ET(B) receptor activates adenylyl cyclase via a c-PLA(2)-dependent mechanism: a novel counterregulatory mechanism of ET-induced contraction in airway smooth muscle.

Endothelin-1 (ET-1) contracted the rabbit tracheal smooth muscle (RTSM), yielding a bell-shaped tension-concentration curve. Moreover, ET-1 induced concentration- and time-dependent increases in cAMP concentrations in RTSM (EC(50), 58 nM; t(1/2), 2.4 min). Pretreatment with the AC inhibitors, SQ-22536, or 2'-5'-dideoxyadenosine, enhanced contraction to ET-1 and converted its bell-shaped tension curve into a sigmoidal one, but left contraction to carbachol and KCl unaltered. The potent ET(B)-receptor agonists, ET-3 or sarafotoxin-c, mimicked ET-1's effects on cAMP levels (EC(50) values 55 and 50 nM). Further, cAMP formation by ETs was inhibited by BQ-788 (selective ET(B) receptor blocker; IC(50), 8 nM), but not by BQ-610 (selective ET(A) receptor blocker). Removal of the epithelium did not prevent ET-induced increases in cAMP levels. Unlike isoproterenol, ETs failed to activate AC in membrane fractions from RTSM. In intact RTSM, the c-PLA(2) inhibitor, AACOCF3, and the cyclooxygenase inhibitor, indomethacin, blocked ET-induced increases in cAMP levels. These findings reveal a novel, nonepithelial, c-PLA(2)-mediated, regulatory mechanism downstream from ET(B) receptors.

Lipid mediators in oxygen-induced airway remodeling and hyperresponsiveness in newborn rats.

We examined whether lipid mediators have a causal role in neonatal hyperoxia-induced lung damage, specifically, airway remodeling and hyperresponsiveness. Newborn rat pups were exposed to hyperoxia (> 95% O2 from Days 4 to 14 and 65% from Days 14 to 32) or normoxia. The 5-lipoxygenase inhibitor, LTD4 receptor antagonist, and inhibitor of platelet-activating factor synthesis, Wy-50,295 (30 mg/kg), or vehicle was administered daily from Days 3 to 32. Oxygen exposure significantly increased (p < 0.05) the production of one potential lipid mediator group, peptido-LTs, from explanted lung slices and large airways from 2-wk-old rat pups. At 4 wk, only the large airway tissue output showed significant elevation because of oxygen exposure. At both ages, Wy-50,295 significantly decreased (p < 0.05) the production of peptido-LTs in the lung and large airways of oxygen-exposed pups. Pulmonary function and airway wall morphometry were studied in 5-wk-old rat pups 2 to 3 d after oxygen exposure and drug administration ceased. The resistance change in response to methacholine (0 to 20 microg/kg body weight given intravenously) was greater (p < 0.02) in oxygen-exposed animals. Oxygen exposure caused significant (60% increase) smooth muscle thickening (p < 0.05). Wy-50,295 prevented the oxygen-induced airway hyperresponsiveness and smooth muscle thickening. We conclude that chronic hyperoxic exposure causes an increase in pulmonary production of at least one lipid mediator, peptido-LTs, from newborn rats and that this is associated with airway smooth muscle layer thickening and, consequently, airway hyperresponsiveness.

Passive immunization with an anti-substance P antibody prevents substance P- and neurokinin A-induced bronchospasm in anesthetized guinea-pigs.

In a guinea-pig model of asthma, active immunization against substance P (SP) prevented the development of airways' hyperresponsiveness and reduced bronchospastic responses to SP (i.v.). The rat-mouse heterohybridoma NC1/34 secretes a specific, rat IgG1, anti-substance P antibody (alpha-SP Ab) which was isolated and purified by passing supernatant from cultures through thiophilic gel. Purity of antibody was about 50% (SDS-PAGE). The relative affinities of the alpha-SP Ab for SP, neurokinin A (NKA) and calcitonin gene-related peptide (CGRP) were estimated by ELISA using a constant amount of SP coupled (glutaraldehyde) to bovine serum albumin (BSA) to capture the antibody, alone and in the presence of increasing concentrations of the neuropeptides. At alpha-SP Ab dilutions of 1 in 5,000 to 1 in 32,000, CGRP did not prevent antibody binding to SP-BSA conjugate bound to the plates, but both SP and NKA prevented binding. In this system, the relative affinity of the alpha-SP Ab, at dilutions of 1 in 5,000 and 1 in 10,000, was about 50 times greater for SP than NKA. Whether passive immunization with alpha-SP Ab prevented bronchospastic responses to SP and NKA (i.v.), in vivo, was determined in groups of anesthetized guinea-pigs by recording pulmonary flow resistance (RL) and dynamic pulmonary elastance (EL). Injection of alpha-SP Ab (i.v., 5:1 molar ratio: alpha-SP Ab:SP total dose) did not alter baseline values of RL and EL, but markedly inhibited increases in RL and EL induced by SP and NKA (i.v.) without affecting responses to methacholine (i.v.). A control, "irrelevant" rat IgG-type antibody at a similar concentration had no effect on responses to SP or NKA. These findings indicate that passive immunization with a monoclonal alpha-SP Ab can prevent the bronchospastic effects of exogenous SP and NKA in guinea-pigs.

