Analysis of receptor reserves in canine tracheal smooth muscle

The receptor reserves for acetylcholine, 5-hydroxytryptamine, and histamine in canine tracheal muscle were evaluated. Muscle strips were dissected free of epithelial and connective tissue and suspended for isometric tension recording in modified Krebs-Ringer solution. Dissociation constants for all three agonists were determined by analysis of their concentration-response curves under control conditions and after partial inactivation of receptors by phenoxybenzamine dihydrochloride. The values of KA for acetylcholine, 5-hydroxytryptamine, and histamine were 1.8 X 10(-5) M, 1.35 X 10(-6) M, and 5.0 X 10(-5) M, respectively. Dissociation constants were used to determine receptor occupancy-response relationships. Maximal responses to acetylcholine were obtained by activation of only 4.0 +/- 1.0% of receptors, indicating the presence of a very large receptor-reserve. In contrast, a maximal response to 5-hydroxytryptamine and histamine required activation of 78.0 +/- 11.0 and 87.7 +/- 1.6% of the receptors, respectively, indicating very modest receptor reserves. The differences in receptor-reserve characteristics for these agonists in airway muscle might contribute to the differential effects of inhibitory and facilitory influences on contractions elicited by them.     

Effects of extracellular calcium on canine tracheal smooth muscle

Strips of canine tracheal smooth muscle were studied in vitro to determine the effects of changes in the extracellular calcium (Cao) concentration on tonic contractions induced by acetylcholine and 5-hydroxytryptamine. Strips were contracted with graded concentrations of the above agents in 2.4 mM Ca, after which CaCl2 was administered to achieve final concentrations of 5.0, 10.0, and 20.0 mM. Increases in Cao to 5 mM or above caused significant relaxation of muscles contracted with 5-hydroxytryptamine but did not significantly relax muscles contracted with acetylcholine. Increases in Cao also caused significant relaxation of muscles contracted with low concentrations of K+ (20 or 30 mM). However, in 60 or 120 mM K+, increases in Cao resulted predominantly in muscle contraction. Inhibition of the Na+-K+-ATPase by ouabain (10(-5) M) or K+ depletion reversed the effects of Cao from relaxation to contraction in tissues contracted with 5-hydroxytryptamine. Increases in Cao also caused contraction rather than relaxation in the presence of verapamil (10(-6) M). We conclude that calcium has both excitatory and inhibitory effects on the contractile responses of canine tracheal smooth muscle. The inhibitory effects of Ca2+ appear to be linked to the activity of the membrane Na+-K+-ATPase.     

Interaction of contractile responses in canine tracheal smooth muscle

Concentration-response curves for norepinephrine, acetylcholine, and 5-hydroxytryptamine were obtained in vitro alone and after precontraction with histamine, 5-hydroxytryptamine, or acetylcholine. Responses obtained to each agonist after precontraction were greater than responses to the agonist alone after subtraction of the force due to the precontracting stimulus. Augmentation of responses after precontraction was the greatest for norepinephrine, less for 5-hydroxytryptamine, and least for acetylcholine. Verapamil had no significant effect on the augmentation of responses to either 5-hydroxytryptamine or acetylcholine caused by precontraction. When the efficacy of acetylcholine was decreased by receptor alkylation with phenoxybenzamine, the augmentation of responses to acetylcholine caused by precontraction with histamine was significantly enhanced. Differences in the magnitude of the effect of precontraction on responses to different agonists may reflect differences in their efficiency of stimulus-response coupling in canine tracheal smooth muscle, or they may result from an increased expression of distinct receptors or receptor-mediated effects uncovered by the facilitory stimuli.     

