Antagonism of relaxation to isoproterenol caused by agonist interactions

The interaction of contractile agonists on the relaxation elicited with isoproterenol (ISO) was studied in 112 tracheal smooth muscle (TSM) strips from 20 dogs in vitro. Strips were contracted to the same active target tension (TT) with acetylcholine (ACh), histamine (HIS), serotonin (5-hydroxytryptamine, 5-HT), potassium chloride (KCl), or the combinations of ACh + HIS, ACh + 5-HT, HIS + KCl, HIS + 5-HT (50% TT from each agonist). Although a less potent agonist, adding HIS to cause 50% of the TT reduced the concentration of ACh to elicit the remaining 50% TT and substantially altered relaxation by ISO compared with HIS alone [concentration required to achieve 50% relaxation (RC50) = 9.2 +/- 2.4 X 10(-8) vs. 9.0 +/- 4.4 X 10(-9) M to HIS alone; P less than 0.003]. Relaxation for TSM strips contracted with ACh + HIS was comparable to that elicited from the same TT with ACh alone, although concentrations required in combination were lower than for either agonist alone. Trachealis strips contracted equivalently with KCl + HIS also had augmented contraction and attenuated relaxation (RC50 = 3.7 +/- 0.8 X 10(-8) M; P less than 0.015 vs. HIS alone). However, combinations of 5-HT + ACh and 5-HT + HIS did not alter relaxation to ISO from that elicited by the weaker agonist alone. We demonstrate that TSM relaxation depends on the combination of agonists eliciting contraction and may be inhibited substantially by interactions among contractile agonists.     

Expression of airway contractile properties and acetylcholinesterase activity in swine

We studied the effect of maturation on contractile properties of tracheal smooth muscle from seventeen 2-wk-old swine (2ws) and fifteen 10-wk-old swine (10ws) in situ and in vitro. The response to parasympathetic stimulation was studied in situ in isometrically fixed segments. Contraction was elicited at lower frequencies [half-maximal response to electrical stimulation (ES50) = 6.7 +/- 0.05 Hz] in 2ws than in 10ws (ES50 = 9.1 +/- 0.4 Hz; P less than 0.01). Despite substantial differences in morphometrically normalized cross-sectional area in 2ws (0.012 +/- 0.003 cm2) and 10ws (0.028 +/- 0.001 cm2; P less than 0.01), maximal active tension elicited by parasympathetic stimulation was similar (12.4 +/- 3.2 g/cm in 2ws vs. 13.3 +/- 2.3 g/cm in 10ws; P = NS). In separate in vitro studies in 25 tracheal smooth muscle strips from 10 swine, concentration-response curves generated with potassium-substituted Krebs solution (KCl) were similar in 2ws and 10ws. In 58 other strips (10 swine), maximal active force elicited with acetylcholine (ACh) in 2ws was significantly greater than for 10ws (P less than 0.001). Removal of the epithelium had no effect. However, cholinesterase inhibition with 10(-7) M physostigmine augmented the response to ACh in 10ws (P less than 0.02) but not 2ws. We demonstrate increased force generation and sensitivity to vagal stimulation in 2ws vs. 10ws, which corresponds to increased reactivity to ACh in vitro. The relative hyperresponsiveness in 2ws is specific for cholinergic response and is attenuated at least in part by maturation of the activity of acetylcholinesterase enzyme.     

Extracellular Ca2+ mobilization in potential-dependent contraction of trachealis of maturing swine.

