Inhibitory action of atrial natriuretic factor on cholinergic and nonadrenergic, noncholinergic neurotransmission in guinea pig small intestine

We studied the effect of synthetic rat atrial natriuretic factor (ANF) (Ser 99-Tyr 126) on the isolated guinea pig proximal ileum. This preparation contained about one-third of the endogenous tissue ANF content which, for the most part, comes from the blood. ANF inhibited, in a dose-dependent manner, cholinergic twitch contractions (EC50 = 4.2 nM), nonadrenergic, noncholinergic (NANC) primary and rebound contractions and histamine-induced sustained tonic contraction (but not carbachol induced contraction) of the longitudinal muscle. Ascending enteric reflex (AER) contractions of the circular muscle were inhibited though not dose-dependently. We suggest pre- and post-synaptic actions of sustained intestinal tissue and blood ANF levels which may play a role in regulating motor activity and muscle tone of the small intestine.     

Adenosine selectively inhibits noncholinergic transmission in guinea pig bronchi

The neuromodulatory action of adenosine and ATP was investigated in isolated guinea pig bronchial strip chain preparations contracted with electrical field stimulation. The tissues were placed in organ baths containing physiological salt solution and stimulated at 8-Hz frequency, 0.5-ms pulse duration, and 30 V (approximately 100 mA) for 5 s. Electrical field stimulation evoked a biphasic contraction of bronchial muscle, consisting of an initial contraction followed by a sustained contraction, which was mediated by intramural cholinergic and noncholinergic nerve stimulations, respectively. Adenosine, at concentrations greater than M, caused a concentration-dependent inhibition in the height of the noncholinergically mediated contraction, accompanied by a very weak inhibition on the cholinergically mediated response. ATP (10(-5) to 3 x 10(-3) M) also produced a similar inhibitory effect on the noncholinergically mediated contraction, but the inhibitory potency was less than that of adenosine. The inhibitory response to adenosine was enhanced by the pretreatment with dipyridamole (2 x 10(-6) M) but antagonized with aminophylline (10(-5) M). Contractions of bronchial muscle evoked by exogenous acetylcholine (2 x 10(-6) M) or substance P (2 x 10(-7) M) were significantly inhibited by the adenosine (3 x 10(-4) M) pretreatment. These data suggest that in isolated guinea pig bronchi adenosine selectively inhibits noncholinergic neurotransmission through prejunctional P1-purinoceptors.     

Arginase attenuates inhibitory nonadrenergic noncholinergic nerve-induced nitric oxide generation and airway smooth muscle relaxation

Background

Recent evidence suggests that endogenous arginase activity potentiates airway responsiveness to methacholine by attenuation of agonist-induced nitric oxide (NO) production, presumably by competition with epithelial constitutive NO synthase for the common substrate, L-arginine. Using guinea pig tracheal open-ring preparations, we now investigated the involvement of arginase in the modulation of neuronal nitric oxide synthase (nNOS)-mediated relaxation induced by inhibitory nonadrenergic noncholinergic (iNANC) nerve stimulation.

Methods

Electrical field stimulation (EFS; 150 mA, 4 ms, 4 s, 0.5 – 16 Hz)-induced relaxation was measured in tracheal preparations precontracted to 30% with histamine, in the presence of 1 μM atropine and 3 μM indomethacin. The contribution of NO to the EFS-induced relaxation was assessed by the nonselective NOS inhibitor L-NNA (0.1 mM), while the involvement of arginase activity in the regulation of EFS-induced NO production and relaxation was investigated by the effect of the specific arginase inhibitor nor-NOHA (10 μM). Furthermore, the role of substrate availability to nNOS in EFS-induced relaxation was measured in the presence of various concentrations of exogenous L-arginine.

Results

EFS induced a frequency-dependent relaxation, ranging from 6.6 ± 0.8% at 0.5 Hz to 74.6 ± 1.2% at 16 Hz, which was inhibited with the NOS inhibitor L-NNA by 78.0 ± 10.5% at 0.5 Hz to 26.7 ± 7.7% at 8 Hz (P < 0.01 all). In contrast, the arginase inhibitor nor-NOHA increased EFS-induced relaxation by 3.3 ± 1.2-fold at 0.5 Hz to 1.2 ± 0.1-fold at 4 Hz (P < 0.05 all), which was reversed by L-NNA to the level of control airways in the presence of L-NNA (P < 0.01 all). Similar to nor-NOHA, exogenous L-arginine increased EFS-induced airway relaxation (P < 0.05 all).

Conclusion

The results indicate that endogenous arginase activity attenuates iNANC nerve-mediated airway relaxation by inhibition of NO generation, presumably by limiting L-arginine availability to nNOS.     

