Segment-dependent expression of muscarinic acetylcholine receptors and G-protein coupling in the equine respiratory tract.

Muscarinic receptors are considered to be of comparable clinical importance in chronic obstructive pulmonary disease (COPD) in equines and in humans. At present, data are scarce on the expression and distribution of probable subtypes of these receptors and their signalling pathways in airway segments, including lung parenchyma and bronchial and tracheal epithelium with the underlying smooth muscle in horses. Specific [N-methyl-3H]scopolamine chloride ([3H]NMS) binding to all three tissues was saturable and of high affinity, with KD values ranging between 1.6+/-0.7 and 1.9+/-0.3 nmol/L. [3H]NMS binding identified a higher density of total muscarinic receptors (fmol/mg protein) in the trachea (720+/-59 nmol/L) than in bronchi (438+/-48 nmol/L) or lung (22 +/- 3 nmol/L). Competitive binding studies using [3H]NMS and the unlabelled subtype-selective antagonists pirenzepine and telenzepine (M1), methoctramine and himbacine (M2), 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) (M3), tropicamide (M4) and mamba toxin (MT-3) (M4) indicated the presence of at least three muscarinic receptor subtypes in peripheral lung tissue (50:40:24-28%: M2>M3>M1), whereas in bronchus and trachea M2 subtypes (87-90%) predominated over M3 (14-22%), and M1 subtypes were lacking. No differences were found between tissues in high-affinity binding sites for carbachol in the absence (31-36%) or presence of guanosine 5'-triphosphate (GTP) (approximately 100%). Western blotting for G-protein alpha-subunits showed a much more robust expression of G(alphai1/2) in the trachea (with highest receptor density) than in the lung or bronchi, whereas G(alphas)-protein was dominantly expressed in bronchus. Concomitantly, carbachol inhibited isoproterenol- and GTP-stimulated adenylyl cyclase activity with increasing muscarinic receptor expression (trachea > bronchi > lung). We conclude that the expression and signalling pathways of muscarinic receptors in the equine respiratory tract are segment-dependent. These receptors might contribute to the pathogenesis of COPD in the horse and could provide potential drug targets for the therapeutic use of anticholinergics in this species.

Migrastatin acts as a muscarinic acetylcholine receptor antagonist.

Migrastatin and its analogs have various biological activities such as inhibition of cell migration and anchorage-independent growth of cancer cells. Although its biosynthesis and chemical synthesis have been under investigation, little is known about the biological target of migrastatin. Here, we found that migrastatin inhibited intracellular calcium mobilization induced by carbachol in neuroblastoma SK-N-SH cells without affecting Ca2+ mobilization and cAMP accumulation induced by ligands of other receptors. The binding of [3H] N-methylscopolamine, an antagonist for muscarinic receptor was also inhibited by migrastain. Functionally, migrastatin inhibited Ca2+ mobilization induced by carbachol in primary cultures of smooth muscle cells of rat bladder. This study reveals that migrastatin acts as a muscarinic acetylcholine receptor antagonist.

Effects of saw palmetto extract on micturition reflex of rats and its autonomic receptor binding activity.

PURPOSE: We examined the effects of saw palmetto extract (SPE) on the rat micturition reflex and on autonomic receptors in the lower urinary tract. MATERIALS AND METHODS: The effect of SPE was examined on cystometrograms of anesthetized rats induced by intravesical infusion of saline or 0.1% acetic acid. SHR/NDmc-cp (cp/cp) rats received repeat oral administration of SPE and nighttime urodynamic function was determined. The autonomic receptor binding activity of SPE in the rat bladder and prostate was examined by radioligand binding assay. RESULTS: Intraduodenal administration of SPE (60 mg/kg) in anesthetized rat cystometry caused a significant increase in the micturition interval, micturition volume and bladder capacity during intravesical saline infusion. Also, similar administration of SPE at doses of 12 and 20 mg/kg significantly reversed the shortened micturition interval as well as the decreased micturition volume and bladder capacity due to 0.1% acetic acid infusion in a dose dependent manner. In conscious SHR/NDmc-cp (cp/cp) rats repeat oral administration of SPE (6 mg/kg daily) constantly increased the micturition interval and concomitantly decreased voiding frequency. SPE inhibited specific binding of [H]NMS ([N-methyl-H]scopolamine methyl chloride) (bladder) and [H]prazosin (prostate) with IC50 values of 46.1 and 183 microg/ml, respectively. CONCLUSIONS: SPE significantly alleviates urodynamic symptoms in hyperactive rat bladders by increasing bladder capacity and subsequently prolonging the micturition interval. Our data may support the clinical efficacy of SPE for the treatment of lower urinary tract symptoms.

Vinburnine decelerates [3H]N-methylscopolamine binding to recombinant human muscarinic M1-M4 acetylcholine receptors.

The kinetics of [3H]N-methylscopolamine binding to membranes of Chinese hamster ovary (CHO) cells expressing muscarinic M(1)-M(4) acetylcholine receptors was studied. [3H]N-methylscopolamine dissociation was used for the "single-point" analysis of allosteric modulation by vinburnine (L-eburnamonine). [3H]N-methylscopolamine dissociation was decelerated by vinburnine with EC(50) values of 29.5, 4.1, 9.5 and 15.0 microM for muscarinic M(1)-M(4) receptors, respectively. Acetylcholine doubled the EC(50) of vinburnine for muscarinic M(3) receptors. These kinetic EC(50) values correlated with equilibrium binding constants, supporting the ternary allosteric model. Vinburnine also decelerated the association of [3H]N-methylscopolamine binding, resulting in opposite cooperativity for muscarinic M(1) and M(2) receptors.

Muscarinic receptors in equine airways.

The distribution of muscarinic receptors in equine airways was investigated using autoradiography. Frozen sections of tissue from six different levels in the bronchial tree, from the trachea to the distal bronchioles, were incubated in vitro with 1.5 nmol/L of the muscarinic receptor antagonist 1-[N-methyl-3H]scopolamine methyl chloride (3H-NMS). In addition, the subtype pattern of muscarinic receptors was investigated in equine tracheal smooth muscle using radioligand binding with methoctramine, tripinamidc, 4-DAMP-methiodide and pirenzipine as competitors against the binding of 1.3 nmol/L 3H-NMS. The autoradiograms showed specific labelling indicating a high density of muscarinic receptors in smooth-muscle tissue in all levels of the airway tree investigated. Besides muscle tissue, subepithelial glands were the only structures specifically labelled. The dominating subtypes in tracheal smooth muscle investigated with radioligand binding studies were found to be M2 and M4, as both methoctramine (pKd = 8.5) and tripinamide (pKd = 8.6 and 6.7 for two different sites) showed high affinity. The density of the M3-muscarinic receptor subtype was low, but this subtype could be detected with statistical significance when methoctramine was used as the competitor against 3H-NMS binding.