Isolated Rat Trachea Research Articles

Effects of VIP and Related Peptides on Airway Mucus Secretion from Isolated Rat Trachea

Wagner, U., D. Bredenbröker, B. Storm, B. Tackenberg, H-C. Fehmann and P. von Wichert. Effects of VIP and related peptides on airway mucus secretion from isolated rat trachea. Peptides 19(2) 241-245, 1998.—Vasoactive intestinal polypeptide (VIP) is known as an important regulator of airway function. It has been suggested that VIP is involved in the pathogenesis of asthma due to its relaxant effects on smooth muscles. The present study was designed to characterize the effects of the peptides of the VIP family on airway mucus secretion. The peptides VIP, PHI, PACAP-27, PACAP-38, GLP-I, exendin-4, helodermin, helospectin I and helospectin II were investigated using isolated rat trachea. Data show that PACAP-27 is the most potent stimulator of airway mucus secretion (225% stimulation). The rank order of potency was PACAP-27 > VIP > helospectin II > PHI > exendin-4 = helodermin = helospectin I = PACAP-38. The addition of the protease inhibitor thiorphan enhanced the effects of PHI and helodermin, but not of the other peptides. These data show that the peptides of the VIP family stimulate airway mucus secretion differently.

P-670 - Effects of optical isomers of ephedrine and methylephedrine on contractile responses to electrical stimulation and agonists in isolated rat trachea

P-670 - Effects of optical isomers of ephedrine and methylephedrine on contractile responses to electrical stimulation and agonists in isolated rat trachea

Effects of selective and non-selective phosphodiesterase inhibitors on tracheal mucus secretion in the rat

The present study was designed to characterize the effects of unselective and isoenzyme-selective phosphodiesterase inhibitors on airway mucus secretion. The isolated rat trachea was incubated in a modified Ussing chamber. Mucus macromolecules were metabolically labelled with 35S. The inhibitors were applied at the luminal side. The unselective phosphodiesterase inhibitors theophylline, enprofylline and 3-isobutyl-methylxanthine stimulated mucus secretion in a concentration-dependent manner with half-maximum effects (EC 50 values) at 690 μM, 400 μM and 46 μM, respectively. The adenosine antagonist 8-phenyltheophylline did not significantly stimulate mucus output, suggesting a negligible role of adenosine in the cellular mechanisms of mucus secretion. Adenosine itself did not increase radiolabel output. Rolipram, an inhibitor of phosphodiesterase isoenzyme IV, and zardaverine, which inhibits the isoenzymes III and IV, increased potently macromolecule output with EC 50 values of 40 nM and 6 μM, respectively. The selective inhibitors of phosphodiesterase isoenzymes III and V, motapizone and zaprinast, did not influence airway mucus release, suggesting a relatively low activity of isoenzymes III and V in glands of rat trachea. The stimulatory effect of theophylline on airway mucus secretion may contribute to its beneficial action in chronic obstructive airway disease. Our data suggest that this effect is mediated predominantly by phosphodiesterase isoenzyme IV.

Effects of liposome-entrapped platelet-activating factor in the isolated rat trachea

The effects of platelet-activating factor (PAF, 1- O-alkyl-2-acetyl- sn-glycero-3-phosphocholine)-filled liposomes upon rat tracheal rings in vitro were examined. The capture of liposomes by the smooth muscle cells of the isolated tracheal rings as well as the release of their content into the cytoplasm was shown by using Evans blue (5 × 10 −4 M)-loaded liposomes. Administration of PAF (10 −3 M)-filled liposomes contracted the preparations, in contrast with extracellular administration of PAF and control liposomes, which had no effect. Administration during the plateau or pretreatment with liposomes containing BN 52021 (3- t-butylhexahydro-4,7b-trihydroxy-8-methyl-9 H-1,7 a-epoxymethano-1 H,6 aH-cyclopenta[ c]furo(2,3- b)furo[3′,2′:3,4]cyclope ((10 −3 M, a selective PAF receptor antagonist) or heparin (5 × 10 −5 M) blocked this contraction. BN 52021 and heparin, not entrapped in liposomes, had no such effect. Our data suggest an intervention of PAF in the mechanisms of contraction of tracheal smooth muscle, involving a direct or indirect intervention (intracellular receptors for PAF cannot be excluded). At the same time, the rat trachea contraction induced by PAF-loaded liposomes could be linked to the PtdIns(1,4,5)P 3-dependent Ca 2+ channels from the endoplasmic reticulum and/or to the interaction with G proteins, as shown by the blocking effects of heparin-containing liposomes.