Release of monokines by pulmonary macrophages following antigen challenge in sensitized guinea pigs.

Guinea pigs were passively sensitized with immune serum to ovalbumin (OA), control serum, or saline. Twenty-four hours later, they inhaled aerosols of OA (2% in saline), saline, or lipopolysaccharide (LPS). Following anesthesia, bronchoalveolar lavage (BAL) was performed at 30, 60, 90 and 120 min postinhalation. Alveolar macrophages (AM) were isolated from the BAL fluid and incubated (18 h) in medium alone or with zymosan (1 mg/ml). Supernatants were collected and levels of interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) determined by bioassays. Unstimulated AM from animals that inhaled OA, saline, or LPS secreted similar amounts of IL-1 at 30, 60, and 90 min postinhalation. Zymosan (1 mg/ml) significantly increased IL-1 secretion by AM collected at 60 and 90 min from OA-sensitized animals that inhaled OA or saline. AM from guinea pigs sensitized to OA that inhaled OA or LPS secreted significantly increased amounts of IL-6 at 30, 60, 90, and 120 min postchallenge compared to saline sensitized controls. In all groups, AM from LPS-treated animals secreted large amounts of TNF-alpha at all sampling times postchallenge; AM from OA-sensitized and challenged animals secreted increasing amounts of TNF-alpha with time postchallenge, spontaneously and in response to zymosan. By contrast, AM from saline sensitized and challenged guinea pigs did not release detectable amounts of TNF-alpha spontaneously and secreted very low amounts in the presence of zymosan. These findings show that antigen challenge results in a rapid activation of AM isolated from BAL and suggest AM may initiate the development of inflammatory processes associated with antigen challenge.

Release of tumour necrosis factor alpha into bronchial alveolar lavage fluid following antigen challenge in passively sensitized guinea-pigs.

Five groups of ten female guinea-pigs were passively sensitized against ovalbumin (OA) (n = 9) or control guinea-pig serum (n = 1). 24 h later, they received mepyramine (0.5 mg/kg, i.p.) and 30 min later inhaled aerosols of: (A) OA (2 in 0.9% saline, 8 min, n = 4/9); (B) saline (40 min, n = 4/9); (C) LPS (40 min, Escherichia coli 0111:B4, 150 ng/kg in PBS, n = 1/9); and (D) the control animal was treated as in (C) (n = 1). Their tracheas were cannulated under pentobarbital anaesthesia and bronchial alveolar lavage (BAL) was performed with 2 x 5 ml PBS containing BSA (1%) (n = 1 group), or BSA (1%) and aprotinin (1000 KIU/ml) (n = 4 groups), at 30, 60, 90 or 120 min post-inhalations. BAL fluids recovered were centrifuged, the supernatants recovered and frozen until assayed for tumour necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) and interleukin-6 (IL-6). No TNF-alpha could be detected unless aprotinin was present in the lavaging solution. BAL fluid from OA-sensitized and control animals that had inhaled LPS contained high levels of TNF-alpha that peaked at 90 min. BAL fluid from OA sensitized animals that inhaled OA aerosols contained no detectable TNF-alpha at 30 min, but it was found in increasing amounts at 60, 90 and 120 min; TNF-alpha was not detected in fluid from any of the animals that inhaled saline. As BAL fluids were toxic to the cells used in the assays, neither IL-1 nor IL-6 could be measured. We conclude that the monokine TNF-alpha is released into BAL fluid following anaphylactic challenge of passively sensitized guinea-pigs. The presence of the antiprotease, aprotinin, in the lavaging solution is essential for the detection and measurement of TNF-alpha in BAL fluid.

Muscarinic-receptor functioning in tracheas from normal and ovalbumin-sensitive guinea pigs.

Responses to bilateral vagal nerve stimulation, to field stimulation, and to exogenous methacholine and histamine were compared in tracheas isolated from (a) saline injected (i.p.) and saline-aerosol exposed guinea pigs (control), (b) ovalbumin-sensitized and saline-aerosol exposed (sensitized) guinea pigs, and (c) ovalbumin-sensitized and 2% ovalbumin-aerosol exposed (challenged) guinea pigs. Tracheal pressor responses (cmH2O; 1 cmH2O = 98.1 Pa) to nerve and field stimulation, and maximal responses to methacholine and histamine were significantly increased in animals from group c compared with groups a and b. Dose-response lines in response to the two agonists, expressed as percent maximal contraction, did not differ among the groups. The M1 antagonist pirenzepine (0.1-10 nM) selectively reduced responses to nerve stimulation in all three groups. The M2 antagonist gallamine potentiated responses to nerve or field stimulation in all three groups. We conclude that M1, M2, and M3 muscarinic receptor functioning is similar in control and ovalbumin-sensitized guinea pigs. Changes in post-receptor transduction mechanisms may mediate the increased responsiveness noted in animals from group c.

