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1 We presently characterized the tachykinin receptor subtypes, using tachykinin receptor agonists and selective antagonists, that induce submucosal gland fluid flux (J(G)) from porcine tracheal explants with the hillocks technique. We also investigated the effects of the tachykinin receptor agonists on the electrophysiologic parameters of the tracheal epithelium in Ussing chambers. 2 The NK(1) tachykinin receptor agonist substance P (SP, 1 microM) and the NK(3) tachykinin receptor agonist [MePhe(7)]neurokinin B ([MePhe(7)]NKB, 1 microM) induced gland fluid fluxes of 0.29+/-0.03 microl min(-1) cm(-2) (n=26) and 0.36+/-0.05 microl min(-1) cm(-2) (n=24), respectively; while the NK(2) tachykinin receptor agonist [betaAla(8)]neurokinin A (4-10) ([betaAla(8)]NKA (4-10), 1 microM) had no effect on J(G) (n=10). 3 The NK(1) receptor antagonist CP99994 (1 microM, n=9) blocked 93% of the SP-induced J(G), whereas the NK(3) receptor antagonist SB223412 (1 microM, n=12) had no effect on the SP-induced J(G). However, SB223412 (1 microM, n=9) blocked 89% of the [MePhe(7)]NKB-induced J(G) while CP99994 (1 microM, n=10) did not affect the [MePhe(7)]NKB-induced J(G). The NK(2) receptor antagonist SR48968 (1 microM) did not block the J(G) induced by either the NK(1) (n=4) or NK(3) (n=13) receptor agonists. 4 The nicotinic ganglionic acetylcholine receptor antagonist hexamethonium (1 microM) and the muscarinic acetylcholine receptor antagonist atropine (1 microM) also decreased the NK(3) receptor agonist-induced J(G) by 67% (n=10) and 71% (n=12), respectively. 5 The potential difference (PD), short-circuit current (I(SC)), and membrane resistance (R(M)) of the porcine tracheal epithelial membranes were not significantly affected by any of the neurokinin agonists or antagonists (1 microM, basolateral) used in this study, although SP and [betaAla(8)]NKA (4-10) induced a slight transient epithelial hyperpolarization. 6 These data suggest that NK(1) and NK(3) receptors induce porcine airway gland secretion by different mechanisms and that the NK(3) receptor agonists induced secretion is likely due to activation of prejunctional NK(3) receptors on parasympathetic nerves, resulting in acetylcholine-release. We conclude that tachykinin receptor antagonists may have therapeutic potential in diseases with pathophysiological mucus hypersecretion such as asthma and chronic bronchitis.

We describe a computer-based image acquisition and analysis system designed for the quantitation of airway submucosal gland fluid flux by the hillocks technique. This technique is based on the detection of the increase in surface area of hillocks formed above gland duct openings by secretions captured under a thin layer of tantalum powder. The advantages of the system are: (a) ability to detect and measure individual submucosal gland secretions, (b) computer assisted data acquisition and analysis, and (c) decreased propagated error in hillock volume measurements. Test results of a practical implementation of the system demonstrate a swine tracheal submucosal gland flux induced by the muscarinic agonist acetylcholine (50 microM) of 0.65 microl/min/cm(2).

Furosemide pretreatment greatly reduces the severity of an asthmatic response to several types of bronchoconstrictor challenge. Indirect evidence suggests that furosemide exerts its protective effects by dilating the airway vasculature during thermal stress. To test the hypothesis that furosemide dilates airway microvessels, the tracheas of anesthetized rats were surgically exposed and continuously suffused with Krebs Ringer bicarbonate warmed to 37 degrees C. Tracheal adventitial arterioles (13.0 to 41.0 microns initial diameter, n = 47) and venules (50.0 to 99.0 microns initial diameter, n = 46) were visualized with a videomicroscope, and vessel diameters were measured using videocalipers. When vessels were preconstricted with 10(-4) M phenylephrine, a selective alpha 1-adrenergic agonist, and then treated with 10(-4) M furosemide, significant (p < 0.05) dilation was observed in both arterioles (from 64.6 to 79.5% of their initial diameter) and venules (from 52.1 to 65.4% of their initial diameter). When vessels were preconstricted with 10(-4) phenylephrine, after pretreatment with the cyclooxygenase inhibitor indomethacin (5.0 mg/kg), 10(-4) M furosemide significantly dilated arterioles (from 77.5 to 93.0% of their initial diameter) and venules (from 58.5 to 80.1% of their initial diameter). In vessels preconstricted with 10(-3) M L-NAME, an inhibitor of nitric oxide synthesis, addition of 10(-4) M furosemide to the suffusion still caused significant dilation in arterioles, from 77.4 to 88.8% of their initial diameter, and in venules from 79.5 to 86.7% of their initial diameter. These data confirm that furosemide, when applied topically, dilates tracheal arterioles and venules by cyclooxygenase- and nitric oxide-independent mechanisms.

