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.     

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.     

Location of PHM/VIP mRNA in human gastrointestinal tract detected by in situ hybridization

The expression of the gene for vasoactive intestinal polypeptide (VIP) and peptide histidine methionine (PHM) in the human gastrointestinal tract was studied by in situ hybridization and Northern blotting for PHM/ VIP mRNA and immunocytochemistry using specific antisera against the bioactive peptides PHM and VIP. In the colon sigmoideum, antisera against all five putative processing products of the VIP precursor (prepro-VIP) were used, namely prepro-VIP 22–79, PHM, prepro-VIP 111–122, VIP and prepro-VIP 156–170. Furthermore, RNA extracted from various regions of the gastrointestinal tract was examined by Northern blots and hybridization to a VIP-cDNA probe. Throughout the gastrointestinal tract, PHM/VIP mRNA was found in neurons only. Using single-or double-staining methods, we demonstrated both PHM/VIP mRNA and the corresponding peptides PHM and VIP in the neurons. In the sigmoideum, the single-staining methods were extended to investigate whether the neurons simultaneously contained PHM/VIP mRNA and each of the five prepro-VIP-derived peptides. Only one major band of PHM/VIP mRNA (1.9 kb) was found by Northern blotting in the tissue of the gastrointestinal tract.     

Capsaicin-induced bronchodilation in mild asthmatic subjects: possible role of nonadrenergic inhibitory system

We investigated whether stimulation of vagal afferent nerve fibers with inhaled capsaicin could induce a nonadrenergic inhibitory reflex in nine mild asthmatic subjects. Changes in total respiratory resistance (Rrs) were measured with a forced oscillation technique. First we induced a rise of 71 +/- 15% in Rrs (P less than 0.001) after leukotriene D4 aerosol. Subsequent inhalation of capsaicin (2 nmol) caused no significant change in mean Rrs of -1.1 +/- 8.2%. After the muscarinic receptor antagonist ipratropium bromide (120 micrograms) was inhaled, leukotriene D4 increased Rrs by 103 +/- 9% (P less than 0.001). Capsaicin subsequently caused bronchodilation in all subjects (Rrs = -22.3 +/- 2.7%, P less than 0.001). Ethanol-saline (diluent) alone caused a nonsignificant fall in Rrs (-9.9 +/- 4.7%) but a deep breath and coughing resulted in bronchodilation (-16.9 +/- 6.1%, P less than 0.05 and -11.6 +/- 2.9%, P less than 0.01, respectively). As observed in normal subjects, capsaicin may initiate an inhibitory reflex, presumably of nonadrenergic origin. This reflex could not be distinguished from that caused by coughing or by deep inhalation. A defect in nonadrenergic mechanisms, at least in mild asthma, seems unlikely.     

Parasympathetic denervation increases responses to VIP in isolated rat parotid acini

Vasoactive intestinal peptide (VIP) is a putative neurotransmitter found in the salivary glands of many species, including the rat parotid gland. Parasympathetic denervation has been reported to deplete VIP in the rat parotid gland and to lead to supersensitivity to this peptide in vivo. We have compared the effects of VIP on acini isolated from parasympathetically denervated and unoperated parotid glands to examine possible supersensitivity to the peptide in vitro. VIP normally produced responses similar to those obtained with a low concentration of the beta adrenergic agonist isoproterenol (ISO), but strikingly different from the effects obtained with the muscarinic agonist carbachol (CARB). In parotid membrane preparations, VIP stimulated adenylate cyclase activity. Dissociated acini treated with VIP showed increases in cAMP accumulation and amylase release which were potentiated by forskolin and also by inhibition of phosphodiesterase. After parasympathetic denervation, maximal effects of VIP on adenylate cyclase, cAMP accumulation and amylase release in intact cells were increased two- to five-fold over contralateral control (or unoperated) parotid responses. The increase in adenylate cyclase-mediated responses after denervation was specific to VIP; there was no increased response nor increased sensitivity of any of these responses to ISO. Specific [125I]VIP binding to parotid acini increased two-fold per gland and three-fold per mg of protein after denervation; this probably explains the observed increases in the response to VIP.     