We studied the effect of maturation on potassium-induced parasympathetic activation and Ca2+ entry in tracheal smooth muscle (TSM) from fifteen 2-wk-old (2ws) and sixteen 10-wk-old (10ws) male domestic farm swine. Atropine (10(-7) M) caused inhibition of the maximal contraction elicited by potassium to 50.3 +/- 2.6% maximum of control response (P less than 0.001) in TSM from 2ws but had no significant effect in TSM from 10ws (94.6 +/- 4.2% maximum; P = NS vs. control). Verapamil (10(-7) M) plus 10(-7) M atropine reduced contraction elicited by potassium in both 2ws (23.7 +/- 5.8% maximum; P less than 0.001 vs. control) and 10ws (50.6 +/- 6.3% maximum; P less than 0.001 vs. control, P less than 0.05 vs. 2ws); 10(-6)M verapamil caused greater than 95% blockade of contraction caused by potassium in both 2ws and 10ws. In separate studies, atropine-treated strips were equilibrated with extracellular Ca2+ concentrations ([Ca2+]o) ranging from normal (1X [Ca2+]o) to four times normal (4x [Ca2+]o). Increasing [Ca2+]o increased maximal contractile response in atropine-treated TSM strips from 68.7 +/- 3.8% maximum for 1x [Ca2+]o to 100.8 +/- 4.8% maximum for 4x [Ca2+]o (P less than 0.001) in 2ws. Neither atropine nor [Ca2+]o affected maximal responses of TSM in 10ws (103.5 +/- 3.0% maximum for 1x [Ca2+]o; P = NS vs. control). However, in the presence of atropine and verapamil, 4x [Ca2+]o augmented KCl-elicited contraction of TSM from both 2ws (46.9 +/- 6.3% maximum; P less than 0.01 vs. control) and 10ws (78.6 +/- 2.3% maximum; P less than 0.005 vs. control).(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta-adrenergic relaxation of dog trachealis: contractile agonist-specific interaction

The phenomenon of contractile agonist-dependent relaxation by isoproterenol (ISO) of active tension elicited by acetylcholine (ACh), histamine (HIS), serotonin (5-HT), and potassium chloride-substituted Krebs-Henseleit solution (KCl) was studied in 210 tracheal smooth muscle (TSM) strips from 28 mongrel dogs in vitro. All TSM strips were contracted to similar active tensions [target tension (TT) = 50% of the maximal active tension elicited by 127 mM KCl] with ACh, HIS, 5-HT, or KCl and relaxed with either ISO, forskolin (FSK), N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (db-cAMP), or 3-isobutyl-1-methylxanthine (IMX). The concentrations of ISO causing 50% relaxation from TT (RC50) were ACh (2.9 +/- 1.1 x 10(-6) M) greater than 5-HT (8.4 +/- 1.5 x 10(-8) M) approximately KCl (8.1 +/- 2.1 x 10(-8) M) greater than HIS (1.6 +/- 0.2 x 10(-8) M). FSK and IMX relaxed TSM in the same rank order of potency as ISO. In contrast to the contractile agonist-dependent relaxation elicited by ISO, FSK, and IMX, db-cAMP was nearly equipotent in relaxing similarly contracted strips. These results are consistent with contractile agonist-specific interaction with cAMP production by ISO and FSK. These data demonstrate that the phenomenon of contractile agonist-dependent relaxation by ISO is not related specifically to the beta-adrenoceptor.     

Caffeine effects on buccal muscles of Aplysia

1. Caffeine (35-70 mM) elicited contractions of Aplysia buccal muscle El. In a Ca2+-free medium, in which ACh-elicited contractions rapidly fail, caffeine elicited contractions of approximately the same size as in normal medium. 2. 5-HT (10(-8) M and 10(-7) M) did not enhance caffeine-elicited contractions. 3. Lower concentrations (1-10 mM) of caffeine inhibited ACh-elicited contractions. Caffeine (7 mM) reduced the contraction by 80%. 4. Caffeine (7 mM) reduced ACh-elicited depolarization by 60%. 5. Caffeine (7 mM) increased 45Ca2+ influx into Aplysia buccal muscle I5. The stimulation of influx of 45Ca2+ by 10(-3) M ACh was non-additive with the stimulation caused by caffeine, and 7 mM caffeine reduced the influx caused by 10(-3) M ACh.     

