Osmotic regulation of airway reactivity by epithelium

Inhalation of nonisotonic solutions can elicit pulmonary obstruction in asthmatic airways. We evaluated the hypothesis that the respiratory epithelium is involved in responses of the airways to nonisotonic solutions using the guinea pig isolated, perfused trachea preparation to restrict applied agents to the mucosal (intraluminal) or serosal (extraluminal) surface of the airway. In methacholine-contracted tracheae, intraluminally applied NaCl or KCl equipotently caused relaxation that was unaffected by the cyclo-oxygenase inhibitor, indomethacin, but was attenuated by removal of the epithelium and Na+ and Cl- channel blockers. Na+-K+-2Cl- cotransporter and nitric oxide synthase blockers caused a slight inhibition of relaxation, whereas Na+,K+-pump inhibition produced a small potentiation. Intraluminal hyperosmolar KCl and NaCl inhibited contractions in response to intra- or extraluminally applied methacholine, as well as neurogenic cholinergic contractions elicited with electric field stimulation (+/- indomethacin). Extraluminally applied NaCl and KCl elicited epithelium-dependent relaxation (which for KCl was followed by contraction). In contrast to the effects of hyperosmolarity, intraluminal hypo-osmolarity caused papaverine-inhibitable contractions (+/- epithelium). These findings suggest that the epithelium is an osmotic sensor which, through the release of epithelium-derived relaxing factor, can regulate airway diameter by modulating smooth muscle responsiveness and excitatory neurotransmission.     

Hyperosmolar solution effects in guinea pig airways. I. Mechanical responses to relative changes in osmolarity

In the guinea pig isolated perfused trachea contracted with serosal methacholine (MCh), increasing the osmolarity of the mucosal bathing solution elicits relaxation of smooth muscle mediated by epithelium-derived relaxing factor (EpDRF). The present study was undertaken to determine whether a specific modality of the hyperosmolar stimulus induced the relaxation response. Mucosal hyperosmolar challenge with D-mannitol, N-methyl-D-glucamine (NMDG)-chloride, NMDG-gluconate (NMDG-Glu), or urea elicited relaxation with equal potency. In contrast, hyperosmolar solutions at the serosal surface induced diverse, osmolyte-specific responses. In tracheae contracted with MCh, abrupt replacement of the mucosal modified Krebs-Henseleit solution (MKHS) with isosmolar osmolyte solutions to stimulate cell shrinkage elicited five discrete response patterns related to the membrane permeance of the solute, but increasing the osmolarity of the isosmolar solution via the further addition of the same solute always induced relaxation. Similarly, perfusion of the lumen with water induced a transient contraction, but subsequent addition of MKHS, or isosmolar D-mannitol, urea, NMDG-Glu, NaCl, or KCl induced relaxation. Subsequent hyperosmolar addition of the same osmolyte-evoked relaxation. Compatible osmolytes had no effect on smooth muscle tone and did not affect responses to hyperosmolar challenge. The results suggest that the airway epithelium acts as an osmolarity sensor, which communicates with airway smooth muscle through EpDRF. The mechanical responses of the smooth muscle resulting from changes in the osmotic environment are associated with discrete modalities of the osmolar stimulus, including membrane reflection of the particles, incremental change in osmolarity and directionality, but not cell shrinkage.     

Hyperosmolar solution effects in guinea pig airways. IV. Lipopolysaccharide-induced alterations in airway reactivity and epithelial bioelectric responses to methacholine and hyperosmolarity

