Differential susceptibility of tracheal contraction to nonadrenergic noncholinergic relaxation.

Studies of nonadrenergic noncholinergic inhibitory (NANCi) nerve-induced relaxations routinely examine relaxations in airway tissue in which tone has been established. Little is known about the ability of NANCi nerve stimulation to prevent airway smooth muscle contraction. The present study compares the capacity of NANCi nerve stimulation to prevent or reverse airway smooth muscle contraction. NANCi nerves in the trachea from ovalbumin-sensitized guinea pigs were subjected to electrical field stimulation (EFS, 10 Hz, 0.3 ms, 10 V, 35 min) initiated before or after induction of tone with antigen or histamine. In tissues precontracted with histamine or antigen, EFS elicited a rapid relaxation which peaked within the first 5 min and stabilized by 20 to 35 min. The peak relaxation was smaller in tissues precontracted with antigen, an effect that was not prevented by tissue treatment with a nitric oxide synthase inhibitor. In contrast, the stabilized level of NANCi relaxation did not differ between histamine- or antigen-contracted tissues. Activation of NANCi nerves prior to induction of tone also resulted in inhibition of the contractile actions of histamine and antigen. However, the stabilized level of tone induced by a contractile agonist added after initiation of EFS was greater than the stabilized tone caused by EFS in tissues already contracted with the same agonist. Relaxations elicited by S-nitrosoglutathione were reduced in antigen-precontracted tissues whereas vasoactive intestinal peptide-induced relaxant responses were similar in antigen- and histamine-precontracted tissues. Results of this study suggest that NANCi nerve activation is more effective at relaxing established airway smooth muscle tone than at preventing airway smooth muscle contraction. Further, the results suggest that the difference in NANCi activity in antigen-precontracted tissues cannot be ascribed solely to reductions in the nitric oxide-dependent component of the response.

Characterization of acetylcholinesterase in rabbit intrapulmonary arteries.

Acetylcholine (ACh) acts on the pulmonary vasculature to evoke vasodilation and, in some species, vasoconstriction. The actions of ACh are terminated by its rapid hydrolysis by cholinesterases. Aside from histochemical localization studies, there is little information on cholinesterase enzymes in pulmonary blood vessels. The present study addresses the hypothesis that pulmonary blood vessels contain sufficient cholinesterase activity to regulate the action of ACh in these tissues. Accordingly, studies were undertaken to characterize and quantify cholinesterase activities in pulmonary arteries and veins, quantify inhibition of enzyme activity, and investigate functional physiological consequences of cholinesterase inhibition. Cholinesterase activities in aorta and trachea also were examined for comparison. Kinetic studies showed that the lobar pulmonary arterial enzyme has a Michaelis constant of 55.3 +/- 17.0 microM and a maximum velocity of 8.6 +/- 2.7 nmol/min/mg protein similar to cholinesterases found in other peripheral tissues. Studies with selective inhibitors revealed that > 98% of total enzyme activity was attributable to acetylcholinesterase. Similar levels of enzyme activity were found in homogenates of lobar branch intrapulmonary arteries, intrapulmonary veins, and aorta. The majority of enzyme activity was localized to the membrane fraction, although a moderate amount was found in the cytosol. Studies in intact intrapulmonary lobar arteries showed that these vessels had cholinesterase activity comparable with that found in intact trachealis muscle and that neostigmine (10 nM to 10 microM) caused concentration-dependent inhibition of enzyme activity. In isolated intrapulmonary lobar arteries, functional studies showed that 1 and 10 microM neostigmine significantly potentiated ACh-induced contractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological characterization of muscarinic receptors mediating acetylcholine-induced contraction and relaxation in rabbit intrapulmonary arteries.

