AndréB.M. Klaassen

Autoradiographic visualization of muscarinic receptors in pulmonary nerves and ganglia

We investigated autoradiographically the distribution of muscarinic receptors in bovine airways using (-)-[3H]quinuclidinyl benzilate as radioligand. The autoradiographs demonstrated the presence of muscarinic receptors in smooth muscle as well as neuronal muscarinic receptors in pulmonary nerves and ganglia. It is reasonable to believe that the neuronal muscarinic receptors participate in the regulation of neurotransmitter release at the peripheral nerve terminals innervating the bronchial smooth muscle.

Autonomic Innervation of the Nasal Mucosa

The nasal passages play a crucial role in the protection and functioning of the lower airways. Consequently the nerve supply of the nasal mucosa is extensive, which is related to an immediate and adequate reaction upon a variety of external and internal stimuli. A brief review of the present knowledge on nasal autonomic innervation and pharmacology will be given. There is special attention to more advanced methods, such as radioligand receptor binding techniques, which might augment our insight in the significance of the nervous system in nasal (patho)physiology. Furthermore data on the secretory activity of the nasal glands in the rat and its neural regulation will be accentuated.

Alterations of adrenoceptors in the nasal mucosa of allergic patients in comparison with nonallergic individuals

Nasal hyperreactivity in nasal allergy may be due to changes of the characteristics in adrenergic receptors. Radioligand receptor-binding studies with the antagonists, 3H-prazosin (alpha 1-adrenoceptor), 3H-rauwolscine (alpha 2-adrenoceptor), and 125I-(-)-Cyanopindolol (beta-adrenoceptor) were performed in homogenates of nasal mucosa of allergic and nonallergic (NA) patients to investigate this hypothesis. The heterogeneous NA group was subdivided into control individuals and patients with chronic sinusitis and vasomotor rhinitis. No significant differences in affinities or densities of alpha 1- and alpha 2-adrenoceptors could be demonstrated in allergic patients in comparison with NA and control individuals. The beta-adrenoceptor density was significantly reduced in allergic patients in comparison with that of control individuals. Neither changes in agonist binding or in the effect of Gpp(NH)p on the agonist binding to beta-adrenoceptors could be observed in allergic patients. The subtype selective antagonist, LK203-030, demonstrated the presence of a homogeneous population of beta 2-adrenoceptors in human nasal mucosa of both NA and allergic patients. In vitro, autoradiography demonstrated specific 125I-(-)-Cyanopindolol labeling of the epithelium in NA and allergic patients. In conclusion, no changes in characteristics of alpha 1- or alpha 2-adrenoceptors in the nasal mucosa could be demonstrated in nasal allergy. However, a decreased number of beta-adrenoceptors may reflect a beta-adrenergic abnormality in nasal allergy.

Cholinergic muscarinic receptors in rat cochlea.

Specific 3H-1-quinuclidinylbenzilate (3H-1-QNB) binding to rat cochlea homogenates occurs to a homogeneous class of binding sites with Kd = 0.13 +/- 0.01 nM and Bmax = 0.57 +/- 0.07 fmol per cochlea. Binding is stereoselectively inhibited by benzetimide enantiomers. Dexetimide was more effective than levetimide in displacing 3H-1-QNB from its binding sites (Ki = 4 x 10(-10) M and 6.5 x 10(-6) M, respectively). Pirenzepine inhibits 3H-1-QNB binding with low affinity (Ki = 2 x 10(-6) M), classifying the binding sites as muscarinic M2 receptors.

ALTERATIONS OF MUSCARINIC ACETYLCHOLINE RECEPTORS IN THE NASAL MUCOSA OF ALLERGIC PATIENTS IN COMPARISON WITH NONALLERGIC INDIVIDUALS