Mucosa-dependent release of prostaglandins from isolated rat trachea by muscarine receptor activation

Mucosa-dependent release of prostaglandins from isolated rat trachea by muscarine receptor activation

Characterization of muscarinic receptors involved in tracheal CGRP release.

Application of acetylcholine (ACh) to isolated rat trachea induces an increase in calcitonin gene-related peptide (CGRP) outflow in the perfusates. The elevation of CGRP release by ACh was absent in capsaicin-desensitized preparations, suggesting that the release of peptide is derived from capsaicin-sensitive afferent nerves. ACh-induced release was not altered by hexamethonium, but was significantly attenuated by atropine, indicating involvement of the muscarinic receptor. Effects of three selective muscarinic subtype antagonists, pirenzepine (M1), methoctramine (M2) and 4-DAMP (M3) on ACh-evoked release were examined. The ordering of antagonist potency was: 4-DAMP (ED50 = 14 nM) > pirenzepine (3.8 microM) > methoctramine (> 10 microM). The results suggest that the muscarinic receptor mediating tracheal CGRP release resembles the M3 receptor subtype.

Chymotryptic activity in perfusates of isolated rat trachea: correlation with mucosal and connective tissue mast cell secretion.

Serine proteinases participate in many inflammatory events in the airway. We therefore screened perfusates of isolated rat tracheas for tryptic, elastolytic, and chymotryptic serine proteinases. Only chymotryptic activity, indicated by hydrolysis of the synthetic substrate N-succinylalanylalanylprolylphenylalanyl p-nitroaniline (AAPF), was consistently detected in these perfusates. Basal levels of chymotryptic activity were not increased significantly by electrical field stimulation (EFS) (mean change +/- SEM: -0.05 +/- 0.05 m o.d. units, n = 4) or by 10(-7) M substance P (SP) (+0.04 +/- 0.02 m o.d. units, n = 14). However, the mean change after the stimuli were jointly administered (0.17 +/- 0.06 m o.d. units, n = 12) was significantly greater than control or after EFS (P = 0.01, one-way ANOVA). The SP + EFS-induced chymotryptic activity was inhibited by PMSF, soybean trypsin inhibitor, and chymostatin and was associated with an increase in histamine concentration and immunoreactivity to rat mast cell proteases (RMCP), indicating that the activity is due to mast cell degranulation. However, the activity was not significantly decreased by pretreating rats with systemic compound 48/80. SP + EFS-induced chymotryptic activity peaked rapidly and was associated with modest histamine release and an immediate peak in immunoreactivity to RMCP II, a marker of mucosal mast cells. Immunoreactivity to RMCP I, a marker of connective tissue mast cells, also increased after SP + EFS, but this immunoreactivity was either delayed or more sustained and did not coincide with the peak of chymotryptic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The stimulatory effect of amylin on mucus secretion in isolated rat trachea.

A modified Ussing chamber technique was used to characterize the effects of amylin on rat tracheal mucus secretion. The effects of amylin were studied at both the mucosal and the submucosal side of isolated rat trachea. Applied at the mucosal side, amylin had no direct effect on mucus secretion, but significantly decreased the acetylcholine (1 mM)-induced tachyphylaxia when this substance was presented a second time (first stimulation: 490%; second stimulation: 334%; second stimulation plus amylin: 407%). At the submucosal side, amylin significantly stimulated mucus secretion (controls: 100%; 1 microM amylin: 193%; 100 nM amylin: 170%; 10 nM amylin: 127%; 1 nM amylin: 127%). Together with the recent observation of specific amylin receptors in rat lung membranes, these data suggest a physiological role for amylin in the regulation of tracheal mucus secretion.