Ca2+-dependent facilitated shortening in isotonic contraction of trachealis muscle

We compared isotonic shortening with isometric force generation as a function of external Ca2+ in 166 tracheal smooth muscle (TSM) strips from 27 mongrel dogs in vitro. Concentration-response curves were generated with muscarinic stimulation (acetylcholine, ACh), alpha-adrenergic receptor activation (norepinephrine after beta-adrenoceptor blockade, NE), serotonin (5-HT), and KCl-substituted Krebs-Henseleit solution. The concentrations of 5-HT causing half-maximal shortening (ECS50, 1.54 +/- 0.14 X 10(-7) M) and half-maximal active isometric tension (ECT50, 1.72 +/- 0.30 X 10(-7) M) were similar (P = NS). Likewise, ECS50 (21.9 +/- 0.7 mM) and ECT50, (22.0 +/- 0.9 mM) were similar for KCl. In contrast, facilitated isotonic shortening (i.e., greater isotonic shortening for comparable degrees of force generation) was elicited with ACh and NE for all levels of force generation between 15 and 85% of maximum and for all concentrations of ACh from 3 X 10(-8) to 3 X 10(-5) M (P less than 0.05 for all points). Facilitated isotonic shortening also was elicited for all concentrations of NE from 10(-8) to 10(-6) M (P less than 0.05 for all points). Removal of Ca2+ from the perfusate substantially reduced the potency of ACh (P less than 0.001) and abolished differences between ECS50 (2.23 +/- 0.28 X 10(-5) M) and ECT50 (2.50 +/- 0.46 X 10(-5) M, P = NS). We demonstrate that for comparable degrees of force generation, muscarinic and alpha-adrenergic receptor activation cause greater isotonic shortening than KCl or 5-HT and that this facilitated shortening is associated with the concentration of external Ca2+.     

Homeostatic regulation of airway smooth muscle tone by catecholamine secretion in swine

We studied the homeostatic secretory response of catecholamine secretion elicited by progressive bronchoconstriction in 18 swine in vivo. The potential reserve of the sympathetic nervous system (SNS) was first assessed by exogenous nicotinic stimulation with 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP). A dose of 250 micrograms/kg iv DMPP caused an increase in plasma norepinephrine (NE) concentration from 207 +/- 86 (basal) to 2,625 +/- 448 pg/ml (P less than 0.02) and in plasma epinephrine (EPI) from 10 +/- 5.0 to 1,410 +/- 432 pg/ml (P less than 0.05) in four swine. In four other swine, bronchoconstriction induced by aerosolized prostaglandin F2 alpha caused approximately a fivefold increase in airway resistance without hemodynamic changes. No increase in plasma EPI was observed. However, plasma NE increased from 330 +/- 131 to 1,540 +/- 182 pg/ml (P less than 0.02). In five swine receiving aerosolized acetylcholine (ACh), similar changes in airways resistance were not associated with significant changes in catecholamine concentration when mean arterial blood pressure (MAP) was unchanged. However, inhalation of sufficient ACh to cause a greater than 10% decrease in MAP caused progressive increase in catecholamine secretion. Plasma EPI increased from 32 +/- 16 (MAP = 124 +/- 7 Torr) to 1,165 +/- 522 pg/ml (MAP = 94 +/- Torr). Hypoxemia that occurred with bronchoconstriction (greater than or equal to 50 Torr) did not cause catecholamine secretion. However, severe hypoxemia (PO2 less than 30 Torr) caused large increases in plasma EPI concentrations from 84 +/- 27 to 1,463 +/- 945 pg/ml (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of substance P on neurally mediated contraction of rabbit airway smooth muscle

