Comparison of early and late responses to antigen of sensitized guinea pig parenchymal lung strips.

The peripheral lung parenchyma has been studied as a component of the asthmatic inflammatory response. During induced constriction, tissue resistance increases in different asthma models. Approximately 60% of the asthmatic patients show early and late responses. The late response is characterized by more severe airway obstruction. In the present study, we evaluated lung parenchymal strips mechanics in ovalbumin-sensitized guinea pigs, trying to reproduce both early and late inflammatory responses. Oscillatory mechanics of lung strips were performed in a control group (C), in an early response group (ER), and in two late response groups: 17 h (L1) and 72 h (L2) after the last ovalbumin challenge. Measurements of resistance and elastance were obtained before and after ovalbumin challenge in C and ER groups and before and after acetylcholine challenge in all groups. Using morphometry, we assessed the density of eosinophils and smooth muscle cells, as well as collagen and elastin content in lung strips. The baseline and postagonist values of resistance and elastance were increased in ER, L1, and L2 groups compared with C (P < or = 0.001). The morphometric analysis showed an increase in alveolar eosinophil density in ER and L2 groups compared with C (P < 0.05). There was a significant correlation between eosinophil density in parenchymal strips of C, L1, and L2 groups and values of resistance and elastance postacetylcholine (r = 0.71, P = 0.001 and r = 0.74, P < 0.001, respectively). The results show that the lung parenchyma is involved in the late response, and the constriction response in this phase is related to the eosinophilic inflammation.     

Thermoelastic properties of uniaxially deformed lung strips

We examined the temperature dependence of small degassed hamster lung strip mechanics to develop insights into the molecular basis of lung elasticity. Quasi-static length-tension curves of adapted lung strips were generated at 10, 23, 37, 50, and 80 degrees C; quasi-static tension-temperature plots (QSTT) at strains of 0.5, 0.75, and 1.0 were then formulated. Static tension-temperature (STT) plots at strain 1 were independently generated from other strips. Stress relaxation was evaluated as a function of temperature at different strains; hysteresis ratio was calculated as a parameter of mechanical efficiency. Between 23 and 37 degrees C, the slopes of the QSTT plots at the different strains were close to zero. The slope of the STT plot was slightly positive, indicating that the tension developed by a stretched strip was primarily due to entropic changes with length, suggesting that strips behave like rubber polymers near physiological temperature. Between 10 and 23 degrees C, the slope of the QSTT curve was zero at the two lowest strains but was negative at strain 1; and slope of the STT curve was zero at strain 1. These data indicated that collagen fiber and possibly glycosaminoglycan function was abnormally affected at 10 degrees C. Between 50 and 80 degrees C at strain 1, the slopes of both the QSTT and STT plots at all strains were positive. These data suggested that elastic fiber function was altered between 50 and 80 degrees C such that both internal energetic and entropic contributions to the tension were changed. Stress relaxation and hysteresis data were consistent with these findings.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mechanism of action of leukotrienes C4 and D4 in guinea-pig isolated perfused lung and parenchymal strips of guinea pig, rabbit and rat

The biological actions of pure slow-reacting substance of anaphylaxis (SRS-A) from guinea-pig lung, pure slow-reacting substance (SRS) from rat basophilic leukaemia cells (RBL-1) and synthetic leukotrienes C4 (LTC4) and D4 (LTD4) have been investigated on lung tissue from guinea pig, rabbit and rat. In the guinea pig, the leukotrienes released cyclo-oxygenase products from the perfused lung and contracted strips of parenchyma. The effects of SRS-A, SRS and LTD4 were indistinguishable. LTC4 and LTD4 had similar actions although LTD4 was more potent than LTC4. Indomethacin (1 microgram/ml) inhibited the release of cyclo-oxygenase products from perfused guinea-pig lung and caused a marked reduction in contractions of guinea-pig parenchymal strips (GPP) due to LTC4 and LTD4. The residual contraction of the GPP was abolished by FPL 55712 (0.5 - 1.0 microgram/ml). It appears, therefore, that a major part of the constrictor actions of LTC4 and LTD4 in guinea-pig lung are mediated by myotropic cyclo-oxygenase products, i.e. thromboxane A2 (TxA2) and prostaglandins (PGs). In rabbit and rat lung, however, SRS-A, SRS and the leukotrienes were much less potent in contracting parenchymal strips and there was little evidence of the release of cyclo-oxygenase products. FPL 55712 at a concentration of 1 microgram/ml failed to antagonise leukotriene-induced contractions.     