We investigated the in vivo and in vitro effects of lipopolysaccharide (LPS) treatment (4 mg/kg i.p.) on guinea pig airway smooth muscle reactivity and epithelial bioelectric responses to methacholine (MCh) and hyperosmolarity. Hyperosmolar challenge of the epithelium releases epithelium-derived relaxing factor (EpDRF). Using a two-chamber, whole body plethysmograph 18 h post-treatment, animals treated with LPS were hyporeactive to inhaled MCh aerosol. This could involve an increase in the release and/or actions of EpDRF, because LPS treatment enhanced EpDRF-induced smooth muscle relaxation in vitro in the isolated perfused trachea apparatus. In isolated perfused tracheas the basal transepithelial potential difference (Vt) was increased after LPS treatment. The increase in Vt was inhibited by amiloride and indomethacin. Concentration-response curves for changes in Vt in response to serosally and mucosally applied MCh were biphasic (hyperpolarization, <3 x 10(-7)M; depolarization, >3 x 10(-7)M); MCh was more potent when applied serosally. The hyperpolarization response to MCh, but not the depolarization response, was potentiated after LPS treatment. In both treatment groups, mucosally applied hyperosmolar solution (using added NaCl) depolarized the epithelium; this response was greater in tracheas from LPS-treated animals. The results of this study indicate that airway hyporeactivity in vivo after LPS treatment is accompanied by an increase in the release and/or actions of EpDRF in vitro. These changes may involve LPS-induced bioelectric alterations in the epithelium.     

Hyperosmolar solution effects in guinea pig airways. II. Epithelial bioelectric responses to relative changes in osmolarity

Osmotic challenge of airways alters the bioelectric properties of the airway epithelium and induces the release of factors that modulate smooth muscle tone. Recent studies in our laboratory suggested that methacholine-contracted airways relax in response to incremental increases in osmolarity, rather than from cell shrinkage or absolute solute concentration. In the present study, guinea pig tracheae were mounted in Ussing chambers to elucidate the bioelectric effects of challenge of the epithelium with hyperosmolar and isosmolar solutions. Transepithelial short-circuit current (Isc) across tracheae stimulated with basolateral methacholine was inhibited by apical amiloride, apical 5-nitro-2-(3-phenylpropylamino)benzoic acid, basolateral bumetanide, basolateral ouabain, and Cl(-)-free solution, but not by basolateral iberiotoxin. Apical hyperosmolar challenge with NaCl variably decreased or increased Isc, but D-mannitol (D-M) always inhibited Isc; bumetanide attenuated decreases in Isc. The effects of the transport blockers depended upon whether Isc was initially decreased or increased. Unique concentration-dependent changes in Isc and transepithelial resistance (Rt) were observed when ionic (NaCl and KCl), nonionic impermeant (D-M and sucrose), and nonionic permeant (urea) osmolytes were added to the apical and basolateral baths. At concentrations that doubled the osmolarity of the apical bath, D-M, urea, and N-methyl-D-glucamine-gluconate (NMDG-Glu) decreased Isc. Apical isosmolar NMDG-Glu solution decreased Isc, and additional NMDG-Glu caused a further decrease in Isc. Inclusion of one permeant ion, either Na+,K+, or Cl-, reversed the response to apical isosmolar and hyperosmolar solutions. Thus, bioelectric responses of the airway epithelium to hyperosmolar solution are induced by incremental increases in osmolarity.     

Comparison of drug responses in vivo and in vitro in airways of dogs with and without airway hyperresponsiveness

Basenji-greyhound (BG) dogs demonstrate marked nonspecific airway hyperresponsiveness. To assess the possible contribution of an abnormal sensitivity of airway smooth muscle to this phenomenon, we studied the in vitro contractile responses to methacholine and histamine and the relaxant response to isoproterenol in trachealis muscle from five BG dogs with airway hyperresponsiveness in vivo and from five greyhound dogs that served as a control population. Isoproterenol responses were determined against a half-maximal methacholine contraction. Aerosol methacholine concentrations required to produce a 2-fold increase in pulmonary resistance were 0.07 +/- 0.02 (+/- S.E.) mg/ml in BG dogs and 0.67 +/- 0.26 mg/ml in greyhounds; pD2 values for methacholine-induced contraction of cervical trachealis muscle were 7.03 +/- 0.11 in BG dogs and 7.50 +/- 0.11 in greyhounds. A significant (P less than .01) negative correlation was found between methacholine sensitivity in vivo and in vitro. Aerosol concentrations of histamine required to produce a 2-fold increase in pulmonary resistance were 0.19 +/- 0.06 mg/ml in BG dogs and 1.44 +/- 0.43 mg/ml in greyhounds; pD2 values for histamine were identical in BG dogs (4.95 +/- 0.08) and greyhounds (5.05 +/- 0.19). Isoproterenol pD2 values were less in the trachealis muscle (cervical) of BG dogs (6.76 +/- 0.10) than in that of greyhounds (7.93 +/- 0.16), but this is probably a consequence of the higher concentration of methacholine needed to contract BG muscles. We conclude that the airway hyperresponsiveness of BG dogs does not reflect an increased sensitivity of airway smooth muscle per se.     