The neuromodulatory action of substance P (SP) was investigated in isolated rabbit tracheal smooth muscle (TSM) segments contracted with electrical field stimulation (ES). The tissues were placed in organ baths containing modified Krebs-Ringer solution and stimulated at a constant voltage (8 V; 24.5 mA) and pulse duration (2 ms) with ES frequencies ranging from 1 to 100 Hz. In the presence of SP, there occurred a dose-dependent augmentation of the TSM contractile response to any given ES, with the maximal effect of SP obtained at a dose of 10(-7) M. Accordingly, with the administration of 10(-7) M SP, the ES frequency-response relationship was altered so that 1) the mean (+/- SE) maximal tension (Tmax) induced by ES significantly increased (P less than 0.02) from a base-line value of 273 +/- 53 to 402 +/- 45 g/g TSM; and 2) the mean (+/- SE) log ES frequency producing 50% of Tmax (ES50) significantly decreased from a base-line value of 1.278 +/- 0.069 to 1.102 +/- 0.070 Hz (P less than 0.01). In contrast to these effects on ES-induced contraction, SP administration did not affect the TSM contractile response to administered methacholine chloride (10(-8) to 10(-3) M). On the other hand, the effects of SP on ES-induced contraction were independently blocked by the cholinergic antagonist, atropine (10(-6) M); the neurotoxin, tetrodotoxin (10(-6) g/ml); and the SP antagonist, D-Arg1,D-Pro2,D-Trp7,9,Leu11-SP (10(-5) M).(ABSTRACT TRUNCATED AT 250 WORDS)     

Bronchial reactivities in guinea pigs to acetylcholine or histamine exposure

The bronchial reactivities in Hartley guinea pigs to acetylcholine (ACh) or histamine (Hist) were investigated, and the following results obtained; 1. Eight-week-old animals were exposed to ACh and Hist. A significant relationship was observed between the concentrations of the chemicals and the time needed to produce falling down (TNPFD) due to the asthmatic reaction to ACh and Hist. 2. Eight-week-old animals were exposed to 0.1% ACh and 0.05% Hist, for which the mean TNPFD +/- standard error were 377 +/- 33 sec and 122 +/- 5 sec respectivity. However, no difference in reactivity between male and female animals was noted. 3. Eight- and 9-week-old animals were exposed to 0.01% ACh and 0.025% Hist. A positive correlation was observed (r = 0.736, p less than 0.01) between the TNPFD for ACh and that for Hist. 4. Growing animals from 2 weeks to 20 weeks old were exposed to 0.08% ACh and 0.025% Hist. After inhalation of both chemicals, 6-week-old animals showed the greatest prolongation of mean TNPFD (lowest sensitivity). 5. Eight-week-old animals were exposed to 0.08% ACh and 0.025% Hist. With both of these chemicals, a positive correlation was observed between TNPFD and dose threshold (ACh r = 0.886, p less than 0.001; Hist r = 0.891, p less than 0.001).     

Contractile properties of bronchial smooth muscle with and without cartilage

The majority of in vitro studies on airway smooth muscle have used the trachealis (TSM) as a convenient substitute for muscle from airways that constitute the flow-limiting segment. The latter are technically difficult to work with. However, because the site of maximum resistance to airflow is at the third to seventh generations of the bronchial tree, the trachealis preparation is of limited value. Length-tension and force-velocity properties were therefore studied at optimal length (lo) of canine bronchial smooth muscle (BSM) from which cartilage had been carefully removed. Normalized maximum isometric tension or stress (Po x 10(4) N/m2) for BSM was 7.1 +/- 0.19 (SE), which was similar to that of BSM with cartilage (BSM+C, 6.8 +/- 0.21) but lower than for TSM (18.2 +/- 0.81). At length greater than lo, the BSM+C was stiffer than the BSM. The values of maximum shortening capacity (delta Lmax), obtained directly from isotonic shortening at a load equal to the resting tension at lo, were 0.76 lo +/- 0.03, 0.41 lo +/- 0.02, and 0.24 +/- 0.02 lo for TSM, BSM, and BSM+C, respectively. The BSM and BSM+C delta Lmaxs were different (P less than 0.05). Maximal shortening velocities (Vo) for BSM, elicited at 2, 4, and 8 s by quick release in the course of an isometric contraction were significantly higher than for the BSM+C. Vos showed gradual decreases in all three groups in the later phase of contraction, suggesting the operation of latch bridges.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronic exercise training improves ACh-induced vasorelaxation in pulmonary arteries of pigs.