Cyclical elongation regulates contractile responses of isolated airways.

Bronchoconstrictor responses are quantitatively different when they are evoked under static conditions and during or after periods of deep inspiration. In vivo, deep inspirations produce bronchodilation and protect the lung from subsequent bronchoconstriction (termed bronchoprotection). These effects may be due in part to dynamic stretch on airways produced by cyclical expansion of airway diameter. However, airways also lengthen cyclically during breathing. The effects of cyclical airway elongation on evoked bronchoconstriction have not been examined. This study recorded evoked contractions of pig bronchial segments 1) at different airway lengths, 2) after a period of cyclical lengthening in relaxed airways, and 3) during cyclical lengthening in pretoned airways. Airway segments were mounted in organ baths and bathed in Krebs solution luminally and on the adventitia. Airways were cyclically lengthened by 5-30% of their deflated length at 0.5-2 Hz for 5 min. Contractions were evoked by electrical field stimulation or carbachol and were recorded under isovolumic conditions. Under static conditions, there was a blunt relationship between length and response to electrical field stimulation. After a period of airway length cycling, electrical field stimulation-induced contractions were increased. In airways pretoned with carbachol, cyclical lengthening produced a transient bronchodilation and a sustained increase in contraction. Contractile responses were not blocked by indomethacin. The results show that isolated airways respond actively to dynamic changes in length. Our results indicate that cyclical lengthening of airways could contribute to lung function in vivo but does not appear to account for the phenomenon of bronchoprotection.     

Evidence for common precursors but differential processing of VIP and PHM in VIP-producing tumors

To elucidate the biosynthesis of vasoactive intestinal polypeptide (VIP) and investigate the suggestion that the prepro-VIP contains another peptide designated PHM (the peptide with N-terminal histidine and C-terminal methionine amide) in its sequence, the concentration and molecular forms of immunoreactive VIP and PHM in 14 human VIP producing tumors (VIP-omas) were determined. Elevated quantities of both peptides were found in all tumor extracts but the concentration of PHM did not correlate with that of VIP and the ratio VIP/PHM varied from 0.5 to 8.5. Gel chromatography showed that in addition to peaks corresponding to VIP and PHM, two larger molecular forms with Kd values of 0.31 and 0.36 which displayed both VIP and PHM immunoreactivity were present. While the proportions between the various PHM molecular forms varied considerably, the relative contribution of the VIP immunoreactive peaks was rather constant from tumor to tumor. The molecular pattern was unaffected by protein denaturing with guanidine hydrochloride and cleavage of sulfide bonds with dithiothreitol. The findings indicate that VIP and PHM are co-produced in VIP-omas probably from common larger molecular forms and that differences in the post-translational processing between tissues exist.     

Effects of changes in osmolarity on isolated human airways

The effects of hypo- and hyperosmolarity on the function of isolated human airways were studied. Changes in osmolarity induced an increasing bronchoconstriction that was proportional to the magnitude of the change in osmolarity. Hypertonicity-induced airway narrowing resulted when buffer was made hypertonic with sodium chloride or mannitol but not with urea. The airways showed no tachyphylaxis to repetitive exposure to hypo- and hypertonic buffer of 200 and 600 mosM, respectively. The bronchoconstriction was not secondary to stimulation of H1 or leukotriene C4/D4 receptors or the release of prostaglandins in the preparation. The bronchoconstriction in hypotonic buffer was totally dependent on extracellular calcium, whereas in hypertonic buffer the bronchoconstriction seemed partially dependent on intracellular calcium release. Isoprenaline prevented the bronchoconstriction in hyper- or hypotonic buffer of 450 and 250 mosM but not in buffer of 600 and 150 mosM. It is concluded that hypo- and hypertonic buffers lead to bronchoconstriction via different mechanisms, which relate to influx of extracellular calcium in hyposmolar buffer and probably to release of calcium from intracellular stores in hypertonic buffer. In strongly hypertonic buffer, part of the bronchoconstriction may be due to osmotic shrinkage. The relevance of our data for the mechanism of bronchoconstriction after inhalation of hypo- or hypertonic saline depends on whether changes in osmolarity around the airway smooth muscle occur in asthmatics but not in normal subjects, and this has not yet been established.     