Rabbit pulmonary arteries exhibit a biphasic response to acetylcholine consisting of an endothelium-dependent contraction in tissues at resting tone and an endothelium-dependent relaxation in vessels with elevated tone. Each response was studied separately by treating the arteries with inhibitors of nitric oxide synthase to block the relaxant response or with inhibitors of cyclooxygenase to inhibit the contractile response. In the present study, experiments in isolated pulmonary arteries were undertaken to characterize the muscarinic receptor subtypes mediating the two separate responses by utilizing subtype-selective antagonists and determining pA2 values of the antagonists through Schild analysis. Both the relaxant and the contractile responses were inhibited most potently by atropine and by the M3-selective antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide. The pA2 values for inhibition of the contractile and relaxant responses were 9.44 and 8.79 for atropine and 8.92 and 9.29 for 4-diphenylacetoxy-N-methylpiperidine methiodide, respectively. The M1-selective antagonist pirenzepine and the M2-selective antagonist (11-([2-[(diethylamino)methyl]-1-piperidinyl]- acetyl)-5, 11-dihydro-6H-pyrido[2,3-b][1,4]-benzodiazepine-6-one) displayed much lower affinities for the muscarinic receptors mediating these responses. The pA2 values for inhibition of the contractile and relaxant responses were 6.77 and 6.74 for pirenzepine and 6.06 and 5.70 for (11-([2-[(diethylamino)methyl]-1-piperidinyl] acetyl)-5, 11-dihydro-6H-pyrido[2,3-b][1,4]-benzodiazepine-6-one), respectively.

Atypical presynaptic alpha-adrenoceptor modulation of neurally-mediated cholinergic responses in guinea-pig tracheal smooth muscle.

Cholinergic excitatory nerves in guinea-pig trachea are subject to inhibitory control by presynaptic alpha 2-adrenoceptors. Recently, the nature of these receptors has come into question insofar as the presynaptic inhibitory effects of the alpha 2-adrenoceptor agonist, clonidine, in the guinea-pig trachea have been shown to be antagonized by the alpha 2-adrenoceptor antagonist, yohimbine, as well as the alpha 1-adrenoceptor antagonist, prazosin. This inhibitory action of prazosin had not been described previously in the airways and may relate to the use of norepinephrine rather than clonidine as the alpha-adrenoceptor agonist in earlier studies. The present study evaluates the susceptibility of norepinephrine-induced inhibition of neurally-mediated cholinergic excitatory responses to antagonism by prazosin and yohimbine under conditions identical to those which showed clonidine to be sensitive to these antagonists. In tissues pretreated with guanethidine, propranolol and indomethacin, norepinephrine (1 microM) induced a 37-fold rightward shift of the frequency-response curve for neurally-mediated cholinergic contractions which was reversed partially by pretreatment of tissues with yohimbine. Norepinephrine also caused a concentration-dependent inhibition of cholinergic 'twitch' responses induced by intermittent (1 Hz) nerve stimulation. This action of norepinephrine was antagonized in a concentration-dependent manner by yohimbine but was unaffected by prazosin. These results indicate that in guinea-pig trachea the presynaptic inhibitory actions of norepinephrine on cholinergic nerves are mediated via classical alpha 2-adrenoceptors, i.e. receptors that can be blocked by yohimbine but not by prazosin.(ABSTRACT TRUNCATED AT 250 WORDS)

Omega-conotoxin-sensitive calcium channels modulate autonomic neurotransmission in guinea pig airways.

omega-Conotoxin GVIA, a peptide derived from the marine snail Conus geographus, is an antagonist of the neuronal N type voltage-sensitive calcium channels associated with neurotransmitter release. The present study investigated effects of this peptide on neurally mediated responses in airways isolated from the guinea pig to determine whether airway nerves are modulated by omega-conotoxin-sensitive calcium channels. Electrical field stimulation was used to induce neurally mediated tachykininergic excitatory responses in guinea pig bronchus and cholinergic excitatory and nonadrenergic noncholinergic inhibitory responses in guinea pig trachea. Exogenous agonists were administered to induce contractile (acetylcholine, substance P) or relaxation (sodium nitroprusside) responses. Tissues were incubated with omega-conotoxin (1 microM) or its vehicle (10 mM acetic acid) for 30 min before establishing frequency- or concentration-response relationships to the various stimuli. Frequency-response curves for neurally mediated cholinergic, nonadrenergic nocholinergic inhibitory and tachykininergic responses were shifted to the right by omega-conotoxin to a similar extent (4- to 5-fold). omega-Conotoxin had no effect on contractile responses elicited by exogenous acetylcholine or substance P or on relaxations induced by sodium nitroprusside. These findings indicate that neurotransmission in afferent tachykininergic kininergic nerves and in efferent cholinergic excitatory and nonadrenergic noncholinergic inhibitory nerves in the airways is modulated by a prejunctional omega-conotoxin-sensitive mechanism.

Enzymatic modulation of vasoactive intestinal peptide and nonadrenergic noncholinergic inhibitory responses in guinea pig tracheae.