Arachidonic acid metabolites and airway epithelium-dependent relaxant factor

Exogenous arachidonic acid (10(-8) to 10(-4) M) contracted epithelium-free guinea pig tracheal strips. Intact tracheal strips were contracted slightly by low concentrations of arachidonic acid (10(-8) to 10(-5) M), but higher concentrations relaxed them. In contrast, when tracheal strips were precontracted with histamine or carbachol, exogenous arachidonic acid had no effect on epithelium-free preparations but induced concentration-dependent (10(-8) to 10(-4) M) relaxation of intact tracheal strips. The effects of arachidonic acid both in epithelium-free and epithelium-containing trachea were blocked by either indomethacin (10(-6) M) or aspirin (10(-4) M). Studies on the effects of exogenous arachidonic acid, performed with a "sandwich protocol," demonstrated that the postulated airway epithelium-dependent relaxant factor released by an intact tracheal strip relaxes an adjacent epithelium-free strip in the same organ bath. This relaxation is antagonized by indomethacin suggesting the involvement of a cyclooxygenase product in this phenomenon. Comparison of concentration-response curves for contractile agonists in epithelium-free preparations and in one containing epithelium suggests the mobilization of airway epithelium-dependent relaxant factor by histamine but not by carbachol. The effects of cyclooxygenase and lipoxygenase inhibitors indicated that both relaxant and contractile arachidonic acid metabolites are generated by epithelial and nonepithelial cells alike in response to contractile agonists.     

Involvement of endothelial cells in relaxation and contraction responses of the aorta to isoproterenol in naive and streptozotocin-induced diabetic rats

Experiments were designed to investigate the importance of the endothelium in the relaxation of isolated rat aorta caused by a beta adrenoceptor agonist. Mechanical removal of the endothelium attenuated the relaxation induced by isoproterenol (ISO) and did not affect the relaxation produced by forskolin and by sodium nitroprusside. High concentrations of ISO produced an increase in the resting tension of aortic strips with and without endothelium in a concentration-dependent manner. Mechanical removal of the endothelium or treatment with methylene blue enhanced the maximal contraction induced by ISO. Phentolamine antagonized the contractile responses induced by ISO. In the case of streptozotocin-induced diabetic rats, both aortic strips with and without endothelium generated concentration-response curves for ISO-induced relaxation that were shifted to the right. The relaxant responses to forskolin and sodium nitroprusside were not significantly different between vessels from diabetic and age-matched control rats. In both aortic strips with and without endothelium, the maximal contraction in response to high concentrations of ISO was significantly enhanced in strips from diabetic rats. These results suggest that ISO-induced relaxation of aortic strips with endothelium is mediated by beta adrenoceptors on both the endothelium and the smooth muscle, and high concentrations of ISO produce an increase in the resting tension through alpha adrenoceptors. It is further suggested that the decreased relaxant response of the aorta to ISO in diabetes may be due to decreased density or affinity of beta adrenoceptors on the smooth muscle.     

Hyperosmolarity-induced dilation and epithelial bioelectric responses of guinea pig trachea in vitro: role of kinase signaling