Cholinergic and nonadrenergic mechanisms in human and guinea pig airways

Electrical field stimulation (70 V, 1 ms, 0.2-500 Hz) of human bronchial strips and guinea pig tracheal chains produced contractile and relaxant responses. Contractions were blocked by atropine, 10(-6) M, and tetrodotoxin (TTX), 0.1-1.0 micrograms/ml, demonstrating a cholinergic excitatory neural component. Frequencies causing half-maximal contractile response to field stimulation (EFc 50) were 10 +/- 2 Hz for guinea pig and 13 +/- 1 Hz for human airways. Relaxations were unmasked by atropine 10(-6) M and slightly diminished by propranolol in guinea pig but not human airways, demonstrating a predominantly nonadrenergic inhibitory pathway in both species. Relaxation of intrinsic tone occurred at stimulation frequencies of 1 Hz or more. Frequencies causing half-maximal relaxation (EFi 50) were 3.5 +/- 0.3 Hz for guinea pig trachealis and 38 +/- 6 Hz for human bronchi. Following 1 microgram/ml TTX, EFi 50 values increased to 104 +/- 12 and 70 +/- 14 Hz, respectively. Frequencies of field stimulation that were inhibitable by TTX (less than or equal to 20 Hz) induced greater relaxation in guinea pig than human airways (70 vs. 10% of the maximal relaxation to 10(-2) M theophylline, respectively). The methods of analysis outlined in this study can be used to compare relative degrees of functional innervation between tissues from the same or different species.     

Absence of nonadrenergic noncholinergic relaxation in the cat cervical trachea

Published in vivo experiments have not supported in vitro reports of the presence of nonadrenergic noncholinergic (NANC) inhibitory pathways in the cat trachea. We therefore examined these pathways, measuring tension in an innervated tracheal segment, flow resistance in more distal airways, and dynamic compliance, in 10 anesthetized mechanically ventilated cats. Initially, cervical vagal stimulation evoked contraction followed by relaxation of smooth muscle of trachea and lower airways; sympathetic stimulation evoked relaxation only. After muscarinic blockade and restoration of smooth muscle tone with 5-hydroxytryptamine (5-HT) applied topically to the tracheal mucosa, vagal stimulation did not affect tracheal segment tension, whereas sympathetic-evoked relaxation was preserved. Similar results were found when tone was restored with intravenous 5-HT, with vagal stimulation also decreasing resistance and increasing compliance. We conclude that NANC pathways are present in lower airways but not in the cervical trachea of the cat. We hypothesize that parasympathetic constriction of cat airway smooth muscle can occur without simultaneous NANC activation, whereas NANC activity occurs only in tandem with parasympathetic stimulation.     

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.     

Protective role of epithelium in the guinea pig airway

We developed an in vitro system to assess the role of the epithelium in regulating airway tone using the intact guinea pig trachea (J. Appl. Physiol. 64: 466-471, 1988). This method allows us to study the response of the airway when its inner epithelial surface or its outer serosal surface is stimulated independently. Using this system we evaluated how the presence of intact epithelium can affect pharmacological responsiveness. We first examined responses of tracheae with intact epithelium to histamine, acetylcholine, and hypertonic KCl when stimulated from the epithelial or serosal side. We then examined the effect of epithelial denudation on the responses to these agonists. With an intact epithelium, stimulation of the inner epithelial side always caused significantly smaller changes in diameter than stimulation of the outer serosal side. After mechanical denudation of the epithelium, these differences were almost completely abolished. In the absence of intact epithelium, the trachea was 35-fold more sensitive to histamine and 115-fold more sensitive to acetylcholine when these agents were applied to the inner epithelial side. In addition, the presence of an intact epithelium almost completely inhibited any response to epithelial side challenge with hypertonic KCl. These results indicate that the airway epithelial layer has a potent protective role in airway responses to luminal side stimuli, leading us to speculate that changes in airway reactivity measured in various conditions including asthma may result in part from changes in epithelial function.     

Properties of synaptic currents during nonadrenergic inhibition of smooth-muscle cells of the guinea pig large intestine

Using a double sucrose gap method, inhibitory junction potentials (IJP) appeared in muscles of the circular layer of the large intestine in response to intramural stimulation in the presence of atropine. Under voltage clamp conditions, an inhibitory junction current (IJC) in the outward direction appeared in response to the same stimulus, declining exponentially 100–150 msec after the peak. The amplitude of IJC was a linear function of membrane potential; the reversal potential of the peak IJC was in the region of the potassium equilibrium potential. The time constant of decay (τ) depended exponentially on membrane potential, falling by a factor ofe on hyperpolarization by 120 mV. A decrease or increase in quantum composition of IJC caused a corresponding change in τ of IJC decay. Meanwhile apamine (5×10^?7 g/ml) reduced the amplitude of IJC without affecting its kinetics. The action of ATP (10^?3 M) led to a decrease in amplitude and τ of decay of IJC, evidently on account of occupation of some postsynaptic receptors by ATP. It is suggested that ATP facilitates the delayed diffusion of releasing mediator, by occupying synaptic receptors. Since an increase in the quantity of secreted mediator caused only a very small increase in the amplitude of IJC, it was postulated that under normal conditions the postsynaptic effect of the released mediator is close to maximal.     