Neuropeptide γ‐(l‐9)‐Peptide: A Major Product of the Posttranslational Processing of γ‐Preprotachykinin in Rat Tissues

gamma-Preprotachykinin mRNA is the most abundant tachykinin mRNA in rat tissues, but the pathway of post-translational processing of its translation product is unknown. An antiserum was raised against the synthetic peptide Asp-Ala-Gly-His-Gly-Gln-Ile-Ser-His [neuropeptide gamma-(1-9)-peptide, equivalent to gamma-preprotachykinin-(72-80)-peptide], that showed < 1% reactivity with intact neuropeptide gamma and other tachykinins. Neuropeptide gamma-(1-9)-peptide was detected by radioimmunoassay in relatively high concentrations in extracts of regions of rat brain and gastrointestinal tract. These concentrations correlated with (r = 0.99), but were significantly (p < 0.05) less than, the concentrations of neurokinin A-like immunoreactivity. The neuropeptide gamma-(1-9)-like immunoreactivity in an extract of rat brain was eluted from a reverse-phase HPLC column in a single fraction with the same retention time as synthetic neuropeptide gamma-(1-9)-peptide. The synthetic peptide did not contract or relax isolated rat trachea, superior mesenteric artery, stomach fundus, or ileum, and the peptide did not affect the ability of neuropeptide gamma to contract the rat fundus. It is concluded that, in rat tissues, Lys70-Arg71 in gamma-preprotachykinin is a major site of posttranslational processing, but the resulting product, neuropeptide gamma-(1-9)-peptide, is neither an agonist nor an antagonist at the neurokinin-2 (NK-2) receptor.

Pharmacology of the effects of bradykinin, serotonin, and histamine on the release of calcitonin gene-related peptide from C-fiber terminals in the rat trachea

The effects of inflammatory substances, bradykinin (BK), 5-HT, and histamine (HIS), on the release of calcitonin gene-related peptide (CGRP) from the peripheral terminals of sensory afferents in the rat trachea were examined ex vivo. With intralumenal perfusion, the isolated rat trachea displays low but measurable secretion of CGRP (32 +/- 4.6 fmol/10 min fraction). The addition of BK (10(-6) to 10(-4) M) to the superfusate resulted in an immediate, concentration-dependent increase in the level of CGRP (5-30-fold increase above baseline) in the perfusates, and this effect showed a concentration-dependent tachyphylaxis. [Des-Arg10]-kallidin, a B1 receptor agonist, at concentrations of up to 10(-4) M did not induce any significant increase in CGRP outflow from the rat trachea. HIS at 10(-4) M caused a modest but progressive augmentation in the release of CGRP. 5-HT at 10(-4) M had no effect upon the resting efflux of CGRP, but at a concentration of 10(-6) M significantly enhanced the release of CGRP evoked by capsaicin (10(-6) M). Similar conditioning studies carried out with HIS and BK showed no augmentation. BK-evoked CGRP efflux was significantly inhibited by [D-Arg0, Hyp3, Thi5,8, D-Phe7]-BK (B2 antagonist) and indomethacin. While [Des-Arg9, Leu8]-BK (B1 antagonist) also caused a reduction of BK-induced release, its effect did not reach statistical significance.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of muscarine m3 receptors stimulates arachidonic acid release from epithelium of isolated rat tracheae

Activation of muscarine m3 receptors stimulates arachidonic acid release from epithelium of isolated rat tracheae

Relaxing action of oligopeptides isolated from the tracheal mucosa and lung parenchyma on smooth musculature of the isolated rat trachea

Relaxing action of oligopeptides isolated from the tracheal mucosa and lung parenchyma on smooth musculature of the isolated rat trachea

Muscarinic stimulation of prostanoid synthesis by the isolated rat trachea: calcium dependency and effect of cortisol and cigarette smoke

Tracheal prostanoid synthesis was stimulated by parasympathomimetics: arecoline > carbachol = methacholine > acetylcholine ⪢ arecaidine. McNA343, dimethyl phenyl piperazinium (DMPP), nicotine, potassium and isoprenaline were without effect. Prostanoid synthesis was also stimulated by Ca 2+ ionophore A23187 and arachidonic acid (AA). Carbachol-stimulated prostanoid synthesis was inhibited antagonists (atropin > ipratropium bromide ⪢ gallamine > pirenzepine); adrenaline and isoprenaline were without effect. Carbachol-stimulated prostanoid synthesis was also inhibited by the Ca 2+-channel blockers, nifedipine, diethylstilboestrol and TMB-8. Hydrocortisone and betamethasone inhibited carbachol- and A23187-stimulated, but not AA-stimulated, prostanoid synthesis following an 18 h tissue culture. Cigarette smoke extracts had a biphasic effect on carbachol-, A23187- and AA-stimulated prostanoid synthesis (potentiation at low concentrations, inhibition at high concentrations of extracts). These data demonstrate (1) that rat tracheal prostanoid synthesis is stimulable by activation of muscarine receptor-linked Ca 2+ mobilisation, and (2) that tracheal prostanoid synthesis may be involved in secretion of mucus, the disruption of which by cigarette smoking may be related to the pathophysiology of airway disease.