Exercise-induced airway obstruction is thought to involve evaporative water loss and hyperosmolarity of the airway surface liquid. Hyperosmolar challenge of the epithelium of isolated, perfused guinea pig trachea rapidly alters transepithelial potential difference (V(t)), and it elicits smooth muscle relaxation mediated by epithelium-derived relaxing factor (EpDRF). In many cell types, protein kinases mediate responses to hyperosmolarity and regulatory volume increase. In this study, inhibitors were used to investigate the involvement of kinases and phosphatases in bioelectric responses of epithelium to hyperosmolarity and their possible relationship to EpDRF-mediated relaxation. After contraction of the perfused trachea with extraluminal methacholine, D-mannitol applied intraluminally (< or = 80 mosM) increased V(t) and elicited dilation of the smooth muscle with a similar concentration-dependence; higher concentrations decreased V(t). In tracheas exposed to 30 mosM D-mannitol (approximately EC(50)), 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB 203580) and SKF 86002 [6-(4-fluorophenyl)-2,3-dihydro-5-(4-pyridyl)imidazo[2,1-b]thiazole] (p38 inhibitors) potentiated the dilation, whereas SP 600125 [anthra[1,9-cd]pyrazol-6(2H)-one-1,9-pyrazoloanthrone] and dicumarol [c-Jun NH(2)-terminal kinase (JNK) inhibitors], chelerythrine [nonselective protein kinase C (PKC) inhibitor], and NaAsO(2) (mitogen-activated protein kinase stress inducer) and Na(3)VO(4) (protein tyrosine phosphatase inhibitor) inhibited the hyperpolarization. Large increases in the phosphorylation of p38 and JNK occurred at concentrations higher than those needed to elicit functional responses. The phosphatidylinositol 3-kinase inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002) and Na(3)VO(4) did not affect the V(t) responses, but they inhibited methacholine-induced constriction; SP 600125 and dicumarol potentiated, and chelerythrine inhibited, methacholine-induced epithelial hyperpolarization. These results suggest that JNK, PKC, and phosphatase(s) are involved in hyperosmolarity-induced hyperpolarization of the tracheal epithelium but that p38 is involved in EpDRF-mediated relaxation.     

The alterations of vascular smooth muscle reactivity in vitro by human chorionic gonadotrophin

The study tests the hypothesis that human chorionic gonadotrophin (hCG) alters vascular smooth muscle reactivity by examining the effect of hCG administration on the contractility and relaxation of isolated vascular smooth muscle. Aortic rings from rats pre-treated with intraperitoneal administration of 5,000 I.U of hCG and control animals were contracted to phenylephrine, angiotensin II, CaCl₂ and KCl. The experiments with phenylephrine were repeated with rings that were either de-endothelialized, incubated with L-NMMA, or incubated with calcium ionophore A23187. Aortic rings precontracted with phenylephrine were relaxed to acetylcholine (endothelium-dependent), sodium nitroprusside, hydralazine (endothelium-independent) or in the presence of A23187. The contractile responses of aortic rings from hCG-treated animals to phenylephrine, angiotensin II, CaCl₂ and KCl were significantly attenuated. This effect was not reversed by pre-treatment with L-NMMA or by de-endothelialisation. In aortic rings from hCG-treated animals, there was almost total inhibition of acetylcholine-induced relaxation, but unaltered relaxation responses to sodium nitroprusside and hydralazine. The inhibitory effects of hCG-treatment on both the contraction and relaxation responses were either fully or partially reversed in the presence of calcium ionophore A23187. These observations suggest that hCG attenuates both contractile and endothelium-dependent relaxation responses by a mechanism which involves inhibition of extracellular calcium ion influx and may indicate a new role for the hormone in the altered vascular responses of both normal and abnormal pregnancies.     

Regional differences in relaxation of electric field-stimulated canine airway smooth muscle by verapamil, isoproterenol and prostaglandin E2