Nonadrenergic noncholinergic vasodilation of guinea pig pulmonary arteries is mediated by nitric oxide.

Nonadrenergic noncholinergic (NANC) mediated vasodilation may contribute to the maintenance of low pulmonary vascular tone. The NANC neurotransmitters, nitric oxide (NO) and the sensory neuropeptides, substance P and calcitonin gene related peptide (CGRP), were investigated as possible mediators of NANC vasodilation in guinea pig pulmonary arteries. Fresh guinea pig pulmonary artery rings, with and without an intact endothelium, were mounted in organ baths containing Krebs solution and precontracted with the prostaglandin F2alpha analogue U44069. In both endothelium-intact and denuded vessels, electrical field stimulation (1-12 Hz) in the presence of guanethidine and atropine resulted in a frequency-dependent vasodilation. The peptide fragment hCGRP8-37, a competitive antagonist of the CGRP receptors, the peptide fragment NK1 antagonist SP4-11, and the nonpeptide NK1 antagonist RP67580 had no effect on NANC vasodilation. In both endothelium-intact and denuded vessels, N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthesis, inhibited NANC vasodilation, an effect that was reversible with L-arginine. We conclude that NANC vasodilation in guinea pig pulmonary arteries is mediated predominantly through NO activity.     

Glucose does not activate nonadrenergic, noncholinergic inhibitory neurons in the rat stomach

We reported previously that intravenously administered d-glucose acts in the central nervous system to inhibit gastric motility induced by hypoglycemia in anesthetized rats. The purpose of this study was to determine whether this effect is due to inhibition of dorsal motor nucleus of the vagus (DMV) cholinergic motoneurons, which synapse with postganglionic cholinergic neurons, or to excitation of DMV cholinergic neurons, which synapse with postganglionic nonadrenergic, noncholinergic (NANC) neurons, particularly nitrergic neurons. Three approaches were employed: 1) assessment of the efficacy of d-glucose-induced inhibition of gastric motility in hypoglycemic rats with and without inhibition of nitric oxide synthase [10 mg/kg iv nitro-l-arginine methyl ester (l-NAME)], 2) assessment of the efficacy of intravenous bethanechol (30 mug.kg(-1).min(-1)) to stimulate gastric motility in hypoglycemic rats during the time of d-glucose-induced inhibition of gastric motility, and 3) determination of c-Fos expression in DMV neurons after intravenous d-glucose was administered to normoglycemic rats. Results obtained demonstrated that l-NAME treatment had no effect on d-glucose-induced inhibition of gastric motility; there was no reduction in the efficacy of intravenous bethanechol to increase gastric motility, and c-Fos expression was not induced by d-glucose in DMV neurons that project to the stomach. These findings indicate that excitation of DMV cholinergic motoneurons that synapse with postganglionic NANC neurons is not a significant contributing component of d-glucose-induced inhibition of gastric motility.     

Histamine, endogenous prostaglandins and cyclic nucleotides in the regulation of airway muscle responses in the guinea pig

Indomethacin (30 mg/kg, i.p.) reduced pulmonary resistance in guinea pigs but did not affect their sensitivity to histamine. This treatment preferentially reduced the generation of PGE2 by isolated tracheal preparations. The ratios of PGF2 alpha/PGE2 before and after treatment were 1/1 and 6/1, respectively. Chronic indomethacin treatment (30 mg/kg, i.p., twice a day for 4 days) increased histamine sensitivity in vivo 2 fold while a longer treatment (10 days) was without effect. The efficacy of histamine and the potency of isoproterenol in tracheal tissues were unaffected by either treatment. Indomethacin (17 microM for 30 min) relaxed tracheal tissues but not bronchial tissues. Responses of both tissues to contractile agonists were potentiated after indomethacin treatment. The efficacy of histamine was smaller in bronchi than in tracheas. Similarly, PGE2, PGI2 and isoproterenol were less potent in bronchi. Basal amounts of cyclic AMP were higher in bronchi than in tracheas; indomethacin did not affect the basal amounts of cyclic AMP in tracheal tissues but reduced them in bronchial preparations. Histamine elevated cyclic AMP content in both preparations; this elevation was reduced by indomethacin. While prostaglandins play a role in modulating airway responses in vitro, their role in airways in normal animals in vivo is more difficult to demonstrate.     