We hypothesized that exercise training would lead to enhanced endothelium-dependent vasodilation in porcine pulmonary arteries. Pulmonary artery rings (2- to 3-mm OD) were obtained from female Yucatan miniature swine with surgically induced coronary artery occlusion (ameroid occluder). Exercise training was performed for 16 wk, and vasomotor responses were studied by using standard isometric techniques. Contractile responses to 80 mM KCl, isosmotic KCl (10-100 mM), and norepinephrine (10(-8) to 10(-4) M) did not differ between sedentary (Sed) and exercise-trained (Ex) pigs. Relaxation was assessed to endothelium-dependent and endothelium-independent vasodilators after norepinephrine contraction. Pulmonary arteries of Ex pigs exhibited greater maximal relaxation to ACh (61.9 +/- 3.5%) than did those of Sed pigs (52.3 +/- 3.9%; P < 0.05). Endothelium-independent relaxation to sodium nitroprusside did not differ. Inhibition of nitric oxide synthase significantly decreased acetylcholine-induced relaxation, with greater inhibition in arteries from Ex pigs (P < 0.05). Inhibition of cyclooxygenase enhanced relaxation to acetylcholine in arteries from Sed pigs. We conclude that exercise training enhances endothelium-dependent (ACh-mediated) vasorelaxation in pulmonary arteries by mechanisms of increased reliance on nitric oxide and reduced production of a prostanoid constrictor.     

Determination of Acetylcholine Release in the Striatum of Anesthetized Rats Using In Vivo Microdialysis and a Radioimmunoassay

A vertical-type in vivo microdialysis probe and a sensitive, specific radioimmunoassay (RIA) were used to study the mechanism of acetylcholine (ACh) release in the striatum of anesthetized rats. Without the use of physostigmine, a cholinesterase inhibitor, our RIA could still detect the amount of ACh present in the perfusate (5.6 +/- 0.6 fmol/min, n = 16). Tetrodotoxin (1 microM) produced a significant decrease in the amount of ACh collected in the perfusate, suggesting that basal ACh determined under the present experimental conditions was related to cholinergic neural activity. Atropine (0.1-1 microM) applied topically via the dialysis probe did not affect the amount of ACh recovered in the perfusate in the absence of physostigmine. Addition of physostigmine (10 microM) to the perfusion fluid produced about a 100-fold increase in the amount of ACh collected. In the presence of physostigmine, topical administration of atropine and pirenzepine (0.01-1 microM) through a dialysis probe produced a further three- to fourfold increase in ACh output, whereas a slight increase was produced by AF-DX 116 at the highest concentration (1 microM). These results indicate that presynaptic modulation of ACh release in the striatum does not occur under basal conditions, and that presynaptic M1 muscarinic receptors are involved in the modulation of ACh release when the ACh concentration is raised under certain conditions.     

Effect of endogenous prostaglandins on acetylcholine release from dog trachealis muscle