Dissociation between hysteresivity and tension in constricted tracheal and parenchymal strips

The object of this study was to investigatehow changes in the contractile state of smooth muscle would modifyoscillatory mechanics of tracheal muscle and lung parenchyma duringagonist challenge. Guinea pig tracheal and parenchymal lung strips were suspended in an organ bath. Measurements of length(L) and tension (T) were recordedduring sinusoidal oscillations under baseline conditions and afterchallenge with 1 mM ACh. Measurements were also obtained in stripspretreated with the calmodulin inhibitor calmidazolium (Cmz) orstaurosporine (Stauro), a protein kinase C inhibitor. Elastance (E) andresistance (R) were calculated by fitting changes in T,L, andL/tto the equation of motion. Hysteresivity () was obtained from thefollowing equation: = (R/E)2f,where f is frequency. Finally, maximalunloaded shortening velocity during electrical field stimulation wasmeasured in Cmz-pretreated and control tracheal strips. In trachealstrips, pretreatment with Cmz caused a significant decrease in the  response to ACh challenge and in maximal unloaded shortening velocitymeasured during electrical field stimulation; Stauro decreased the T,E, and R response to ACh. In parenchymal strips, Cmz decreased the  response, whereas Stauro had no effect. These results suggest thatmodifications in the contractile state of the smooth muscle arereflected in changes in the hysteretic behavior and that T and  maybe controlled independently. Second, inasmuch as changes in  weresimilar in parenchymal and tracheal strips, the contractile element isimplicated as the structure responsible for constriction-induced changes in the mechanical behavior of the lung periphery.

     

Postnatal maturation of the parenchymal cell types in the rabbit pineal gland.

An ultrastructural study on the maturation of the parenchymal rabbit pineal cell types from the first postnatal day up to 120 days is presented. Two main cell types are distinguished from the first 24h of postnatal life. Pinealocytes of the types I and II display different developmental degrees. Both immature cell types are arranged in groups. In addition, type II pinealocytes form rosette-like structures. Both cell types progressively become isolated and display cell processes. The nucleus and the cytoplasm of type I pinealocytes are barely electrondense. During the postnatal period, the number of cytoplasmic organelles, cell processes and terminal clubs increase progressively. Terminal clubs are frequently seen near blood vessels. After 30 days, type I pinealocytes show characteristics of adult pinealocytes. However, the maturation of most type I pinealocytes does not complete until the 90th postnatal day. Type II pinealocytes present a fairly electrondense nucleus and cytoplasm. Mature forms can be seen after the 5th postnatal day. During the postnatal period, a close relationship is determined among type II pinealocytes and cell processes and terminal clubs of type I pinealocytes.     

Effects of postnatal maturation on energetics and cross-bridge properties in rat diaphragm.

The effects of maturation on cross-bridge (CB) properties were studied in rat diaphragm strips obtained at postnatal days 3, 10, and 17 and in adults (10-12 wk old). Calculations of muscle energetics and characteristics of CBs were determined from standard Huxley equations. Maturation did not change the curvature of the force-velocity relationship or the peak of mechanical efficiency. There was a significant increase in the total number of CBs per cross-sectional area (m) with aging but not in single CB force. The turnover rate of myosin ATPase increased, the duration of the CB cycle decreased, and the velocity of CBs decreased significantly only after the first week postpartum. There was a linear relationship between maximum total force and m (r = 0.969, P < 0.001), and between maximum unloaded shortening velocity and m (r = 0.728, P < 0.001). When this study in the rat and previous study in the hamster are compared, it appears that there are few species differences in the postnatal maturation process of the diaphragm.     