Effects of VIP, PHM and substance P on blood vessels and secretory elements of the human submandibular gland

The effects of the neuropeptides VIP, PHM and substance P (SP) on vascular smooth muscle tone, K+ secretion from exocrine elements and tissue content of cyclic AMP (cAMP) in the human submandibular gland were studied in vitro. All three peptides caused relaxation of noradrenaline contracted human submandibular arteries at nM concentrations. SP was slightly more active than VIP and PHM which had a similar potency as vasodilators. Only carbachol but not VIP, PHM or SP stimulated K+ secretion from exocrine elements of the human submandibular gland. Principally similar in vitro effects on K+ secretion were obtained on the cat submandibular gland, but in the rat not only carbachol but also SP stimulated K+ secretion. VIP and PHM increased cAMP production of exocrine elements in the human submandibular gland in nM concentrations. VIP was about 5-fold more potent than PHM with regards to cAMP production. In conclusion, VIP, PHM and SP relaxed human submandibular arteries in vitro. Both VIP and PHM stimulated cAMP production in glandular tissue but none of the three peptides induced K+ secretion from human submandibular gland tissue. This suggests that, in contrast to the situation in the rat, SP does not cause watery salivation in man, while VIP and PHM may modulate protein e.g. amylase content of the saliva.     

Prejunctional inhibitory muscarinic receptors on cholinergic nerves in human and guinea pig airways

We have investigated whether prejunctional inhibitory muscarinic receptors ("autoreceptors") exist on cholinergic nerves in human airways in vitro and whether guinea pig trachea provides a good model for further pharmacological characterization of these receptors. Pilocarpine was used as a selective agonist and gallamine as a selective antagonist of these autoreceptors. Acetylcholine (ACh) release from postganglionic cholinergic nerves was elicited by electrical field stimulation (EFS) (40 V, 0.5 ms, 32 Hz). In human bronchi, pilocarpine inhibited the contractile response to EFS in a dose-related fashion; the dose inhibiting 50% of the control contraction was 2.2 +/- 0.4 x 10(-7) (SE) M (n = 22), and the inhibition was 96% at 3 x 10(-5) M. The inhibitory effects of pilocarpine were antagonized by gallamine in a dose-related fashion. The results were qualitatively the same in the guinea pig. Gallamine significantly enhanced the contractile response to EFS in the guinea pig, whereas pirenzepine failed to do so, which suggests that M2-receptors are involved. We conclude that prejunctional muscarinic receptors that inhibit ACh release are present on cholinergic nerves in human airways and that guinea pig trachea is a good model for further pharmacological characterization of these receptors, which appear to belong to the M2-subtype.     

Passive sensitization of human airways induces myogenic contractile responses in vitro