Regional differences in contraction produced by methacholine and electric field stimulation (EFS) and in relaxation produced by isoproterenol, prostaglandin E2 and verapamil were studied in isolated canine airway smooth muscle in vitro. Low-frequency EFS (3 Hz, 0.5 msec, 50 V) contracted thoracic trachealis to 43% of maximal EFS response, whereas cervical trachealis contracted to only 14% of maximum. EFS at 10 Hz produced 75% of the maximal response in both regions of the trachea. These EFS responses were abolished by 0.1 microM tetrodotoxin and 1.0 microM atropine. Contraction produced by EFS was also matched in each tissue by contraction with methacholine. The concentrations of methacholine that matched EFS at 10 Hz were 52 +/- 7, 378 +/- 84 and 66 +/- 11 nM for cervical and thoracic trachealis and lobar bronchi, respectively. Both EFS and matched methacholine contractions of cervical trachealis and lobar bronchi were completely relaxed by isoproterenol, whereas thoracic trachealis relaxed maximally to only 60% of induced tone. When verapamil was used to relax EFS and matched methacholine contractions, cervical trachealis was completely relaxed whereas thoracic trachealis relaxed to 15% of induced tone. Although there was a regional difference in the relaxant potency of isoproterenol and, to some extent, verapamil, there was no difference in isoproterenol or verapamil EC50 values for EFS vs. matched methacholine contractions within each region. In contrast, EFS contractions of thoracic trachealis were more sensitive to prostaglandin E2-induced relaxation than were matched methacholine contractions. These data demonstrate marked differences in cholinergic and beta adrenergic receptor-mediated responses between regions of the tracheobronchial tree.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vascular muscarinic receptors: pharmacological characterization in the bovine coronary artery

The goal of this study was to make functional comparisons between muscarinic receptors mediating endothelial-dependent relaxation responses in the rabbit ear artery and receptors mediating endothelial-independent contractile responses. Ring segments of the bovine coronary artery with the endothelium removed proved to be an excellent model for studying the properties of muscarinic receptors mediating vascular smooth muscle contraction. Although endothelial-dependent relaxation responses could be seen with the calcium ionophore A-23187, no relaxation responses to cholinergic agonists were seen in the bovine coronary artery, whether or not the endothelium was present or in the presence or absence of smooth muscle tone. In ring segments of the bovine coronary artery or the rabbit ear artery, the cholinergic agonists, acetylcholine, methacholine and carbachol, proved to be approximately equipotent in evoking contraction or relaxation, respectively. In contrast, the putative M1 selective agonist McN-A-343 did not produce any effect in either tissue; nor did McN-A-343 have any effect on a perfused rabbit ear artery segment. Measurement of antagonist affinities indicated that the bovine coronary artery muscarinic receptors show low affinity for both pirenzepine (pKB = 6.9) and AF-DX 116 (11-2-[[2-[diethylaminomethyl]-1-piperidinyl]acetyl]-5,11- dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one) (pKB = 6.3). Pirenzepine affinity was also low in the perfused rabbit ear artery preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective inhibition of relaxation of guinea-pig trachea by charybdotoxin, a potent Ca(++)-activated K+ channel inhibitor.

Airway smooth muscle plasma membranes are rich in K+ channels of various types. Charybdotoxin (ChTX) is a potent blocker of the high-conductance Ca(++)-activated K+ channel in smooth muscle and produces a concentration-dependent contraction of guinea pig trachea. In the present study, pharmacologic experiments were performed on carbachol-contracted (0.34 microM) guinea-pig trachea contracted further with ChTX in order to determine if Ca(++)-activated K+ channels play a role in the responses to cAMP-dependent and cAMP-independent bronchodilators. Relaxation concentration response curves to the beta-agonists, isoproterenol and salbutamol; the phosphodiesterase inhibitor, aminophylline; the cAMP mimic, N6-2'-O-adenosine 3':5'-cyclic monophosphate the guanylate cyclase activator, sodium nitroprusside; and the K+ channel agonists, BRL-34915 and pinacidil, were obtained in the absence and presence of ChTX. The concentration response curves to isoproterenol and salbutamol were shifted to the right (approximately 27-fold and greater than 40-fold, respectively) by 180 nM ChTX, whereas concentration response curves to N6-2'-O-adenosine 3':5'-cyclic monophosphate and aminophylline were affected significantly less (shifted approximately 7.5-fold). Concentration response curves to the cGMP-dependent relaxant sodium nitroprusside were also altered by ChTX (17-fold rightward shift at 180 nM). In the presence of 60 nM ChTX, the concentration response curves to the above relaxants were shifted only 3- to 5-fold. In contrast, ChTX (60 and 180 nM) failed to produce a significant rightward shift in the concentration response curves to BRL-34915 or pinacidil. Relaxation to BRL-34915 was however, blocked by glybenclamide, suggesting differences in the mechanism of relaxation. Contraction of tissues with depolarizing concentrations of KCl (20-80 mM) inhibited responses to all bronchodilators. These results suggest that hyperpolarization of tracheal smooth muscle as a result of opening various types of K+ channels can lead to relaxation of carbachol-contracted tracheal smooth muscle.     