Induction of rhythmic contraction in canine tracheal smooth muscle

Na(+)-K+ ATPase activity of the canine tracheal smooth muscle membrane is responsible for the electrogenic pumping of Na+ and K+ ions. It has been shown that this activity results in muscle relaxation. Based on the results of the current study, we suggest that prolonged electrical stimulation induces increased Na(+)-K+ ATPase activity in isolated tracheal smooth muscle. Tracheal smooth muscle pretreated with prolonged electrical stimulation developed graded mechanical activity when subsequently treated with histamine, serotonin, acetylcholine, or 80 mM K+. This increased isometric tension was interrupted by rhythmic activity, which was elicited by histamine or serotonin but not by acetylcholine or 80 mM K+ stimulation. The spontaneous phasic activity was not inhibited by atropine or propranolol but was totally inhibited by 10(-6) M ouabain. These results suggested that the relaxation phase of rhythmic contraction in response to histamine and serotonin stimulation could be the result of stimulated Na(+)-K+ ATPase activity.     

Endogenous prostaglandins modulate histamine-induced contraction in canine tracheal smooth muscle

We studied the role of endogenous prostaglandins in modulating the histamine response of canine tracheal smooth muscle (TSM) in vitro. Indomethacin (INDO) (10(-7) - 10(-5) M), a cyclooxygenase and prostaglandin synthesis inhibitor, significantly increased maximum histamine-induced tension (Tmax) and decreased the concentration of histamine required to produce 50% of Tmax (EC50). Acetylsalicylic acid (10(-5) -5 X 10(-4) M), another less potent cyclooxygenase inhibitor, also decreased EC50. Neither the lipoxygenase inhibitor nordihydroguaiaretic acid nor the leukotriene antagonist FPL 55712 had any effect on histamine-induced tension in INDO-pretreated TSM. INDO reduced the standard deviation of EC50 from 0.47 in control TSM (n = 51) to 0.26 in INDO-pretreated TSM (n = 31) (P less than 0.02). High-pressure liquid chromatography established prostacyclin (PGI2), through its degradation product 6-oxo-PGF1 alpha, as the predominant prostaglandin produced by canine TSM. Exogenous PGI2 caused a concentration-dependent relaxation of histamine-contracted TSM. In the tissue bath, spontaneous efflux of 6-oxo-PGF1 alpha from TSM, as measured by radioimmunoassay, averaged 4.7 ng . g muscle-1 . min-1 and increased to 10 ng/g muscle (n = 10, P less than 0.001) with administration of histamine. The isometric tension produced by histamine (10(-4) M) was inversely linearly correlated with the log concentration of endogenous 6-oxo-PGF1 alpha (r = 0.81, P less than 0.01). Our results are consistent with an important role for endogenous bronchodilating prostaglandins, probably prostacyclin, in determining both the histamine sensitivity of canine TSM in vitro and its variability among individual animals.     

Effect of Na-K adenosinetriphosphatase activity on relaxation of canine tracheal smooth muscle

The effect of Na-K adenosinetriphosphatase (ATPase) on relaxation induced by isoproterenol, prostaglandin E2, sodium nitroprusside, and forskolin, a specific stimulant of adenylate cyclase, was investigated in canine tracheal smooth muscle strips. Relaxation in response to isoproterenol, prostaglandin E2, and forskolin was significantly decreased after inhibition of the Na-K ATPase by ouabain or a potassium-free medium, but relaxation to sodium nitroprusside was not affected. Relaxation to isoproterenol was greater in muscles contracted by 5-hydroxytryptamine than in those contracted by acetylcholine. The stimulation of Na-K ATPase activity with potassium also caused differences in relaxation between tissues contracted with 5-hydroxytryptamine or acetylcholine. Relaxation caused by isoproterenol by activation of the Na-K-ATPase was also decreased by the Ca2+-channel antagonists, verapamil and diltiazem. The results suggest 1) Na-K ATPase activity modulates relaxation caused by isoproterenol, prostaglandin E2, and forskolin in canine tracheal smooth muscle, 2) isoproterenol or activation of the Na-K ATPase may cause relaxation partly by reducing Ca2+ influx through potential-dependent Ca2+ channels, and 3) the differences in the inhibitory effects of isoproterenol and Na-K ATPase activity on muscles contracted by acetylcholine and 5-hydroxytryptamine could be due to differences between these contractile agents in their dependence on extracellular Ca2+ for activation.     

In vivo recording of electrical activity of canine tracheal smooth muscle.