Cholinergic nasal hyperresponsiveness in nasal allergy may be due to changes of the characteristics in muscarinic cholinergic receptors. Radioligand receptor binding and in vitro autoradiographic studies of nasal mucosa in nonallergic (NA) and allergic patients were performed to investigate this hypothesis. The heterogeneous NA group was subdivided into control individuals and patients with chronic sinusitis and vasomotor rhinitis. The 3H-(-)-Quinuclidinylbenzilate binding to muscarinic receptors in human nasal mucosa membranes was saturable and of high affinity in all groups. No significant differences could be demonstrated between the subgroups of the NA patients. In allergic patients the dissociation constants and receptor densities were significantly decreased in comparison with those of NA and with those of control individuals. No differences in agonist binding or coupling of the muscarinic receptor to the effector system via the G protein could be observed in allergic patients. In vitro autoradiographic experiments demonstrated specific 3H-(-)-Quinuclidinylbenzilate labeling of the glandular acini in NA and allergic patients. No specific labeling could be observed in the epithelium, blood vessels, or connective tissue. In conclusion, the increased sensitivity and decreased muscarinic receptor number may reflect the cholinergic-induced hypersecretion in nasal allergy but are probably too small to explain the complex allergic reaction.

Muscarinic receptors in rat nasal mucosa are predominantly of the low affinity agonist type.

Specific [3H]l-quinuclidinyl benzilate binding to rat nasal mucosa homogenates occurs to a homogeneous class of binding sites with Kd = 60 +/- 2 10(-12) M and Bmax = 8.1 +/- 2 pmol/g tissue. Binding is stereoselectively inhibited by benzetimide enantiomers. Pirenzepine inhibits [3H]l-quinuclidinyl benzilate binding with low affinity (Ki = 5.0 10(-7) M), classifying the binding sites as muscarinic M2-receptors. Methylfurtrethonium and methacholine inhibit [3H]l-quinuclidinyl benzilate binding following an almost sigmoid curve at high concentrations pointing to the presence of mainly low affinity agonist binding sites.

Biochemical and autoradiographic analysis of β-adrenoceptors in rat nasal mucosa

The specific binding of 125I-(-)-cyanopindolol (125I-(-)-CYP) to homogenates and cryostat sections of rat nasal mucosa was saturable, stereoselective and of high affinity (Kd = 5.0 +/- 0.4 pM. Bmax = 204 +/- 12 fmol/mg protein and Kd = 7.2 +/- 0.7 pM; Bmax = 15 +/- 1 fmol/mg protein respectively). The subtype-selective antagonists, LK203-030 and ICI118,551, inhibited specific 125I-(-)-CYP binding according to a two-binding site model, indicating the presence of 57 and 45% beta 1-adrenoceptors in homogenates and cryostat sections, respectively. Competition of isoprenaline for antagonist binding to homogenates demonstrated 30 +/- 3% high-affinity agonist binding sites. A steepening of the curve was observed in presence of guanine nucleotides. In vitro labelling of cryostat sections of rat nasal mucosa was combined with autoradiography. The autoradiographs generated after incubation with 20 pM 125I-(-)-CYP showed specific labelling of the epithelium and glandular excretory ducts. It appeared from autoradiographs generated with subtype-selective antagonists in addition to the radioligand that beta 1- and beta 2-adrenoceptors were present in both structures.

Demonstration of β-adrenergic receptors in rat nasal glands by an in vitro autoradiographic technique ☆

The distribution of beta-adrenergic receptors in rat nasal glands has been investigated with the use of an in vitro autoradiographic technique. Radioligand binding studies indicated that [125I]cyanopindolol binds specifically to beta-adrenergic receptors in cryostat sections of these glands. Autoradiograms generated after incubation with 0.02 nM [125I]cyanopindolol and dipping in nuclear K2 emulsion, showed specific labelling of the striated excretory ducts. These in vitro observations suggest a sympathetic control of the ion and water content of the glandular secretion.

Differences in Innervation and Secretory Behaviour of Two Nasal Glandular Parts in the Rat

The submucosal glands in the rat nose are characterized by the presence of both neutral and acid glycoproteins, which are important constituents of nasal and tracheobronchial secretions. This study is an attempt to gain a better insight into the secretion of these two types of glycoproteins and its neural regulation. Radiobiochemical experiments show a higher sensitivity to methacholine of the nasal glandular region producing acid glycoproteins than the area secreting neutral glycoproteins. Radioligand receptor binding suggests that the binding parameters of the muscarinic receptors in these two areas are mutually different. Furthermore, rat nasal glandular muscarinic receptors appear to be different from those in smooth muscle of rat ileum.