ABSTRACT: We have analyzed binding domains of the oxytocin receptor for barusiban, a highly selective oxytocin receptor antagonist, in comparison to the combined vasopressin V1A/oxytocin receptor antagonist atosiban and the agonists oxytocin and carbetocin. For this purpose, chimeric 'gain-in function' oxytocin/vasopressin V2 receptors were expressed in COS-7 cells. These recombinant receptors have been produced by transfer of domains from the oxytocin receptor into the related vasopressin V2 receptor and have already been successfully employed for the identification of ligand binding domains at the oxytocin receptor (Postina, R., Kojro, E., Fahrenholz, F., 1996. Separate agonist and peptide antagonist binding sites of the oxytocin receptor defined by their transfer into the V2 vasopressin receptor. J. Biol. Chem. 271, 31593-31601). In displacement studies with 10 chimeric receptor constructs, the binding profile of barusiban was compared with the binding profiles of the ligands oxytocin, [Arg8]vasopressin, carbetocin, and atosiban. The binding profiles for the agonists oxytocin and carbetocin were found to be similar. For both agonists, important binding domains were the extracellular N-terminus (=E1) and the extracellular loops E2 and E3 from the oxytocin receptor. For the vasopressin V1A/oxytocin receptor antagonist atosiban, none of the receptor constructs were able to provide a binding with higher affinity than the starting vasopressin V2 receptor. In contrast, the binding of barusiban was significantly improved when the transmembrane domains 1 and 2 were transferred from the oxytocin receptor to the vasopressin V2 receptor. The binding domain of barusiban differs from the binding domain of the agonists and the nonselective oxytocin receptor antagonist d(CH2)5[Tyr-(Me)2,Thr4,Orn8,Tyr9]vasotocin that has been used in previous studies. Overall, the data supported the concept of a central pocket site within the oxytocin receptor.
European Journal of Pharmacology 04/2005; 510(1-2):9-16. · 2.52 Impact Factor
ABSTRACT: Recent experimental evidence indicates that non-neuronal acetylcholine is involved in the regulation of basic cell functions. Here we investigated the cholinergic system in the skin of healthy volunteers and patients with atopic dermatitis (AD). The synthesizing enzyme, choline-acetyltransferase (ChAT), was studied by anti-ChAT immunohistochemistry and enzyme assay. Skin biopsies taken from healthy volunteers and from AD patients were separated into the 2 mm superfical (epidermis and upper dermis) and 3 mm underlying portion (deeper dermis and subcutis). ChAT enzyme activity was detected in homogenized skin and subcutaneous fat (about 13 nmol/mg protein/h). ChAT immunoreactivity was expressed in keratinocytes, hair papilla, sebaceous and eccrine sweat glands, endothelial cells and mast cells. In healthy volunteers the superficial and underlying portion of skin biopsies contained 130 +/- 30 and 550 +/- 170 pmol/g acetylcholine (n = 12), respectively. In AD patients (n = 7) acetylcholine was increased 14-fold in the superficial and 3-fold in the underlying biopsy portion. The present study demonstrates the widespread expression of ChAT protein in the vast majority of human skin cells. Tissue levels of acetylcholine are greatly (14-fold) enhanced in the superficial 2 mm skin of AD patients.