Changes in distribution of phosphomannosyl receptors during maturation of rat spermatozoa

The aim of the present work was to study the distribution of the cation-independent (CI) and cation-dependent (CD) mannose-6-phosphate receptors (MPRs) in spermatozoa obtained from either rete testis or three regions of rat epididymis. We observed that both receptors underwent changes in distribution as spermatozoa passed from rete testis to cauda epididymis. CI-MPR was concentrated in the dorsal region of the head in rete testis sperm and that this labeling extended to the equatorial segment of epididymal spermatozoa. CD-MPR, however, changed from a dorsal distribution in rete testis, caput, and corpus to a double labeling on the dorsal and ventral regions in cauda spermatozoa. The percentages of spermatozoa that showed staining for either CI-MPR or CD-MPR increased from rete testis to epididymis. The observed changes were probably the result of a redistribution during transit rather than an unmasking of receptors. The fluorescence corresponding to CD-MPR and CI-MPR on the dorsal region disappeared when caudal spermatozoa underwent the acrosomal reaction. Receptors were localized on the plasmalemma of spermatozoa, as observed by immunoelectron microscopy. Changes in distribution may be related to a maturation process, which suggests new roles for the phosphomannosyl receptors.     

Changes in creatine transporter function during cardiac maturation in the rat

Background  

It is well established that the immature myocardium preferentially utilises non-oxidative energy-generating pathways. It exhibits low energy-transfer capacity via the creatine kinase (CK) shuttle, reflected in phosphocreatine (PCr), total creatine and CK levels that are much lower than those of adult myocardium. The mechanisms leading to gradually increasing energy transfer capacity during maturation are poorly understood. Creatine is not synthesised in the heart, but taken up exclusively by the action of the creatine transporter protein (CrT). To determine whether this transporter is ontogenically regulated, the present study serially examined CrT gene expression pattern, together with creatine uptake kinetics and resulting myocardial creatine levels, in rats over the first 80 days of age.     

Changes in ectonucleotidase activities in rat Sertoli cells during sexual maturation

Sertoli cell maturation is a complex process involving both morphological and biochemical changes. These cells have previously been shown to be targets for extracellular purine structures such as ATP and adenosine. These compounds evoke responses in rat Sertoli cells through the purinoceptor families, P₂X and P₂Y and PA₁. The signals to purinoceptors are usually terminated by the action of ectonucleotidases. In a previous work, we demonstrated that rat Sertoli cells have ecto-ATPdiphosphohydrolase (EC 3.6.1.5), ecto-5-nucleotidase (EC 3.1.3.5) and ecto-adenosine deaminase (ecto-ADA) (EC 3.5.4.4) activities. Here we investigated whether some changes occur during rat Sertoli cell maturation in these activities. Rat Sertoli cells obtained from rats of different ages representing the pre pubertal, mid pubertal and young adult (10-, 18- and 35-day-old, respectively) were cultured and used for different assays. The nucleotide hydrolysis was estimated by measuring the Pi released using a colorimetric method and by HPLC analysis. ATP and ADP hydrolysis was increased 3-fold during sexual maturation. AMP hydrolysis increased 4-fold in 10- to 35-day-old Sertoli cells. Similar results were obtained when we used other substrates to measure the extracellular hydrolysis of nucleotides (GTP, GDP, GMP and IMP). The ecto-ADA activity showed a 2-fold increase in the specific activity (18- to 35-day-old Sertoli cells). The termination of the purine cascade by adenosine degradation was faster in the 35- than in 18-day-old Sertoli cells. Follicle Stimulating Hormone (FSH) influences on the ectonucleotidase activities were investigated in 10- and 18-day-old Sertoli cells and a significant increase in the ATP and ADP hydrolysis was observed. Our results show an increase in the extracellular purine cascade during the Sertoli cell development, indicating a rise in the purine communication inside the seminiferous tubules with rat sexual maturation.     

Effects of collagenase and elastase on the mechanical properties of lung tissue strips

The dynamic stiffness (H), dampingcoefficient (G), and harmonic distortion (k_d)characterizing tissue nonlinearity of lung parenchymal strips fromguinea pigs were assessed before and after treatment with elastase orcollagenase between 0.1 and 3.74 Hz. After digestion, data wereobtained both at the same mean length and at the same mean force of thestrip as before digestion. At the same mean length, G and H decreasedby ~33% after elastase and by ~47% after collagenase treatment.At the same mean force, G and H increased by ~7% after elastase andby ~25% after collagenase treatment. The k_dincreased more after collagenase (40%) than after elastase (20%)treatment. These findings suggest that, after digestion, the fractionof intact fibers decreases, which, at the same mean length, leads to adecrease in moduli. At the same mean force, collagen fibers operate ata higher portion of their stress-strain curve, which results in anincrease in moduli. Also, G and H were coupled so that hysteresivity(G/H) did not change after treatments. However,k_d was decoupled from elasticity and wassensitive to stretching of collagen, which may be of value in detectingstructural alterations in the connective tissue of the lung.