A digital timing system for a small animal respiration pump.

A digital timing system has been developed to control the airflow from a respiration pump used in small animal pulmonary measurements. Three separate periods of up to 10 sec may be selected with millisecond resolution using thumb-wheel switches. A modular design using CMOS technology minimized the number of interconnections among the various parts of the system. A larger number of timing channels, longer time periods, and greater time resolution may be achieved by directly extending the existing design.

Muscarinic receptors and parasympathetic neurotransmission in guinea-pig trachea.

Muscarinic receptors involved in cholinergic neurotransmission were studied in isolated innervated guinea-pig tracheas using preganglionic (nerve) and postganglionic (field) stimulation, after blocking sympathetic effects with bethanidine (5 microM). Neostigmine (10 nM) significantly increased responses to nerve and field stimulation. The M1 antagonist pirenzepine (0.1-100 nM) selectively reduced tracheal responses to nerve stimulation in control and in neostigmine-treated tissues. The M2 antagonist gallamine (0.1-100 microM) significantly increased tracheal responses to nerve and field stimulation in control and in neostigmine-treated preparations. At concentrations that increased baseline tone, oxotremorine, arecoline and pilocarpine decreased responses to nerve and field stimulation. Gallamine (30 microM) selectively reduced the inhibitory effects of these agonists on responses to nerve and field stimulation. The findings indicate that cholinergic neurotransmission in guinea-pig trachea is modulated by facilitatory M1 receptors at parasympathetic ganglia and inhibitory M2 receptors at the postganglionic nerve endings.

The effects of vasoactive intestinal polypeptide on cholinergic neurotransmission in an isolated innervated guinea pig tracheal preparation.

To explore the possibility that VIP modulates cholinergic neurotransmission in the airways, we studied the effects of exogenous VIP on contractile responses of an isolated innervated guinea pig tracheal preparation to vagal nerve stimulation (NS), electrical field stimulation (EFS) and exogenous acetylcholine (ACh). VIP (2.5 x 10(-8), 2.5 x 10(-7) M) caused concentration-dependent reductions in responses to NS and EFS. Low frequency stimulations (1 Hz) were significantly more inhibited than higher frequencies (5 and 20 Hz). VIP inhibited NS significantly more than EFS at 1 and 5 but not 20 Hz. In contrast, VIP (2.5 x 10(-7) M) did not significantly reduce responses to ACh (3 x 10(-7), 10(-6), and 3 x 10(-6) M). The neutral endopeptidase inhibitor phosphoramidon (5 x 10(-6) M) enhanced responses of NS to VIP but did not affect responses to NS in the absence of VIP. We conclude that in the isolated guinea pig tracheal preparation, VIP can modulate cholinergic neurotransmission by actions that are predominantly on post-ganglionic nerves but it has also a small additional effect on ganglionic transmission.

Capsaicin selectively reduces airway responses to histamine, substance P and vagal stimulation.

Airway responsiveness to histamine, substance P, methacholine and bilateral electrical vagal stimulation was assessed in capsaicin-treated and control guinea pigs. In animals treated with capsaicin (50 mg/kg s.c.) 7 days before experiments, airway responsiveness to histamine, substance P and vagal stimulation was significantly reduced but responsiveness to methacholine was unchanged. The findings suggest that histamine and substance P, but not methacholine, require capsaicin-sensitive sensory afferent nerves for part of their actions.

High current pulser for transmural and field stimulation.

The determination of the requirements and the design of a constant current pulse generator with low internal resistance capable of providing the high currents required for transmural and field stimulation of biologic tissues are described. It is concluded that direct measurement of current flowing and voltage applied to electrodes is the best way of monitoring stimulus parameters.

An inexpensive bipolar stimulator.

The design of a pulse generator for electrical transmural stimulation is described. The device produces pulses of alternating polarity with amplitudes continuously variable up to 120 V and output currents limited to 6 mA. Pulse widths may be varied from 1 to 5 ms with repetition rates from 1.7 to 40 pulses per sec. A gate signal may be applied to inhibit output so that bursts of pulses may be generated.

Scanning electron microscopy of elastic networks from the bifurcation region of guinea pig carotid arteries.