Functional implication of spare ATP-sensitive K(+) channels in bladder smooth muscle cells

ATP-sensitive K(+) (K(ATP)) channels play important roles in the regulation of excitability in urinary bladder smooth muscle cells. Patch-clamp studies revealed that the current density was about 9-fold higher in the pig bladder smooth muscle cells, compared with guinea pig, although the rank order of potencies for suppression of electrical field-stimulated contraction of bladder strips by K(ATP) channel openers (KCOs) showed a nearly 1:1 correlation between pig and guinea pig. To investigate the existence of spare K(ATP) channels, P1075-evoked current and membrane potential responses were studied in bladder smooth muscle cells. During a 10-min exposure to P1075 (10 microM), K(ATP) currents ran down by approximately 30.5%, whereas membrane hyperpolarization remained constant. P1075 evoked membrane hyperpolarization with an EC(50) value of 0.20 +/- 0.02 microM, comparable to that required for smooth muscle relaxation (EC(50) = 0.11 +/- 0.01 microM). However, these potencies are 6-fold higher than those required for current activation (EC(50) = 0.73 +/- 0.4 microM). These findings demonstrate that the reduction in membrane excitability by KCOs is associated with membrane hyperpolarization, and that a low amount of K(ATP) channel opening is sufficient to suppress bladder smooth muscle contraction.     

M1 receptor-mediated nitric oxide-dependent relaxation unmasked in stomach fundus from M3 receptor knockout mice

Muscarinic receptors can mediate both contractile and relaxant responses in smooth muscle. The stomach fundus from wild-type mice possesses a neuronal M(1) receptor that mediates relaxation to carbamylcholine and (4-hydroxy-2-butynyl)-1-trimethylammonium-3-chlorocarbanilate chloride (McN-A-343) but is masked by M(3) receptor-mediated contraction to both agonists. When the M(3) receptor was deleted, cholinergic-induced relaxation was unmasked. M(1) receptor antagonism with pirenzepine, nitric oxide (NO) synthase inhibition with N(omega)-nitro-L-arginine methyl ester hydrochloride, and inhibition of neuronal activation with tetrodotoxin abolished relaxation to McN-A-343 in tissues from M(3) receptor knockout mice, supporting the neuronal localization of an M(1) receptor that activated NO release to effect relaxation. However, the cyclooxygenase inhibitor indomethacin did not affect contraction or relaxation to carbamylcholine in stomach fundus from wild-type or M(3) receptor knockout mice, indicating that cyclooxygenase products played no role in these responses. The neuronal M(1) receptor modulated relaxation induced by carbamylcholine and McN-A-343 but not relaxation induced by electric field stimulation of the stomach fundus. These data support the presence of M(1) receptor-mediated relaxation in the stomach and suggest that when the M(3) receptor is eliminated or blocked, M(1) receptor-mediated gastric relaxation may be enhanced, possibly leading to alterations in gastric emptying and subsequent effects on body weight.     

Differential inhibitory effects of forskolin, isoproterenol, and dibutyryl cyclic adenosine monophosphate on phosphoinositide hydrolysis in canine tracheal smooth muscle.

A characteristic feature of airway smooth muscle is its relative sensitivity to relaxant effects of beta adrenergic agonists when contracted by inflammatory mediators, such as histamine, vs. resistance to these relaxant effects when contracted by muscarinic agonists. Because contractions presumably depend upon the hydrolysis of membrane phosphoinositides (PI) and the generation of inositol phosphates (IP), our goal was to test for the effects of forskolin, isoproterenol, and dibutyryl cAMP on histamine- vs. methacholine-induced IP accumulation in canine tracheal smooth muscle. Methacholine (10(-3) M) was a more effective stimulant of IP accumulation (9.6 +/- 2.1-fold increase) than equimolar histamine (3.6 +/- 0.5-fold increase) in this tissue. When responses to equieffective methacholine (4 x 10(-6) M) and histamine (10(-3) M) were compared, neither forskolin, isoproterenol, nor dibutyryl cAMP significantly decreased IP accumulation in response to methacholine. In contrast, each of these three agents significantly decreased responses to histamine (by 56 +/- 9, 52 +/- 2, and 61 +/- 2%, respectively). We concluded that, in canine tracheal smooth muscle, increased cAMP is associated with inhibition of PI hydrolysis in response to histamine but not methacholine. The findings suggest a novel mechanism for selective modulation by cAMP of receptor-mediated cellular activation.     