Potentiation of isoproterenol-induced relaxation of isolated trachea by aminophylline: modulation by desensitization.

Continued exposure of many beta-adrenoceptor-coupled adenylate cyclase systems to high doses of agonist causes diminished responsiveness, a phenomenon called desensitization. After exposure of isolated guinea pig tracheae to a high concentration of isoproterenol for 30 min, relaxation produced by subsequent challenge by a lower concentration was attenuated, as expected. However, potentiation of isoproterenol-induced relaxation by aminophylline was greater after desensitization as compared to that prior to desensitization. This observation was further investigated using a graphical method that allows quantitative and statistical evaluation of combinations of synergistically acting drugs. Concentration-relaxation curves (CRC) for isoproterenol alone and in the presence of a fixed concentration of aminophylline were determined in isolated rat trachea. A theoretical additive curve was constructed from the data obtained, and the displacement of the isoproterenol CRC from the theoretical additive curve caused by aminophylline in tracheae desensitized by 2.5 hr of exposure to 2 X 10(-5) M isoproterenol (DESN) was compared to that in tracheae equilibrated for a similar period in physiologic salt solution (CON). Desensitization had no significant effect on aminophylline-induced relaxation but caused a marked depression and right-shift of the isoproterenol CRC. In the CON group aminophylline shifted the isoproterenol CRC upward and to the left indicating that the synergistic interaction between the two agents was greater than additive. The left-shift and elevation of the ceiling effect of the isoproterenol CRC caused by aminophylline were significantly greater in the DESN group vs the CON group. These observations from intact tissue are compared with published data from biochemical and broken cell studies. The possibility of increased phosphodiesterase activity as an explanation for the observations reported is discussed.

Effects of PCO2, pH and extracellular calcium on contraction of airway smooth muscle from rats

The effect of changes in PCO2 on airway smooth muscle was studied in acetylcholine-induced contractions of isolated rat trachea. Elevation of superfusate PCO2, 38 mm Hg (pH 7.49), to 168 mm Hg (pH 6.74) decreased tension to 68% of control tension; reduction of PCO2 to 19 mm Hg (pH 7.84) increased tension to 104%. Similar effects on tension occurred when pH was altered by varying superfusate bicarbonate concentration at constant PCO2. Modification of the response to changes in PCO2 by varying extracellular calcium (Ca2+) concentration and also by verapamil indicated that changes in PCO2 and pH may alter Ca2+ uptake by the smooth muscle. Calcium uptake was measured by 45Ca2+ and the lanthanum method. At control pH 7.49, net Ca2+ uptake was 5.34 mmol Ca2+/kg trachea 60 min after the onset of contraction; this decreased to 4.26 at pH 6.88, and increased to 6.58 at pH 7.85. The results suggest that the mechanism whereby chages in PCO2 affect airway smooth muscle contraction is a pH-dependent alteration of Ca2+ uptake.

Adenosine-induced contractions of the isolated rat trachea are mediated by 5-hydroxytryptamine.

Adenosine-induced contractions of the isolated rat trachea are mediated by 5-hydroxytryptamine.

PROCEEDINGS OF THE AUSTRALASIAN SOCIETY OF CLINICAL AND EXPERIMENTAL PHARMACOLOGISTS

PROCEEDINGS OF THE AUSTRALASIAN SOCIETY OF CLINICAL AND EXPERIMENTAL PHARMACOLOGISTS

Effect of Acetylcholine on Desensitization of Isolated Rat Trachea to 5-Hydroxytryptamine—A Possible Allosteric Interaction Between Drug Receptors

It is well known that desensitization of smooth muscle preparations occurs by repeated exposure to a stimulating drug. This is called tachyphylaxis. This paper demonstrates the response of isolated rat tracheal muscle to 5-hydroxytryptamine (5-HT) as being diminished by repeated application in almost every instance and that 5-HT applied in the presence of stimulating drugs caused a further contraction when the preparation did not respond to 5-HT alone. For this reason interaction between 5-HT and other stimulating drugs was studied on the rat tracheal muscle after desensitization had occurred.