The airways of the guinea pig are innervated by four types of autonomic nerves: cholinergic excitatory, adrenergic inhibitory, nonadrenergic noncholinergic (NANC) excitatory, and NANC inhibitory. Tachykinins (neurokinins A and B and substance P) are believed to mediate NANC excitatory responses, and vasoactive intestinal peptide (VIP) has been proposed as the chemical mediator of the NANC inhibitory system. Enzymatic degradation represents an important means by which the biologic actions of neurotransmitters are terminated. In the present study, relaxation responses of guinea pig tracheae to NANC nerve stimulation and to exogenous VIP administration were compared in the absence and presence of various peptidase inhibitors. NANC inhibitory responses elicited by electrical field stimulation were unaffected by aprotinin or soybean trypsin inhibitor but were depressed by thiorphan or leupeptin. Concentration-response curves to exogenous VIP were shifted to the left by soybean trypsin inhibitor but were not affected by aprotinin, leupeptin, or thiorphan. After tachykinin depletion with capsaicin, thiorphan also induced a leftward shift in the VIP concentration-response curve. Under the same conditions, thiorphan failed to influence NANC inhibitory responses. These results indicate that the NANC inhibitory neurotransmitter is not metabolized by enzymes susceptible to inhibition by aprotinin, leupeptin, soybean trypsin inhibitor, or thiorphan and, accordingly, distinguish NANC nervous responses from those induced by VIP. The results also suggest that the NANC excitatory system can interact functionally with the NANC inhibitory system, as evidenced by the blunting of NANC relaxation responses following inhibition of tachykinin metabolism and elimination of this effect by capsaicin.

Nonadrenergic bronchodilation induced by high concentrations of sulfur dioxide.

SO2 is an environmental pollutant known to elicit bronchospasm in susceptible subjects. We observed that brief exposure of artificially bronchoconstricted cats to high concentrations of SO2 induces a bronchodilator response. This study assessed the characteristics of this response and examined various mechanisms that might underlie it. Cats were anesthetized with diallylbarbital-urethan, and airway smooth muscle tone, measured by lung resistance and dynamic lung compliance, was elevated with a continuous infusion of 5-hydroxytryptamine. Administration of 10 breaths of SO2 via a tracheostomy induced concentration-dependent bronchodilation in the range 100-1,000 parts/million. Only infrequently was bronchoconstriction observed before bronchodilation. SO2-induced bronchodilator responses were unaffected by pretreatment with intravenous atropine or propranolol, establishing them as nonadrenergic noncholinergic (NANC) in origin. Neither the ganglionic blocking agent hexamethonium nor the nerve toxin tetrodotoxin influenced the SO2-induced bronchodilation, thus excluding a role for central or local autonomic reflexes in the response. Efforts to modulate the response by pretreatment with the cyclooxygenase inhibitor indomethacin or the mediator release inhibitor cromolyn sodium also were unsuccessful. Administration of acidic aerosols failed to mimic the SO2-induced bronchodilator response. Although the mechanism whereby SO2 induces bronchodilation under these experimental conditions remains unclear, release of a NANC inhibitory transmitter from a neural, epithelial, or other cellular source via a mechanism insensitive to both tetrodotoxin and cromolyn is a distinct possibility. An intrinsic NANC inhibitory system may exist in feline airways functioning as a local regulator of bronchomotor tone and possibly serving to override responses to strong, potentially asphyxial bronchoconstrictive stimuli.

Presynaptic alpha adrenoceptor modulation of neurally mediated cholinergic excitatory and nonadrenergic noncholinergic inhibitory responses in guinea pig trachea.

Cholinergic and nonadrenergic noncholinergic (NANC) excitatory nerves in guinea pig trachea are subject to presynaptic alpha-2 adrenoceptor inhibitory control. Although the trachea is also innervated by NANC inhibitory nerves, little is known about their presynaptic regulation. The present study assessed the capacity of alpha-1 and alpha-2 adrenoceptor agonists to modulate NANC inhibitory nerves and for comparison, cholinergic excitatory nerves in guinea pig trachea. To eliminate effects of intrinsic sympathetic nerve stimulation and prostanoid production, tissues were pretreated with guanethidine, propranolol and indomethacin. The alpha-2 adrenoceptor agonist, clonidine (1 microM), induced a 12-fold rightward shift of the frequency-response curve for neurally mediated cholinergic contractions but had no effect on the concentration-response curve for exogenously administered acetylcholine. This action of clonidine was inhibited in a concentration-dependent manner by the alpha-2 adrenoceptor antagonist, yohimbine, and by the alpha-1 adrenoceptor antagonist, prazosin, NANC inhibitory responses were unaffected by clonidine (1 microM). The alpha-1 adrenoceptor agonist, phenylephrine (1 microM), failed to influence responses induced by cholinergic or NANC inhibitory nerve stimulation, acetylcholine or vasoactive intestinal peptide. Furthermore, in tissues treated with propranolol but not subjected to adrenergic neuronal blockade with guanethidine, neurally mediated cholinergic responses were not altered by yohimbine (0.3 microM) treatment. These results indicate that in guinea pig trachea: 1) cholinergic nerves are modulated by presynaptic, prazosin-sensitive inhibitory presynaptic alpha-2 adrenoceptors and 2) NANC inhibitory nerves do not possess presynaptic, modulatory alpha adrenoceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Nicotinic agonist modulation of feline bronchomotor tone.