Mitchell, R. W., K. F. Rabe, H. Magnussen, and A. R. Leff.Passive sensitization of human airways induces myogenic contractile responses in vitro. J. Appl.Physiol. 83(4): 1276-1281, 1997.We assessedeffects of passive sensitization on human bronchial smooth muscle (BSM)response to mechanical stretching in vitro. Bronchial rings were sham(control) or passively sensitized overnight by using sera from donorsdemonstrating sensitivity to Dermatophagoides farinae and having immunoglobulin E (IgE)concentrations of 2,600 ± 200 U/ml. Tissues were fixedisometrically to force transducers to measure responses to electricalfield stimulation (EFS) and quick stretch (QS). The myogenic responseto QS was normalized to the maximal response to EFS (%EFS). Themyogenic response of sensitized BSM was 47.9 ± 10.9 %EFS to a QSof ~6.5% optimal length (L_o);sham-sensitized tissues had a myogenic response of 13.5 ± 6.4 %EFS(P = 0.012 vs. passively sensitized).A QS of ~13% L_o in sensitizedBSM caused a response of 82.8 ± 20.9 %EFS; sham-sensitized tissuesdeveloped a response of 38.2 ± 17.3 %EFS(P = 0.004). BSM incubated with serumfrom nonallergic donors did not demonstrate increased QS response (4.6 ± 1.4 %EFS, P = not significantvs. tissue exposed to atopic sera). However, tissues incubated in serafrom nonatopic donors supplemented with hapten-specific chimeric IgE(JW8) demonstrated augmented myogenic response to QS of ~6.5% L_o (21.9 ± 6.2 %EFS, P = 0.027 vs. nonatopicsera alone). We demonstrate that passive sensitization of human BSMpreparations causes induction and augmentation of myogenic contractionsto QS; this hyperresponsiveness corresponds to the IgE concentration insensitizing sera.

     

Endothelin-induced vascular and bronchial effects in pig airways: role in acute allergic responses.

The effects of endothelin (ET) agonists on airway mechanics and bronchial blood flow were studied as well as the effects of mixed ET-receptor antagonist bosentan on allergen-induced airway reactions in the pig. ET agonists [ET-1, ET-3, and the ET(B) receptor-selective agonist Sarafotoxin 6c (Sf6c)] were given as intravenous injections (0.4-200 pmol/kg) to eight anesthetized pigs. Bosentan (10 mg/kg iv) was then administered, and the injections were repeated. Only Sf6c caused a significant increase in airway resistance, and this response was blocked by bosentan. Sf6c and ET-1 (200 and 400 pmol/kg, respectively) were also given as aerosols to five pigs. Sf6c, but not ET-1, caused bronchoconstriction via this route. All agonists (intravenous) caused increases in bronchial vascular conductance, an effect that was blocked by an NO-synthase inhibitor (N(G)-nitro-L-arginine) but unaffected by a cyxlooxygenase inhibitor (diclofenac). Fourteen pigs were sensitized with ascaris suum antigen. Under anesthesia, eight pigs were pretreated with bosentan, and six pigs were controls. They were all challenged with allergen aerosol resulting in acute bronchoconstriction and elevation of ET-1 in bronchoalveolar lavage fluid. Bosentan did not affect the maximal acute airway obstruction but markedly increased baseline bronchial vascular conductance, suggesting a basal vascular tone regulated by ETs. In conclusion, ETs induce bronchoconstriction primarily via the ET(B) receptor in the pig. However, ETs are probably not involved in the allergen-induced acute bronchoconstriction in this model.     

Interaction of PHM, PHI and 24-glutamine PHI with human VIP receptors from colonic epithelium: comparison with rat intestinal receptors

PHM, the human counterpart of porcine Peptide Histidine Isoleucine amide (PHI), is shown to be a VIP agonist with low potency on human VIP receptors located in colonic epithelial cell membranes. Its potency is identical to that of PHI but by 3 orders of magnitude lower than that of VIP itself in inhibiting 125I-VIP binding and in stimulating adenylate cyclase activity. This contrasts markedly with the behaviour of PHI on rat VIP receptors located in intestinal epithelial cell membranes where PHI is a potent agonist with a potency that is 1/5 that of VIP. In another connection, we show that 24-glutamine PHI has the same affinity as 24-glutamic acid PHI (the natural peptide) for rat or human VIP receptors. These results indicate that while PHI may exert some physiological function through its interaction with VIP receptors in rodents, its human counterpart PHM is a very poor agonist of VIP in human. Furthermore, they show that the drastic change in position 24 of PHI (neutral versus acid residue) does not affect the activity of PHI, at least on VIP receptors.     

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.