomethacin (2 micromol/l) did not affect the inhibitory effect of sivelestat in bronchial ring preparations. In conclusion, epithelium removal or LPS treatment induced hyperreactivity to substance P in the guinea-pig airway. Sivelestat caused epithelium-, nitric oxide- and prostaglandin-dependent inhibition of the substance P-induced contraction of isolated guinea-pig tracheal ring preparations. In contrast, the inhibitory effect of sivelestat on substance P-induced contraction of guinea-pig bronchial ring preparations is mediated by epithelium-, nitric oxide- and prostaglandin-independent mechanisms. Sivelestat may be effective in reducing the airway hyperresponsiveness to tachykinins induced by epithelial injury as occurs in LPS-mediated inflammatory lung diseases.

Decreased sensory neuropeptide release in isolated bronchi of rats with cisplatin-induced neuropathy

We studied if attenuated neurogenic bronchoconstriction was associated with a change in sensory neuropeptide release in preparations from rats with cisplatin-induced neuropathy. Electrical field stimulation (100 stimuli, 20 V, 0.1 ms, 20 Hz) induced an increase in the release of somatostatin, calcitonin gene-related peptide (CGRP) and substance P determined by radioimmunoassay from baseline 0.18±0.01, 0.17±0.01 and 0.86±0.02, to 0.59±0.02, 1.77±0.04 and 5.96 fmol/mg wet tissue weight, respectively, in organ fluid of tracheal tubes from rats. This was significantly attenuated to post-stimulation values of 0.36±0.02, 0.45±0.02, 4.68±0.24 fmol/mg wet tissue weight for somatostatin, CGRP, and substance P, respectively, with a significant decrease in field stimulation-induced contraction of bronchial preparations from animals 11 days after a 5-day treatment period with cisplatin (1.5 mg/kg i.p. once a day). The cisplatin-treated animals developed sensory neuropathy characterized by a 40% decrease in femoral nerve conduction velocity. The results show that a decrease in tracheo-bronchial sensory neuropeptide release associates with feeble bronchomotor responses in rats with cisplatin-induced sensory neuropathy.

Comparison of molecular abnormalities in bronchial brushings and tumor touch preparations

Preneoplastic lung lesions and early-stage lung carcinomas are associated with molecular abnormalities. The authors performed a pilot study to evaluate the use of DNA fluorescence in situ hybridization (FISH) probes to ascertain whether these biomarkers can predict nonsmall cell lung carcinoma (NSCLC). Fourteen bronchial brushings ipsilateral to the tumor (BB/Ts), tumor touch imprints, and touch imprints of the bronchus adjacent to the tumor obtained from 15 patients with early-stage NSCLC were analyzed. The LAVysion multicolor probe set consisting of probes to 5p15, 6, 7p12, and 8q2 and the in-house probes 3p22.1 and 10q22 was used. Using the LAVysion multicolor probe set, 25 epithelial cells were counted and considered positive if > 5 cells were abnormal. Using 3p22.1 and 10q22, > or = 100 nuclei per slide were scored. The results were tabulated as the percentage of cells with deletions compared with the centromeric probes 3 and 10. Greater than 2% of the deletions were positive for 3p22.1 and 10q22. Bronchial washings from patients without lung tumors were used as controls. The BB/Ts were negative for malignant cells by cytologic evaluation and the LAVysion probe set; however, the combined in-house probes for 3p22.1 and 10q22 tested on BB/Ts predicted cancer in 100% of cancer patients. FISH positivity in the lung cancers was 100% for 3p22.1 deletions, 79% for 10q22 deletions, and 57% for LAVysion probes. When compared with the bronchial epithelium, tumor cells showed a 3.7-fold excess of 3p22.1 deletions, a 2-fold excess of 10q22 deletions, and a 12.6-fold excess of abnormal cells. The current study indicated that detection of molecular abnormalities in bronchial epithelial cells via FISH was very useful in identifying patients at high risk for developing lung carcinoma. The molecular abnormalities identified in the BB/Ts were detected at elevated levels in the tumor specimens.

KCl evokes contraction of airway smooth muscle via activation of RhoA and Rho-kinase.