Anesthesia protocol for hyperpnea-induced airway obstruction in the guinea pig

Guinea pigs (GP; Cavia porcellus) are used extensively as an experimental animal model in a wide range of disciplines including respiratory physiology. Guinea pigs are difficult to anesthetize, and many investigators use paralytic agents to eliminate spontaneous respiratory movements; however, strict federal regulations and institutional policies governing use of paralytic agents are few. We report an anesthesia protocol, using the injectable anesthetic agents sodium pentobarbital (SP) and xylazine (XYL) for the GP that induces consistent anesthesia while eliminating use of paralytic agents. Sixty percent of the calculated SP dose (45 mg/kg of body weight) was given for anesthesia induction, followed by 50% of the calculated XYL dose (7 mg/kg) 15 min later. Depth of anesthesia was monitored by response to toe pinch, ECG, and spontaneous respiratory movements. The animals were given additional boosts of SP (5 to 15% of the original dose, i.p. or i.v.) if a change in anesthesia depth was noted. Thirty-one animals completed the hyperpnea-induced bronchoconstriction (HIB) study with no fatalities. Using this protocol, we collected consistent, repeatable, and reliable data without use of propranolol or skeletal muscle paralytics. We believe that this protocol is not restricted to the GP and could be adapted for use in other terminal experiments.     

Mechanism of A23187-induced airway obstruction in the guinea pig

Exposure of conscious guinea pigs to A23187 aerosol produced a concentration-related increase of excised lung gas volume (ELGV), . ., postmortem pulmonary gas trapping. Measurements of ELGV were highly correlated with measurements of dynamic compliance (C_dyn) and total pulmonary resistance (R_L) and were used as an indication of airway obstruction. We pretreated guinea pigs intravenously with the following drugs: atropine; LY163443, a selective LTD₄/E₄ antagonist; indomethacin; propranolol; and pyrilamine. The guinea pigs were exposed for 8 minutes to the A23187 aerosol, and ELGV measurements were then made. Atropine or pyrilamine prevented the A23187-induced gas trapping. Indomethacin or propranolol tended to potentiate the response and when combined, they potentiated the gas trapping by 80%. LY163443 had no effect alone, but when combined with indomethacin, propranolol, and pyrilamine, inhibited A23187-induced gas trapping by 67%. We conclude that cholinergic and histaminergic mechanisms play major roles in the ionophore-induced pulmonary gas trapping of the guinea pig. With appropriate pretreatment, sulfidopeptide leukotrienes may produce a substantial effect.     

Mechanism of A23187-induced airway obstruction in the guinea pig

Exposure of conscious guinea pigs to A23187 aerosol produced a concentration-related increase of excised lung gas volumes (ELGV), i.e., postmortem pulmonary gas trapping. Measurements of ELGV were highly correlated with in vivo measurements of dynamic compliance (Cdyn) and total pulmonary resistance (RL) and were used as an indication of in vivo airway obstruction. We pretreated guinea pigs intravenously with the following drugs: atropine; LY163443, a selective LTD4/E4 antagonist; indomethacin; propranolol; and pyrilamine. The guinea pigs were exposed for 8 minutes to the A23187 aerosol, and ELGV measurements were then made. Atropine or pyrilamine prevented the A23187-induced gas trapping. Indomethacin or propranolol tended to potentiate the response and when combined, they potentiated the gas trapping by 80%. LY163443 had no effect alone, but when combined with indomethacin, propranolol, and pyrilamine, inhibited A23187-induced gas trapping by 67%. We conclude that cholinergic and histaminergic mechanisms play major roles in the ionophore-induced pulmonary gas trapping of the guinea pig. With appropriate pretreatment, sulfidopeptide leukotrienes may produce a substantial effect.     

Membrane fluidity of guinea pig lymphocytes and the dysfunction of the respiratory airway and lymphocyte beta-adrenergic systems of the guinea pig

The beta-adrenergic receptor responsiveness of isolated guinea pig tracheal spirals can be negatively affected by intraperitoneal administration of the Gram-negative bacterium Haemophilus influenzae, four days prior to the experiment. The reduction in tracheal relaxation is accompanied by a decrease in beta-adrenergic receptor binding sites in splenic lymphocyte membranes and by a decrease in the fluidity of these membranes. The H. influenzae-induced dysfunction of both the respiratory airway and lymphocyte beta-adrenergic systems can be mimicked by modulating the amount of linoleic acid in the diet. This linoleic acid induced dysfunction of the beta-adrenergic system is also accompanied by a decrease in the plasma membrane fluidity of the splenic lymphocyte membranes of the guinea pigs. The role for plasma membrane fluidity in asthma is discussed in relation to current concepts for atopy.