Influence of cartilage on reactivity and on the effectiveness of verapamil in guinea pig isolated airway smooth muscle

The authors have examined the effects of cartilage removal on smooth muscle reactivity and the action of verapamil in guinea pig trachealis. In preparations devoid of cartilage, smooth muscle reactivity to both histamine and KCl was reduced. Reactivity to methacholine was unaffected by cartilage removal. In the absence of cartilage, verapamil had a greater depressant effect on the maximum responses to histamine and methacholine than in intact tissues. Similarly, verapamil was more potent against histamine- and methacholine-induced responses in the absence of cartilage where a greater shift to the right was seen in the concentration-response curves when compared with cartilage-containing controls. The spasmolytic action of verapamil on methacholine-induced responses was greater in the absence of cartilage and was greater than its antispasmogenic activity against methacholine (whether or not cartilage was present). Thus, cartilage removal reduces muscle reactivity and increases the potency of verapamil in guinea pig trachealis.     

Involvement of the neurokinin-2 receptor in airway smooth muscle stretch-activated contractions assessed in perfused intact bovine bronchial segments

The airway response to deep inspirations (DIs) in asthmatics has been shown to be ineffective in producing bronchodilation and can even cause bronchoconstriction. However, the manner by which a DI is able to cause bronchoconstriction remains ambiguous. We sought to investigate the pathway involved in this stretch-activated contraction and whether this contraction is intrinsic to airway smooth muscle (ASM). In brief, intact bovine bronchial segments were dissected, and side branches were ligated and then mounted horizontally in an organ bath. Intraluminal pressure was measured under isovolumic conditions. Instantaneously opening and then closing the tap on a column of fluid 5 to 30 cm high evoked a sudden increase in intraluminal pressure (equivalent to the height of the column of fluid) followed by a stress relaxation response of the ASM. When tissues were stimulated with carbachol (10(-8) M) or serotonin (10(-7) M) for 10 min, and the consequent agonist-evoked pressure response was dissipated manually, the response to the same transmural stretch was accompanied by a slowly developing and prolonged increase in intraluminal pressure. This stretch-activated response was significantly diminished by the stretch-activated cation channel blocker gadolinium (10(-3) M), the L-type Ca2+ channel blockers nifedipine (2 x 10(-6) M), diltiazem (10(-5) M), and verapamil (10(-5) M), the sensory neurotoxin capsaicin (10(-5) M), and the neurokinin (NK)(2) receptor antagonists MEN 10376 ([Tyr(I),d-Trp(6,8,9),Lys(10)]-NKA(4-10)) (10(-5) M) and SR48968 (N-[(2S)-4-(4-acetamido-4-phenylpiperidin-1-yl)-2-(3,4-dichlorophenyl)butyl]-N-methylbenzamide) (3 x 10(-6) M). These results show the ability of isolated airways to exhibit stretch-activated contractions and suggest a role for stretch-activated cation channels, sensory afferent neurons, the neurotransmitter NKA, and L-type Ca(2+) channels in these isolated airway responses.     

Mechanism of relaxation induced by K+ and nicotine in dog duodenal longitudinal muscle.