Airway smooth muscle (ASM) cells express voltage-dependent Ca2+ channels, primarily of the L-subtype. These may play a role in excitation-contraction coupling of ASM, although other signaling pathways may also contribute: one of these includes Rho and its downstream effector molecule Rho-associated kinase (ROCK). Although voltage-dependent Ca2+ influx and Rho/ROCK signaling have traditionally been viewed as entirely separate pathways, recent evidence in vascular smooth muscle suggest differently. In this study, we monitored contractile activity (muscle baths) in bronchial and/or tracheal preparations from the pig, cow, and human, and further examined Rho and ROCK activities (Western blots and kinase assays) and cytosolic levels of Ca2+ (fluo 4-based fluorimetry) in porcine tracheal myocytes. KCl evoked substantial contractions that were suppressed in tracheal preparations by removal of external Ca2+ or using the selective L-type Ca2+ channel blocker nifedipine; porcine bronchial preparations were much less sensitive, and bovine bronchi were essentially unaffected by 1 microM nifedipine. Surprisingly, KCl-evoked contractions were also highly sensitive to two structurally different ROCK inhibitors: Y-27632 and HA-1077. Furthermore, the inhibitory effects of nifedipine and of the ROCK inhibitors were not additive. KCl also caused marked stimulation of Rho and ROCK activities, and both these changes were suppressed by nifedipine or by removal of external Ca2+. KCl-induced elevation of [Ca2+]i was not affected by Y-27632 but was reversed by NiCl2 or by BAPTA-AM. We conclude that KCl acts in part through stimulation of Rho and ROCK, possibly secondary to voltage-dependent Ca2+ influx.

Growth factor-induced contraction of human bronchial smooth muscle is Rho-kinase-dependent

Growth factors have been implicated in the pathophysiology of asthma. However, the putative effects of these growth factors on human airway smooth muscle tone are still largely unknown. We performed contraction experiments using human bronchial smooth muscle ring preparations. The growth factor insulin-like growth factor-1 (IGF-1) induced a slowly developing sustained contraction, which was dependent on Rho-kinase, since contraction was almost completely inhibited by (+)-( R)- trans-4-(1-aminoethyl)- N-(4-pyridyl) cyclohexane carboxamide (Y-27632; 1 μM). Angiotensin II, a G q-coupled receptor agonist which can act as a growth factor as well, induced a biphasic contraction, the sustained phase of which was also almost completely inhibited by Y-27632. We conclude that angiotensin II and IGF-1 induce a Rho-kinase-dependent sustained contraction of human bronchial smooth muscle. Since growth factors are associated with pathophysiologiocal conditions such as asthma, inhibition of Rho-kinase could be effective under these conditions.

TNF-alpha augments the expression of RhoA in the rat bronchus.

While nonspecific airway hyperresponsiveness (AHR) is a central feature of allergic bronchial asthma, the mechanism underlying the development of AHR is not clearly understood. We have previously demonstrated in vitro hyperresponsiveness of bronchial smooth muscle to acetylcholine (ACh) in rats that were actively sensitized and repeatedly challenged with aerosolized antigen. It has also been demonstrated that the ACh-induced, RhoA-mediated Ca(2+) sensitization is markedly augmented concomitantly with an increased expression and activation of RhoA protein in the bronchial smooth muscle of the antigen-treated rats. In the present study, we have investigated whether TNF-alpha, a proinflammatory cytokine which is involved in bronchial asthma, causes upregulation of RhoA mRNA and protein in the rat bronchus. Treatment of rat bronchial smooth muscle preparations with TNF-alpha (300 ng/ml for 24 hr) significantly shifted the concentration-response curve to ACh upwards, but did not alter the response to high K(+), when compared to that of control tissues. Levels of RhoA mRNA and protein in the TNF-alpha-treated bronchus were significantly greater than those in the control group. In conclusion, it is suggested that the augmentation of the ACh-induced contractile response evoked by TNF-alpha might be mediated by an upregulation of RhoA in rat bronchial smooth muscle.

Functional identification of epithelial and smooth muscle histamine-dependent relaxing mechanisms in the bovine trachea, but not in bronchi

Theoretically, the overall effect of histamine on respiratory smooth muscle is the result of a subtle balance of contraction and relaxation. The aim of the study was to identify histamine type 2 (H2) and 3 (H3) receptor-dependent relaxing mechanisms in the contractile elements of the bovine tracheobronchial tree. In bronchial preparations, histamine induced very weak contractions, which were not exacerbated with the H2-antagonist cimetidine. Moreover, precontracted bronchial rings never relaxed in response to cumulative doses of histamine or amthamine (H2-agonist). In intact tracheal preparations, histamine induced strong contractions that were exacerbated by cimetidine ( E max: +17.2±6.6%) but not by thioperamide (H3-antagonist). Precontracted tracheal bundles did not relax in response to cumulative doses of the H3-agonist R-α-methylhistamine. The tracheal contractile response was higher in denuded compared to intact preparations (11.0±1.2 vs. 6.0±1.7 g). Cimetidine effect was dramatically potentiated in denuded tracheal strips (+40.0±11.7%). It is concluded that the weak response of bovine bronchi to histamine is due to a relative scarcity of H1 receptors on bronchial smooth muscle rather than to H2- or H3-dependent relaxation. In the bovine trachea, the smooth muscle possesses relaxing H2 but no H3 receptors. The epithelium exercises a relaxation, which is independent from H2 and H3 receptors.