1. The bronchomotor actions of three nicotinic cholinoceptor agonists were investigated in anaesthetized, mechanically ventilated cats. The agonists were administered intravenously after increasing baseline airways smooth muscle tone with an infusion of 5-hydroxytryptamine. 2. Acetylcholine induced a biphasic change in lung resistance, characterized by initial bronchoconstriction followed by bronchodilation. The specific nicotinic cholinoceptor agonists, nicotine and dimethylphenylpiperazinium (DMPP), principally induced bronchodilator responses, although initial bronchoconstrictor phases were observed occasionally. 3. All bronchoconstrictor phases were sensitive to muscarinic cholinoceptor blockage with atropine. DMPP-induced bronchodilator responses were adrenergic in nature, whereas those induced by either nicotine or acetylcholine resulted from a combination of adrenergic and non-adrenergic influences. 4. It is concluded that intravenously administered nicotinic cholinoceptor agonists exert varying actions on feline bronchomotor tone and that these actions result from activation of different autonomic inputs.

Pulmonary clearance of atrial natriuretic peptides.

Pulmonary clearance and metabolism of atrial natriuretic peptides were studied in isolated perfused rat lungs utilizing high performance liquid chromatography and radioimmunoassay analyses. The disappearance rate of atrial peptides from the perfusion medium followed pseudo-first order kinetics and was associated with formation of lower molecular weight products. Pretreatment of the lungs with MK-521, an angiotensin converting enzyme inhibitor, decreased pulmonary clearance values for both atrial natriuretic peptide and atriopeptin III but did not affect clearance of atriopeptin II. Aminopeptidase inhibition also decreased the rate of atrial natriuretic peptide clearance, however carboxypeptidase inhibition did not affect atrial natriuretic peptide, atriopeptin III or atriopeptin II clearance. The results suggest that the lung through uptake and metabolic processes participates in the clearance and enzymatic metabolism of the atrial natriuretic peptides.

Pulmonary vasorelaxant activity of atrial peptides.

Pulmonary vascular relaxant effects of the 28-amino acid atrial natriuretic peptide and atriopeptins I, II and III (21, 23 and 24 amino acid peptides, respectively) were studied in isolated blood vessels and in perfused rat lungs. In isolated tissue studies, intrapulmonary arteries were more responsive to the relaxant effects of atrial peptides than the main pulmonary artery or aorta. In perfused lung preparations, each of the four atrial peptides produced dose-dependent pulmonary vasodilation of PGF2 alpha or hypoxia-induced pulmonary vasoconstriction. Atriopeptin I was the least potent pulmonary vasodilator peptide in all studies. Pretreatment of perfused lungs with various peptidase inhibitors, including the angiotensin converting enzyme inhibitors, captopril and MK-521, the carboxypeptidase inhibitor, 1,10-phenanthroline, and the aminopeptidase inhibitor, bestatin, variably potentiated the pulmonary vasodilator activities of the atrial peptides. The results demonstrate that atrial peptides released from the right heart into the pulmonary circulation can have potent vasorelaxant effects in the pulmonary vascular bed and further suggest that upon passage through the lung atrial peptides may undergo metabolic degradation that alters their pulmonary vasodilator activities.

Epidermal growth factor augments reactivity to angiotensin II in the rat pulmonary circulation.