We used a radioenzymatic technique to measure effects of the prostaglandin synthesis inhibitor indomethacin and of exogenous prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2) on acetylcholine (ACh) efflux from canine tracheal smooth muscle (TSM) during sustained electrical field stimulation (EFS; 2 Hz, 2 ms pulse duration, 50 V for 15 min). ACh efflux from indomethacin (INDO, 10(-6) M)-pretreated and control TSM increased with consecutive stimulations. However, efflux of ACh was greater in INDO-treated than control muscles. INDO increased the tension produced by TSM in response to EFS. Neither PGE2 (10(-8) M) nor PGI2 (10(-6) M) had any effect on ACh efflux from INDO-pretreated TSM during the first of three periods of EFS. However, PGI2 and PGE2 prevented the progressive increase in ACh efflux observed on subsequent stimulations. PGE2 but not PGI2 decreased contractions of TSM caused by EFS. Our results demonstrate that endogenous prostaglandins, probably PGE2, do inhibit EFS-evoked ACh release from canine TSM in vitro, but suggest that these prostaglandins modulate EFS-evoked contractions predominantly by postsynaptic mechanisms.     

Role of platelets in contraction of canine tracheal muscle elicited by PAF in vitro

To elucidate mechanisms of platelet-activating factor (PAF)-induced contraction, we studied the effect of PAF on 203 canine tracheal smooth muscle (TSM) strips from 45 dogs in vitro in the presence and absence of platelets. PAF (10(-11) to 10(-7) M) alone caused no contraction of TSM even in the presence of airway epithelium. In the presence of 2 x 10(5) platelets/microliter, PAF was an extremely potent contractile agonist (threshold 10(-11) M). This response was inhibited by the PAF antagonist, CV-3988 (10(-6) M), and reversed by the serotonin antagonist, methysergide (EC50 = 3.7 +/- 0.79 x 10(-9) M). Neither atropine nor chlorpheniramine (10(-9) to 10(-6) M) attenuated the response to PAF + platelets. In the presence of platelets, 10(-7) M PAF caused an increase in perfusate concentration of serotonin from 0.93 +/- 0.037 x 10(-8) to 1.7 +/- 0.046 x 10(-8) M (P less than 0.001). Tachyphylaxis, previously demonstrated to be irreversible, was shown to be a platelet-dependent phenomenon; contraction could be repeated in the same TSM after addition of fresh platelets. We demonstrate that PAF-induced contraction of canine TSM is caused by the release of cellular intermediates such as serotonin from platelets. We also demonstrate the site of PAF-induced tachyphylaxis in airway smooth muscle contraction.     

Amiloride inhibits contraction and serotonin modulation of Aplysia muscle

1. Serotonin (5-HT) potentiates acetylcholine (ACh)-elicited contractions of Aplysia buccal muscles. Serotonin potentiation was significantly reduced by 0.03 mM, 0.1 mM, and 0.3 mM amiloride. 2. Unpotentiated ACh-elicited contractions were significantly reduced by 0.1 mM and 0.3 mM amiloride. 3. Amiloride reduced ACh-elicited depolarization. The reduction in contraction caused by 0.3 mM amiloride (to 16% of control) was larger than could be explained by the reduction in depolarization (86% of control). 4. Amiloride had no effect on tension in skinned muscle fibers, indicating that amiloride probably did not have a direct effect on contractile mechanisms. 5. Potentiation of contraction produced by zero sodium (Tris substituted, 0 Na-Tris) medium could be abolished by 0.3 mM amiloride. 6. Zero Na-Tris increased 45Ca influx 2.7-fold. In the presence of 0.3 mM amiloride, 0 Na-Tris increased 45Ca influx only 1.4-fold. 7. Amiloride (0.3 mM) reduced the elevation of muscle cAMP caused by 10(-6) M 5-HT by 60%. Zero Na-Tris did not cause a change in muscle cAMP.     

