Pulmonary Pharmacology & Therapeutics (1999) 12, 313–323 Article No. pupt.1999.0213, available online at http://www.idealibrary.com on PULMONARY PHARMACOLOGY & THERAPEUTICS Detection of Endothelin Receptors in Rat and Guinea-pig Airway Nerves by Immunohistochemistry L. B. Fernandes*†, A. C. D’Aprile†, P. J. Henry†, L. J. Spalding†, C. J. Pudney†‡, R. G. Goldie†‡ †Department of Pharmacology and ‡Biomedical Confocal Microscopy Research Centre, University of Western Australia, Perth, Western Australia 6907, Australia SUMMARY: We investigated the existence of endothelin (ET) receptor subtypes in airway neurones from the rat and guinea-pig and determined the ability of these receptors to modulate contractile function. Rat tracheal neuron cultures as well as rat and guinea-pig whole mount preparations were labelled with antibodies to the cholinergic nerve marker choline acetyltransferase (ChAT), the neuron specific marker protein gene product 9.5 (PGP 9.5) and to ET A and ET B receptors. Following incubation with fluorescent secondary antibodies, fluorescence was detected using confocal microscopy with dual emission protocols. Specific fluorescence was detected both in whole mount preparations and neuron cultures, in association with the primary antibodies. Specific fluorescence associated with either ET A and ET B receptors was colocalized with that for PGP 9.5. Despite the presence of ET A and ET B receptors on airway nerves, ET-1 failed to significantly alter cholinergic, excitatory or inhibitory non-adrenergic–non-cholinergic nerve-mediated responses in guinea-pig airways. This is in sharp contrast to ET-1-induced potentiation of responses to cholinergic nerve-evoked contraction in rat trachea. Thus, although ET A and ET B receptors exist in airway cholinergic neurons in whole mount preparations and in primary neuron cultures from rat and guinea-pig trachea, the influence of these receptors on contractile function appears to be species- dependent.  1999 Academic Press KEY WORDS: Endothelin, Airway, Parasympathetic neuron, Neurotransmission, Neuron culture. INTRODUCTION ET receptors were expressed in these tissues. 3,4,5 This is in line with recent evidence that ET-1 and related Endothelin-1 (ET-1) is an endogenous peptide released peptides can enhance cholinergic neurotransmission. 6 into the airway wall. The major sources of ET-1 in the This phenomenon is potentially important in ob- airways are the epithelium and vascular endothelium, structive airway diseases such as asthma, which may with inflammatory cells such as macrophages playing involve increased cholinergic neuronal traffic con- a smaller role. 1,2 The evidence accumulating in favour tributing to bronchoconstriction. 7,8 We have pre- of a significant mediator role for this spasmogenic viously demonstrated that activation of prejunctional peptide in asthma is increasingly compelling. A range ET receptors on cholinergic nerves in rat trachea of biological activities mimicking asthma symptoms induces significant potentiation of cholinergic con- and pathologies have been reported and these are traction, 9 with a similar phenomenon seen in human consistent with the fact that both subtypes of ET isolated bronchus. 10,11 Given that ET peptides have receptor are widely distributed in human lung. 1,2 In- been shown to potentiate cholinergic contractile re- deed, particularly high receptor densities are evident sponses in rat and human airways, it was of interest in bronchial submucosal tissues. Cholinergic ganglia to determine whether ET-1 had a similar effect on and peripheral airway neurones have also been iden- cholinergic nerve function in guinea-pig airways, tified as sites of specific ET-1 binding, suggesting that which are often used as a model for human airways * Author for correspondence. physiology and disease. Furthermore, it is now well 1094–5539/99/050313+11 $30.00/0  1999 Academic Press 313