Dog duodenal longitudinal muscle strips precontracted with bradykinin responded to K+ (10 mM) with a transient relaxation, which was abolished by tetrodotoxin and oxyhemoglobin, but not influenced by atropine, ouabain and apamin. The induced relaxation was suppressed by treatment with 10(-5) M NG-nitro-L-arginine (L-NNA) a nitric oxide synthesis inhibitor, but not by the D-enantiomer. The inhibitory effect was antagonized by L- but not D-arginine. High concentrations (20 mM or higher) of K+ produced a relaxation followed by a sustained contraction; nicardipine abolished the contraction, but did not alter the relaxation. Nicotine produced a contraction, which was converted to a relaxation by atropine. The relaxant response was abolished by tetrodotoxin, hexamethonium and oxyhemoglobin, but was unaffected by timolol and phentolamine. L-NNA suppressed the relaxation, and L-arginine reversed the inhibition. The addition of K+ (20 mM) increased the content of cyclic GMP in the strips, the effect being prevented by tetrodotoxin and L-NNA. These findings suggest that K+ selectively stimulates the nonadrenergic inhibitory nerve, whereas nicotine stimulates both the excitatory cholinergic and inhibitory nerves. Nitric oxide released from the inhibitory nerve appears to transmit information to duodenal smooth muscle by increasing the production of cyclic GMP.     

Endothelium-dependent relaxations in rabbit aorta are depressed in artificial buffered solutions

Contractile and relaxant responses of rabbit aortae in artificial buffered (Tris and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) solutions were compared with those in bicarbonate-buffered solution. EC50 values for responses to KCl in aortae equilibrated in Tris and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid were slightly less than those in bicarbonate; maximum contractions did not differ. There were no significant differences in contractile responses to prostaglandin Fa alpha or histamine in the three buffered solutions. In aortae precontracted with prostaglandin F2 alpha, both acetylcholine and the calcium ionophore, A23187, elicited much less relaxation in artificial buffered solutions than in bicarbonate-buffered solutions. Maximum relaxations to nitroprusside or D600 did not differ among the three solutions. Relaxant responses to isoproterenol were also depressed in artificial buffered solutions. Endothelial damage eliminated relaxant responses to acetylcholine and A23187, as well as the difference in responsiveness to KCl and isoproterenol noted in artificial buffered compared with bicarbonate-buffered solutions. Within the same solution, endothelial damage alone did not alter the responsiveness to KCl or histamine. Total and La -resistant Ca++ binding was similar in all three buffered solutions. Thus, contractile responses are not depressed in Tris- or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered solutions, whereas endothelium-dependent relaxations are attenuated in similar solutions. Overall, the results imply that, in artificial buffered solutions, the production and/or release of an endothelium derived relaxing factor is attenuated or that its action on aortic smooth muscle is, in some manner, functionally antagonized.     

Inhibitory effects of a synthetic atrial peptide on contractions and 45Ca fluxes in vascular smooth muscle

The mechanism of a synthetic atrial peptide (APII)-induced inhibition of smooth muscle contractility was investigated by studying its effects on tension development and 45Ca fluxes in isolated rabbit aorta. APII (10(-9) to 10(-7) M) produced a dose-dependent relaxation of contractions produced by alpha adrenoceptor activation with norepinephrine (NE; 10(-6) M). APII was a potent relaxant of NE contraction with an IC50 = 1.1 X 10(-8) M, with 10(-7) M APII causing a 97% relaxation. APII also produced a dose-dependent inhibition of NE contraction when added to the resting muscle before the exposure to NE. The relaxing effects of APII were found to be endothelium independent. In contrast, APII was only marginally effective in relaxing high-K+ contraction, with 10(-7) M APII causing only 17% relaxation. Furthermore, when a NE contraction was obtained on top of a high-K+ contraction, APII was still capable of relaxing the NE component. APII was similarly more effective in inhibiting NE-stimulated 45Ca influx than high-K+-stimulated 45Ca influx, indicating selective action of APII on the receptor-operated Ca++ channels. This was in contrast to D600, a well known Ca++ antagonist, which had a more selective inhibitory effect on the potential-operated Ca++ channels. The data presented indicate that APII is a potent relaxant of contractions produced by receptor-agonists involving 45Ca influx through receptor-operated Ca++ channels. APII may also prove to be a very useful tool to further distinguish and define receptor-operated Ca++ channels and potential-operated Ca++ channels in vascular smooth muscle.