Electrical activity of the tracheal smooth muscle was studied using extracellular bipolar electrodes in 37 decerebrate, paralyzed, and mechanically ventilated dogs. A spontaneous oscillatory potential that consisted of a slow sinusoidal wave of 0.57 +/- 0.13 (SD) Hz mean frequency but lacked a fast spike component was recorded from 15 dogs. Lung collapse accomplished by bilateral pneumothoraxes evoked or augmented the slow potentials that were associated with an increase in tracheal muscle contraction in 26 dogs. This suggests that the inputs from the airway mechanoreceptors reflexly activate the tracheal smooth muscle cells. Bilateral vagal transection abolished both the spontaneous and the reflexly evoked slow waves and provided relaxation of the tracheal smooth muscle. Electrical stimulation of the distal nerve with a train pulse (0.5 ms, 1-30 Hz) evoked slow-wave oscillatory potentials accompanied by a contraction of the tracheal smooth muscle in all the experimental animals. Our observations in this in vivo study confirm that the electrical activity of tracheal smooth muscle consists of slow oscillatory potentials and that tracheal contraction is at least partly coupled to the slow-wave activity of the smooth muscle.     

Agonist interactions at the calcium pools in skinned and unskinned canine tracheal smooth muscle

We studied the functionally discrete calcium sources used by acetylcholine, 5-hydroxytryptamine, histamine and high K+ in the dog tracheal smooth muscle. The extracellular calcium dependence of their responses was assessed by altering the calcium and by pretreatment with the calcium antagonist, nifedipine. The intracellular calcium pool was assessed by studying the interactions between caffeine and the agonists in both skinned and unskinned preparations. The extent of overlap for the different calcium pools between the various agonists was determined by studying the dose-response relationships of these agents before and after pretreatment with another agonist, i.e., the conditioning agonist, in zero calcium conditions. The rank order of sensitivity to calcium removal and to nifedipine was histamine greater than KCl greater than 5-hydroxytryptamine greater than acetylcholine. Caffeine-induced atenuation of the agonist responses was predominantly through physiological antagonism. However, the caffeine responses in unskinned fibres were augmented by pretreatment with the agonists through both nifedipine-sensitive (as with KCl) and -insensitive (as with acetylcholine) mechanisms. The responses to acetylcholine and caffeine were inhibited by theophylline and forskolin. In the skinned muscle fibres, the pCa-tension relationship suggested high calcium sensitivity, a significant caffeine-sensitive calcium pool, and no evidence of calcium release by exogenous inositol trisphosphate. The results are consistent with multiple extracellular and intracellular calcium sources for the agonist responses. We observed considerable overlap of the calcium sources used by these agonists. Of the four agonists studied, histamine appeared to inhibit the release and sequestration of calcium utilized by the other agonists most effectively.     

Cyclic GMP-dependent protein kinase activation in canine tracheal smooth muscle by methacholine and sodium nitroprusside

Methacholine (3 μM) and sodium nitroprusside (300 μM) increased cGMP-dependent protein kinase activity ratios (activity without cGMP divided by activity with 2 μM cGMP) in canine tracheal smooth muscle from a control value of 0.47 to 0.55 and 0.71, respectively. This correlates with 3-fold and 6-fold increases in cGMP concentrations in response to methacholine and sodium nitroprusside, respectively. Addition of charcoal to the homogenizing buffer prior to homogenization had no significant effect on the cGMP-dependent protein kinase response to either agent, suggesting that activation of the enzyme was not occurring as a result of cGMP release during homogenization. In order to limit cGMP dissociation from cGMP-dependent protein kinase during the assay procedure, it was necessary to perform assays at a reduced temperature (0°C) and with an abbreviated incubation time (2.5 min). When assayed at 30°C, activated cGMP-dependent protein kinase rapidly lost activity. This inactivation occurred whether the enzyme had been activated exogenously, by exposing a supernatant fraction of canine trachealis to 0.1 μM cGMP, or endogenously, by treating intact canine trachealis with methacholine or sodium nitroprusside. By assaying instead at 0°C, the inactivation of cGMP-dependent protein kinase was minimized. Therefore, the activity ratio obtained by this new modified assay provided an estimate of the endogenous activation state of cGMP-dependent protein kinase. The data indicate that cGMP responses in canine trachealis to both methacholine and sodium nitroprusside are functionally linked to activation of cGMP-dependent protein kinase and are consistent with the hypothesis that cGMP, via cGMP-dependent protein kinase activation, regulates smooth muscle contractility.     