Antagonists of cholinergic and serotonergic responses of Aplysia buccal muscle

Cholinergic and serotonergic receptors of Aplysia californica buccal muscles were characterized pharmacologically by determining compounds that effectively inhibited contractile responses to acetylcholine (ACh) and modulatory effects of serotonin (5-HT), respectively. pA50 for ACh to elicit contraction averaged 4.7 ± 0.1 (mean ± SE, equivalent to 2 × 10⁻⁵ M). Both hexamethonium bromide and atropine inhibited ACh-elicited contractions, but neither inhibited the response completely, nor were the two together able to antagonize the response completely. Curare caused inhibition only at low ACh doses, and muscarinic antagonists pirenzapine and 4-diphenylacetoxy-N-methylpiperidine methiodide caused partial inhibition. The most effective blocker of ACh-elicited contractions was the nicotinic antagonist mecamylamine. 10⁻⁴M mecamylamine completely blocked the cholinergic response. ACh contractions were inhibited 90% within 10 min and took >40 min to recover from mecamylamine. Specificity was indicated by the lack of effect of mecamylamine on potassium-elicited contraction. NAN-190 blocked the potentiating effect of 5-HT without having inhibitory or potentiating effects by itself on ACh-elicited contractions. NAN-190 blocked the potentiating effect of 8-OH-DPAT. Cholinergic receptors on Aplysia buccal muscles are most effectively inhibited by mecamylamine and may have mixed nicotinic/muscarinic character. Serotonergic receptors have pharmacological similarities to vertebrate 5-HT_1A receptors and may be closely related to the gastropod 5-HTlym receptor.     

Regulation of calcium influx into buccal muscles of Aplysia by acetylcholine and serotonin

Acetylcholine (ACh) causes contraction of Aplysia buccal muscles E1 and I5, and serotonin (5-hydroxytryptamine, 5-HT) enhances ACh-elicited contractions of these muscles. Possible roles of calcium influx in mediating these responses were examined by studying influx of 45Ca++. 5-HT increased calcium influx into both I5 and E1. Maximal influx occurred at 10(-6) M 5-HT and the increased influx could be sustained in the presence of 5-HT for at least 10 min. ACh also caused calcium influx, and calcium influx increased approximately in proportion to log[ACh] from 10(-5) M to 10(-3) M ACh. 5-HT and ACh probably bring about calcium influx by different mechanisms since the effect of ACh was additive to a maximal 5-HT response, and 10(-4) M hexamethonium bromide inhibited the increased influx caused by ACh but did not affect influx caused by 5-HT. Cyclic AMP analogues and forskolin neither caused an increase in calcium influx nor an increase in the influx caused by ACh. The data support a model in which ACh-elicited contractions of I5 and E1 are due primarily to calcium entry across the extracellular membrane, and 5-HT can "load" an intracellular site by a mechanism different from that activated by ACh. The data do not support a role for cyclic AMP in mediating the calcium influx response to 5-HT.     

Epithelial augmentation of trachealis contraction caused by major basic protein of eosinophils

We studied the effect of epithelial removal and intraepithelial administration of human eosinophil granule major basic protein (MBP) on the contraction of underlying canine tracheal smooth muscle in 23 dogs in vivo. A dual in situ tracheal preparation was utilized that allowed sharp excision of epithelium. The response to intra-arterial acetylcholine (ACh) was augmented substantially in five dogs receiving 200 micrograms MBP by intraepithelial instillation. Active tension elicited by 10(-8) mol intra-arterial ACh was 34.0 +/- 2.2 g/cm before and 46.1 +/- 2.6 g/cm 30 min after MBP (P less than 0.002). There was no change in active tension in the control segment in the same dogs after intraepithelial instillation of vehicle only (34.7 +/- 3.2 vs. 34.4 +/- 2.3 g/cm; P = NS). Instillation of MBP directly into the subepithelial tracheal smooth muscle did not alter contraction. To assess whether this augmentation was caused by inhibition of an epithelial-derived relaxant factor, additional studies were performed in nine other dogs in which the epithelium was excised discretely from one of the two tracheal segments. No significant differences in contractile response to ACh or relaxation response to isoproterenol were observed at 2, 15, 30, or 60 min after epithelial excision. We demonstrate that intraepithelial administration of MBP augments the contraction of underlying canine tracheal smooth muscle elicited by ACh. This augmentation is a direct effect of MBP and does not require antagonism of epithelial inhibition.     