Previous study of adrenergic control of the tracheal vasculature in rats (1) demonstrated that beta-adrenergic blockade heightened arteriolar and large venular contractile responses to norepinephrine, a nonselective alpha-adrenergic agonist. The present study was undertaken to confirm the presence of functional beta-adrenergic receptors and to determine which beta-adrenergic receptor subtypes mediate vasodilation in this tissue. Tracheal adventitial arterioles (12.0 to 47.0 micro m initial diameter, n=39) and venules (48.0 to 98.5 micrometers initial diameter, n=44) were observed through a video microscope, and vessel diameters were measured. Vessels were preconstricted with 10(-4) M phenylephrine (PHE), a selective alpha 1-adrenergic agonist, to achieve sufficient tone for measurement of dilation responses. When vessels were treated only with PHE, arterioles and venules constricted to 55.9% and 67.6% of their initial diameter, respectively, after 15 min of suffusion. When preconstricted vessels were treated with the nonselective beta-adrenergic agonist isoproterenol (10(-5) M), both arterioles and venules significantly dilated from 63.4% to 82.9% and from 71.5% to 81.3% of their initial diameters. At high concentration (10(-5) M), the putative beta 2-adrenergic agonist terbutaline also caused preconstricted arterioles and venules to significantly dilate from 70.8% to 79.8% and from 71.5% to 83.4% of their initial diameters. The selective beta 1-adrenergic antagonist atenolol (10(-6) M) did not affect terbutaline-induced dilation in preconstricted arterioles, but greatly attenuated dilation in preconstricted venules. From these data, we conclude that beta 2-adrenergic receptors are present in and mediate dilation of tracheal arterioles, and also, that the dilation in large tracheal venules is mediated in large part through beta 1-adrenergic receptors.

Although blood flow to the pulmonary airways is known to be largely under sympathetic control, virtually nothing is known about adrenergic regulation of vascular segments within the airway microcirculation. To evaluate the distribution of functional adrenergic receptor subtypes in the microvessels of the large airways, the change in diameter of adventitial vessels in rat trachea was measured following suffusion with selective and nonselective receptor agonists and antagonists. Microvessels were viewed with a video microscope, and vessel diameters were measured using video calipers. Arterioles (11.0 to 40.0 microns, n = 32), small postcapillary venules (11.0 to 26.0 microns, n = 16), medium venules (28.0 to 59.5 microns, n = 40), and large collecting venules (61.0 to 99.0 microns, n = 42) were distinguished. Similar sensitivities to norepinephrine (NE), a mixed alpha 1 and alpha 2 agonist, were observed in arterioles and medium venules with EC50 (agonist concentration needed to produce 50% of the maximal response) for contraction of 2.4 x 10(-7) and 3.3 x 10(-7) M, respectively. Large venules (EC50 of 1.6 x 10(-6) M) were significantly (p < 0.05) less sensitive than arterioles to NE. In the presence of propranolol, a beta receptor antagonist, the EC50 values for NE were not different between the three vessel groups, although the response to low doses of NE was significantly increased in arterioles. When vessels were treated with propranolol and phenylephrine, a selective alpha 1 agonist, arterioles (EC50 of 4.1 x 10(-7) M) were significantly more sensitive than large venules (EC50 of 4.9 x 10(-6) M).(ABSTRACT TRUNCATED AT 250 WORDS)