Cyclic GMP-dependent protein kinase activation in canine tracheal smooth muscle by methacholine and sodium nitroprusside

Methacholine (3 microM) and sodium nitroprusside (300 microM) increased cGMP-dependent protein kinase activity ratios (activity without cGMP divided by activity with 2 microM cGMP) in canine tracheal smooth muscle from a control value of 0.47 to 0.55 and 0.71, respectively. This correlates with 3-fold and 6-fold increases in cGMP concentrations in response to methacholine and sodium nitroprusside, respectively. Addition of charcoal to the homogenizing buffer prior to homogenization had no significant effect on the cGMP-dependent protein kinase response to either agent, suggesting that activation of the enzyme was not occurring as a result of cGMP release during homogenization. In order to limit cGMP dissociation from cGMP-dependent protein kinase during the assay procedure, it was necessary to perform assays at a reduced temperature (0 degree C) and with an abbreviated incubation time (2.5 min). When assayed at 30 degrees C, activated cGMP-dependent protein kinase rapidly lost activity. This inactivation occurred whether the enzyme had been activated exogenously, by exposing a supernatant fraction of canine trachealis to 0.1 microM cGMP, or endogenously, by treating intact canine trachealis with methacholine or sodium nitroprusside. By assaying instead at 0 degree C, the inactivation of cGMP-dependent protein kinase was minimized. Therefore, the activity ratio obtained by this new modified assay provided an estimate of the endogenous activation state of cGMP-dependent protein kinase. The data indicate that cGMP responses in canine trachealis to both methacholine and sodium nitroprusside are functionally linked to activation of cGMP-dependent protein kinase and are consistent with the hypothesis that cGMP, via cGMP-dependent protein kinase activation, regulates smooth muscle contractility.     

Sympathetic innervation promotes vascular smooth muscle differentiation

The sympathetic nervous system (SNS) is an important modulator of vascular smooth muscle (VSM) growth and function. Several lines of evidence suggest that the SNS also promotes VSM differentiation. The present study tests this hypothesis. Expression of smooth muscle myosin (SM2) and alpha-actin were assessed by Western analysis as indexes of VSM differentiation. SM2 expression (normalized to alpha-actin) in adult innervated rat femoral and tail arteries was 479 +/- 115% of that in noninnervated carotid arteries. Expression of alpha-actin (normalized to GAPDH or total protein) in 30-day-innervated rat femoral arteries was greater than in corresponding noninnervated femoral arteries from guanethidine-sympathectomized rats. SM2 expression (normalized to alpha-actin) in neonatal femoral arteries grown in vitro for 7 days in the presence of sympathetic ganglia was greater than SM2 expression in corresponding arteries grown in the absence of sympathetic ganglia. In VSM-endothelial cell cultures grown in the presence of dissociated sympathetic neurons, alpha-actin (normalized to GAPDH) was 300 +/- 66% of that in corresponding cultures grown in the absence of neurons. This effect was inhibited by an antibody that neutralized the activity of transforming growth factor-beta2. All of these data indicate that sympathetic innervation increased VSM contractile protein expression and thereby suggest that the SNS promotes and/or maintains VSM differentiation.     

Relaxation of canine airway smooth muscle

Relaxation of airway smooth muscle is an inadequately understood yet critical process that, if impaired, may have significant implications for asthma. Here we explore why relaxation is an important process to consider, how it may determine airway hyperresponsiveness, and some of the factors that influence relaxation of the airway smooth muscle. These include mechanical and biochemical factors such as deep inspirations or large amplitude oscillation of the muscle, plastic properties of the muscle, the load the muscle experiences, calcium, phosphorylation of the myosin light chain, cytoskeletal proteins, and sensitization.