     

Genetic testing in diffuse parenchymal lung disease

Background

Mutations in SCO2 cause cytochrome c oxidase deficiency (COX) and a fatal infantile cardioencephalomyopathy. SCO2 encodes a protein involved in COX copper metabolism; supplementation with copper salts rescues the defect in patients?? cells. Bezafibrate (BZF), an approved hypolipidemic agent, ameliorates the COX deficiency in mice with mutations in COX10, another COX-assembly gene.

Methods

We have investigated the effect of BZF and copper in cells with SCO2 mutations using spectrophotometric methods to analyse respiratory chain activities and a luciferase assay to measure ATP production..

Results

Individual mitochondrial enzymes displayed different responses to BZF. COX activity increased by about 40% above basal levels (both in controls and patients), with SCO2 cells reaching 75-80% COX activity compared to untreated controls. The increase in COX was paralleled by an increase in ATP production. The effect was dose-dependent: it was negligible with 100 ??M BZF, and peaked at 400 ??M BZF. Higher BZF concentrations were associated with a relative decline of COX activity, indicating that the therapeutic range of this drug is very narrow. Combined treatment with 100 ??M CuCl₂ and 200 ??M BZF (which are only marginally effective when administered individually) achieved complete rescue of COX activity in SCO2 cells.

Conclusions

These data are crucial to design therapeutic trials for this otherwise fatal disorder. The additive effect of copper and BZF will allow to employ lower doses of each drug and to reduce their potential toxic effects. The exact mechanism of action of BZF remains to be determined.     

Antagonism by ETYA of the effects of leukotrienes on ileum and lung parenchymal strips independent of effects on arachidonic acid metabolism

5,8,11,14-Eicosatetraynoic acid (ETYA), a compound which inhibits both the cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism, antagonized the contraction of segments of guinea-pig ileal longitudinal muscle produced by SRS-A (IC₅₀ = 2.73 μM). This activity was unaffected by pretreatment of the tissues with 10 μM indomethacin. Phenidone, another mixed cyclooxgenese-lipoxygenese inhibitor, was inactive. FPL-55712, an SRS-A antagonist, was a very potent inhibitor (IC₅₀ = 0.011 μM).BW755C and NDGA nonselectively inhibited the contractions of the guinea-pig ileal longitudinal muscle induced by SRS-A or histamine.ETYA antagonized the contraction of the guinea-pig ileal strip produced by 6 nM synthetic LTC₄ (IC₅₀ = 9.3 μM). FPL-55712 demonstrated an IC₅₀ of 0.3 μM in a similar series of experiments. ETYA, 1, 3 or 10 μM did not inhibit the contractions elicited by 0.5 μM of histamine.This was not a tissue-selective effect since 100 μM ETYA antagonized the LTC₄-induced contraction of the guinea-pig lung parenchymal strip preparation.These data demonstrate that ETYA antagonized the contractile effect of the leukotrienes on tissues from the gastrointestinal tract and lung. Furthermore, the inability of indomethacin or phenidone to inhibit the contractile response suggests that antagonism by ETYA may occur by a mechanism independent of cyclooxygenase and lipoxygenase enzymes.     

Effects of oligohydramnios on lung growth and maturation in the fetal rat

Oligohydramnios (OH) retards fetal lung growth by producing less lung distension than normal. To examine effects of decreased distension on fetal lung development, we produced OH in rats by puncture of uterus and fetal membranes at 16 days of gestation; fetuses were delivered at 21 or 22 days of gestation. Controls were position-matched littermates in the opposite uterine horn. OH lungs had lower weights and less DNA, protein, and water, but no differences in saturated phosphatidylcholine, surfactant proteins (SP)-A and -B, and mRNA for SP-A, -B, -C, and -D. To evaluate effects on epithelial differentiation, we used RTI(40) and RTII(70), proteins specific in lung to luminal surfaces of alveolar type I and II cells, respectively. At 22 days of gestation, OH lungs had less RTI(40) mRNA (P < 0.05) and protein (P < 0.001), but RTII(70) did not differ from controls. With OH, type I cells (in proportion to type II cells) covered less distal air space perimeter (P < 0.01). We conclude that OH, which retards lung growth, has little effect on surfactant and impedes formation of type I cells relative to type II cells.