We isolated the elastic network from the bifurcation region of guinea pig carotid arteries by treatment with hot alkali and examined its adventitial and adluminal components by SEM. The thickness of networks from common and external carotid arteries averaged 87.5 microns (+/- 6.9 SD) and from the carotid sinus averaged 46.8 microns (+/- 2.4 SD). The networks consisted of a mesh of elastic tissue that became a continuous sheet, 2 microns thick, which formed the internal elastic lamina (IEL). The IEL was fenestrated; the perforations varied in number among the vessels (occipital greater than carotid sinus greater than common = external carotid), and some were spanned by delicate elastic fibers. The IEL's adluminal surface was a smooth membranous sheet, which in some specimens bore unidirectional loose fibers, or was composed of tightly fused bundles of uni- or multidirectional fibers. The interior region of the cranial carotid sinus contained unique blister-like structures and dense clusters of fenestrations, together with a honeycomb-like mesh near the ascending pharyngeal artery. The outer, adventitial elastic layer consisted of a network of loose elastic fibers that were fused with the inner layers. We conclude that the structural differences noted among the common and external carotid arteries and carotid sinus are related to the sinus's unique pressure-sensing functions.

Observations on the fine structure of the baroreceptors and adrenergic innervation of the guinea-pig carotid sinus.

We examined the fine structure of the baroreceptors and the adrenergic innervation of the guinea-pig carotid sinus. The tunica adventitia contained many nerve bundles whose perineuria enclosed unmyelinated nerve fibers, alone or together with myelinated nerve fibers. Baroreceptors, which lay close to elastic and collagen fibers in the adventitia and media, were surrounded by "terminal" cells with ultrastructural features characteristic of Schwann cells and contained inclusions of various types. Morphologic features of the baroreceptors included densely packed mitochondria, osmiophilic lamellated and homogeneous bodies, clear and granular vesicles, lamellar systems, glycogen granules, neurofilaments, neurotubuli, and vacuolated mitochondria. In animals that had been treated with 6-hydroxydopamine, occasional electron-dense endings (or fibers) were observed in the adventitial layer. The baroreceptors in the guinea-pig carotid sinus appear to have most of the morphologic features reported for other species.

Comparison of the bronchoconstrictor and cardiovascular effects of some tachykinins in guinea pigs.

The effects of three tachykinins [substance P (SP), physalaemin (PH), and eledoisin-related peptide (ERP)] were investigated in anesthetized paralyzed guinea pigs. We measured airway resistance (R) and dynamic thoracic elastance (E) with a computerized technique, and blood pressure via a carotid artery. Tachykinins injected iv or intra-aortically (ia) induced dose-dependent increases in R and E, 4 times greater on iv than ia injection. They did not give rise to tachyphylaxis. As a bronchoconstrictor, PH was 5.0X and ERP 1.8X more potent than SP; time to peak response was longer for PH than for ERP and SP; and hypotensive responses, which were of similar magnitude for all three substances, lasted longest after PH. Bronchoconstrictor responses were unaltered by bilateral vagotomy, atropine, mecamylamine, and mepyramine. Morphine reduced PH-induced increases in R (P less than 0.01) and E (P less than 0.05), which were not reversed by naloxone, and capsaicin treatment 1 week before the experiments reduced both SP- and PH-induced increases in E (P less than 0.05). [D-Pro2,D-Trp7,9]-SP reduced ERP-induced increases in R and E, and [D-Arg1,D-Pro2,D-Trp7,9,Leu11]-SP reduced both SP- and PH-induced increases in R and E. We conclude that PH is the most potent bronchoconstrictor of the tachykinins tested. Tachykinin-induced bronchospasm is 'non-reflex' arising via a direct effect on airway smooth muscle; the release of histamine, acetylcholine, or other tachykinins is not involved in the responses. [D-Pro2,D-Trp7,9]-SP is more effective at SP-E receptors, and [D-Arg1,D-Pro2,D-Trp7,9,Leu11]-SP at SP-P receptors; both types of receptor are located all along the airways.

Aspirin, indomethacin, and tartrazine increase carotid-sinus-nerve activity and arterial blood pressure in guinea pigs.

Acetylsalicylic acid (ASA; 0.1-10 mg/kg), indomethacin (IND; 0.1-1.0 mg/kg), and tartrazine (TZ; 0.1-2.0 mg/kg), given intravenously induced dose-dependent increases in carotid-sinus nerve (CSN) activity, accompanied by increases in mean arterial blood pressure (MABP), but only the IND-induced MABP increases were dose-dependent. The MABP and CSN activity responses to all three drugs were not correlated, suggesting a direct action on CSN afferents that is unrelated to the pressor effects of the drugs. Sodium cromoglycate (10 mg/kg) selectively reduced the increases in CSN response to ASA and IND. Phentolamine (0.2 mg/kg) inhibited the increased CSN activity induced by ASA, IND, and TZ. These findings indicate that ASA, IND, and TZ act directly on carotid baroreceptors to increase their activity.