Cytokine profiles in peripheral blood mononuclear cells and lymph node cells from piglets infected in utero with porcine reproductive and respiratory syndrome virus.

The aim of the present study was to investigate at 2, 4, and 6 weeks after birth cytokine expression by peripheral blood mononuclear cells and bronchial lymph node cells from piglets infected in utero with porcine reproductive and respiratory syndrome virus (PRRSV). Technically, by flow cytometry we were able to measure gamma interferon (gamma-IFN), tumor necrosis factor alpha (TNF-alpha), interleukin-4 (IL-4), and IL-8 levels. In general, we found increases in the percentages of IL-4-, gamma-IFN-, and TNF-alpha-producing lymphocytes in the infected piglets compared to the percentages in the uninfected control animals, while there was a decrease in the percentage of IL-8-producing monocytes. We believe that these findings reflect a general lymphocyte activation stage that is created due to the infection and that occurs in combination with impairment of the monocyte function, possibly due to the ongoing viral replication in these cells. Single-cell bronchial lymph node preparations exhibited very much the same cytokine profiles as peripheral blood mononuclear cells except for a lack of IL-8 production. When the levels of the individual cytokines in the three groups of PRRSV-infected piglets were compared, the levels of cytokine expression at 4 weeks diverged from those at 2 and 6 weeks, in that there was a significant decrease in the numbers of lymphocytes producing gamma-IFN and TNF-alpha. This tendency was also observed among blood monocytes and lymph node macrophages. Possible reasons for this temporary immunosuppression in the piglets at 4 weeks are discussed.

Modulation of calcium homeostasis as a mechanism for altering smooth muscle responsiveness in asthma.

Airway hyperresponsiveness remains a defining characteristic of asthma. Traditional views assert that airway smooth muscle is an important structural effector cell in the bronchi that modulates bronchomotor tone induced by contractile agonists. New evidence, however, suggests that abnormalities in airway smooth muscle functions, induced by variety of extracellular stimuli, may play an important role in the development of airway hyperresponsiveness. Studies using isolated bronchial preparations or cultured cells show that inflammatory mediators and cytokines may alter calcium homeostasis in airway smooth muscle and render the cells nonspecifically hyperreactive to agonists.

L-CARNITINE DOES NOT EXERT ANY IN VITRO RELAXANT EFFECT IN GUINEA PIG TRACHEA, LUNG PARENCHYMA AND HUMAN BRONCHIAL TISSUE

The aim of the present study was to investigate the probable in vitro relaxant effect of carnitine in guinea pig trachea, guinea pig lung parenchymal strips, and human bronchial tissue. It was suggested by an in vivo study that carnitine pretreatment prevented the subclinic bronchospasm in children who underwent chronic hemodialysis. Tracheal and lung parenchymal preparations of 10 guinea pigs and 5 human bronchial tissues were prepared and mounted in 20-mL organ baths. In the first series of experiments, contractions to carbachol and histamine (10 -9 to 10 -3 M) were compared after the tissues were incubated with different concentrations of L-carnitine (10 -6 to 10 -4 M). pD 2 values were compared with analysis of variance (ANOVA) and P <. 05 was considered as significant. In the second part of experiments, the inhibitory effect of L-carnitine (10 -9 to 10 -3 M) was investigated on the sustained contractions of preparations to carbachol (10 -6 M) and histamine (10 -5 M). In the first part of the study pD 2 values obtained with carbachol were 6.48 ± 0.09, 5.42 ± 0.05, and 6.48 ± 0.02 for guinea pig trachea, guinea pig lung parenchymal strips, and human bronchial tissues, respectively. pD 2 values obtained with histamine were 5.34 ± 0.10, 5.74 ± 0.06, and 6.32 ± 0.03 for guinea pig trachea, guinea pig lung parenchymal strips, and human bronchial tissues, respectively. No significant difference was observed between the pD 2 values before and after incubation with carnitine (P >. 05). In the second part of the study, only 10 -4 M L-carnitine exerted an insignificant relaxant effect (6.16% ± 1.22% on carbachol induced contractions and 4.48% ± 0.85% on histamine induced contractions) in guinea pig trachea. Our results show that L-carnitine exerts no in vitro relaxant effect in guinea pig trachea, guinea pig lung parenchymal strips, and human bronchial preparations.