Cholinergic neuromodulation by prostaglandin D2 in canine airway smooth muscle

To determine whether prostaglandin D2 (PGD2) modulates cholinergic neurotransmission in airway smooth muscle and, if so, what the mechanism of action is, we studied bronchial segments from dogs under isometric conditions in vitro. PGD2 (10(-8)-10(-5) M) elicited dose-dependent muscle contraction, which was reduced after blockade of muscarinic receptors, so that 50% effective dose (ED50) increased from 1.3 +/- 0.3 X 10(-6) to 3.9 +/- 1.0 X 10(-6) M by atropine (10(-6) M) (mean +/- SE, P less than 0.05). Physostigmine, at a concentration insufficient to alter base-line tension (10(-8) M), enhanced the PGD2-induced contraction and decreased ED50 to 6.4 +/- 0.5 X 10(-7) M (P less than 0.05). When added at the highest doses that did not cause spontaneous contraction (1.9 +/- 0.5 X 10(-7) M), PGD2 increased the contractile response to electrical field stimulation (1-50 Hz) by 21.9 +/- 6.6% (P less than 0.001). In contrast to this effect, the response to administered acetylcholine was not affected by PGD2. On the other hand, PGD2-induced augmentation of the response to electrical field stimulation (5 Hz) was further increased from 23.6 +/- 3.0 to 70.4 +/- 8.8% in the presence of physostigmine (10(-8) M) and was abolished by atropine but not affected by the alpha-adrenergic antagonist phentolamine or the histamine H1-blocker pyrilamine. These results suggest that the contraction of airway smooth muscle induced by PGD2 is in in part mediated by a cholinergic action and that PGD2 prejunctionally augments the parasympathetic contractile response, likely involving the accelerated release of acetylcholine at the neuromuscular junction.     

Regulation of vascular tone in animals overexpressing the sarcolemmal calcium pump

The mechanisms governing vascular smooth muscle tone are incompletely understood. In particular, the role of the sarcolemmal calcium pump PMCA (plasma membrane calmodulin-dependent calcium ATPase), which extrudes Ca2+ from the cytosol, and its importance compared with the sodium/calcium exchanger remain speculative. To test whether the PMCA is a regulator of vascular tone, we generated transgenic mice overexpressing the human PMCA4b under control of the arterial smooth muscle-specific SM22alpha promoter. This resulted in an elevated systolic blood pressure compared with littermate controls. In PMCA-overexpressing mice, endothelium-dependent relaxation of norepinephrine-preconstricted aortic rings to acetylcholine did not differ from wild type controls (76 +/- 8% versus 79 +/- 8% of maximum relaxation; n = 12, n.s.). De-endothelialized aortas of transgenic mice exhibited stronger maximum contraction to KCl (100 mmol/liter) compared with controls (86 +/- 6% versus 68 +/- 7% of reference KCl contraction at the beginning of the experiment; p <0.05). Preincubation of de-endothelialized vessels with the nitric oxide synthase (NOS) inhibitor l-NAME (l-N(G)-nitroarginine methyl ester) (10-5 mol/liter) resulted in a stronger contraction to KCl (p <0.05 versus without l-NAME), thus unmasking vasodilatory effects of inherent NO production. Maximum contraction to KCl after preincubation with l-NAME did not differ between PMCA mice and controls. In analogy to the results in PMCA-overexpressing mice, contractions of de-endothelialized aortas of neuronal NOS-deficient mice to KCl were significantly increased compared with controls (151 +/- 5% versus 131 +/- 6% of reference KCl contraction; p <0.05). In conclusion, our data suggest a model in which the sarcolemmal Ca2+ pump down-regulates activity of the vascular smooth muscle Ca2+/calmodulin-dependent neuronal NOS by a functionally relevant interaction. Therefore, the PMCA represents a novel regulator of vascular tone.