To determine if epidermal growth factor (EGF), a vascular smooth muscle mitogen exhibiting systemic vasoactivity, causes constriction or dilation of the pulmonary vascular bed, this study evaluated the actions of EGF in isolated, buffer-perfused rat lungs and in isolated rat pulmonary arteries. In perfused rat lungs with baseline vasomotor tone, EGF administered at bolus doses of 10(-9) to 3 x 10(-7) M failed to exert either constrictor or dilator actions or to promote edema formation as evidenced by a constant lung wet-to-dry-weight ratio. Elevation of baseline tone with either prostaglandin (PG)F2 alpha or angiotensin II also failed to unmask an effect of EGF on pulmonary vascular resistance. In contrast to these negative observations, pretreatment with 5 x 10(-8) M EGF consistently augmented pressor responses evoked by angiotensin II. Constrictor responses to potassium chloride and to PGF2 alpha were unaffected by EGF pretreatment. In isolated rat extrapulmonary arteries, EGF provoked contraction in 40% of the preparations studied. Responsive vessels exhibited maximal EGF-induced contractions that were approximately 25% of that associated with angiotensin II and were characterized by an ED50 of 7 x 10(-8) M. Relaxant activity of EGF could not be demonstrated in isolated arterial preparations with normal resting tone or with tone elevated by addition of norepinephrine to the tissue bath. Endothelial denudation also failed to unmask contractile or relaxant effects of EGF. Similar to its actions in isolated, perfused rat lungs, EGF potentiated contractions of isolated pulmonary arteries induced by angiotensin II.(ABSTRACT TRUNCATED AT 250 WORDS)

An analysis of the haemodynamic effects of tolbutamide in conscious dogs.

1. In conscious chronically instrumented dogs, tolbutamide (5-45 mg/kg) induced significant dose-related increases in mean arterial pressure and left ventricular end-diastolic pressure. 2. Cardiac output was decreased while heart rate, d(LVP)/dt, and regional myocardial performance at the left ventricle were not significantly affected. Computed total peripheral resistance was increased. 3. Pretreatment with the alpha-antagonist phentolamine (1-1.5 mg/kg) abolished the pressor response. Furthermore, the pressor response to norepinephrine (0.1 microgram/kg) was enhanced by pretreatment with tolbutamide (45 mg/kg). 4. In an isolated tissue preparation using ring segments of canine femoral arteries, neither tolbutamide nor its major hepatic metabolites (carboxytolbutamide, p-toluene-sulfonamide and p-toluene-sulfonylurea) caused any smooth muscle contraction. However, pretreatment of these tissues with 10(-4), 10(-3), or 10(-2) mol/l tolbutamide potentiated the contractile response to norepinephrine by up to 19% and to phenylephrine by up to 8%. 5. It was concluded that the pressor effect of tolbutamide arises by potentiating the alpha-adrenoceptor mediated vasoconstrictor action of circulating endogenous catecholamines.

The effects of antagonists of vasoactive intestinal peptide on nonadrenergic noncholinergic inhibitory responses in feline airways.

Relaxations of the feline intrapulmonary bronchus (IPB) induced by VIP or nonadrenergic noncholinergic (NANC) inhibitory nervous stimulation were unaffected by the VIP receptor antagonist [Ac-Tyr1,D-Phe2]-GRF (1-29) (30 microM). A second VIP antagonist, [pCl-D-Phe6,Leu17]-VIP (30 microM), also had no effect on NANC relaxation responses or IPB sensitivity to VIP. However, responses to three of the four highest VIP concentrations were inhibited by this antagonist. These results indicate that [Ac-Tyr1,D-Phe2]-GRF (1-29) and [pCl-D-Phe6,Leu17]-VIP are not effective competitive antagonists of VIP receptors in feline airways and, hence, have but limited applicability in determining the role of VIP in mediating airway NANC inhibitory responses in this tissue.

The effect of epithelium removal on non-adrenergic, non-cholinergic inhibitory responses in the isolated central airways of the cat and guinea pig.

Epithelium removal from the feline or the indomethacin-treated guinea pig trachea had no effect on tissue sensitivity or responsiveness to the contractile actions of pharmacological agonists or electrical field stimulation. In the feline hilar bronchus, epithelium removal had no effect on tissue sensitivity or responsiveness to acetylcholine or electrical field stimulation but increased bronchial sensitivity to serotonin without affecting responsiveness. Non-adrenergic non-cholinergic (NANC) relaxation responses elicited by electrical field stimulation in airway preparations from either species were unaffected by epithelium removal. These results suggest that the epithelium does not modulate contractile responses in the feline trachea but may modulate the actions of specific contractile agonists in the feline hilar bronchus. Further, NANC relaxation responses appear to occur independently of the airway epithelium.