Life Sciences 04/2003; 72(18-19):2169-72. · 2.53 Impact Factor
ABSTRACT: 1The release of neuronal [3H]acetylcholine (ACh) from isolated human bronchi after labelling with [3H]choline was measured to investigate the effects of prostanoids.2A first period of electrical field stimulation (S1) caused a [3H]ACh release of 320±70 and 200±40 Becquerel (Bq) g−1 in epithelium-denuded and epithelium-containing bronchi respectively (P>0.05). Subsequent periods of electrical stimulation (Sn, n=2, 3, and 4) released less [3H]ACh, i.e. decreasing Sn/S1 values were obtained (0.76±0.09, 0.68±0.07 and 0.40±0.04, respectively).3Cumulative concentrations (1–1000 nM) of EP-receptor agonists like prostaglandin E2, nocloprost, and sulprostone (EP1 and EP3 selective) inhibited evoked [3H]ACh release in a concentration dependent manner with IC50 values between 4–14 nM and maximal inhibition of about 70%.4The inhibition of evoked [3H]ACh release by prostaglandin E2, nocloprost and sulprostone was not affected by the DP-, EP1- and EP2-receptor antagonist AH6809 at a concentration of 3 μM, i.e. a 3–30 times greater concentration than its affinity (pA2 values) at the respective receptors.5Circaprost (IP-receptor agonist; 1–100 nM), iloprost (IP- and EP1-receptor agonist; 10-1000 nM) and U-46619 (TP-receptor agonist; 100–1000 nM) did not significantly affect [3H]ACh release.6Blockade of cyclooxygenase by 3 μM indomethacin did not significantly modulate evoked [3H]ACh release in epithelium-containing and epithelium-denuded bronchi. Likewise, the combined cyclo- and lipoxygenase inhibitor BW-755C (20 μM) did not affect evoked [3H]ACh release.7In conclusion, applied prostanoids appear to inhibit [3H]ACh release in epithelium-denuded human bronchi under the present in vitro conditions, most likely via prejunctional prostanoid receptors of the EP3 subtype.British Journal of Pharmacology (1998) 125, 271–276; doi:10.1038/sj.bjp.0702057
British Journal of Pharmacology 08/1998; 125(2):271 - 276. · 4.41 Impact Factor
ABSTRACT: The cholinergic system in rat and human airways and the effects of glucocorticoids were investigated by assay of choline acetyltransferase activity, by high-pressure liquid chromatography measurement of acetylcholine, and by anti-choline acetyltransferase immunocyto-/histochemistry. Human bronchi were obtained at surgery from patients with lung cancer. Group 1 patients did not suffer from additional lung diseases and had not been treated with glucocorticoids. Group 2 patients, who suffered in addition to lung cancer from chronic obstructive bronchitis, had been treated for at least 6 weeks before surgery with four puffs of flusinolid daily. Isolated bronchial epithelial cells as well as intact surface epithelium of human bronchi expressed choline acetyltransferase immunoreactivity and choline acetyltransferase enzyme activity (3±1 nmol/mg protein per h). Ciliated epithelial cells showed strong choline acetyltransferase immunoreactivity at the basal body and the roolet of cilia. Surface epithelium in group 1 and 2 bronchi contained 23±6 (n=14) and 1.8±0.3 pmol/g acetylcholine) (n=7, P<0.001), respectively, whereas the transmural acetylcholine content did not differ significantly between both groups. The amount of choline acetyltransferase immunoreactivity appeared similar in the surface epithelium of both groups. In an animal (rat) study the effects of oral dexamethasone (3 mg/day, 1 week) on choline acetyltransferase activity and acetylcholine levels were investigated. Dexamethasone treatment reduced epithelial acetylcholine in the airways and small intestine by about 80% and inhibited epithelial choline acetyltransferase activity. In conclusion, epithelial cells of human airways possess components of the cholinergic system, i.e., contain the synthesizing enzyme choline acetyltransferase and store acetylcholine. The data obtained from the animal study indicate that glucocorticoids can inhibit epithelial acetylcholine.
European Journal of Pharmacology 06/1998; · 2.52 Impact Factor
ABSTRACT: Acetylcholine acts as a prominent transmitter in the central and peripheral nervous system. The aim of the present study
was to investigate whether mammalian non-neuronal cells can synthesize and store acetylcholine. A cotton tipped applicator
(Q-tip) was used to collect surface cells from airways and alimentary tract. Histological inspection indicated that rubbing
of the luminal surface of human bronchi did not penetrate the basal membrane. Acetylcholine was measured by an HPLC-method
using substrate-specific enzyme reactor-columns.
Non-neuronal acetylcholine was found in cells covering inner and outer surfaces of rat and man. For example, acetylcholine
was detected in the surface epithelium of human bronchi (33 pmol/g), mouth (female 0.7 and male 8 pmol/sample), small and
large intestine (800 and 16 pmol/g, respectively), gall bladder (12 pmol/g), vagina (6 pmol/sample), skin 1000 (pmol/g) and
in pulmonary pleura (5 pmol/sample). Somewhat higher amounts of acetylcholine were found in rat tracheal and intestinal epithelium
and in rat skin. The synthesizing enzyme choline acetyltransferase (ChAT) was demonstrated in human surface epithelium by
immunohistochemistry and by Western blot analysis. Enzymatic ChAT activity was demonstrated in isolated epithelial cells of
human bronchi and small intestine (3.5 and 28 nmol/mg protein/h, respectively). Applied acetylcholine (in nM concentrations)
increased, whereas inhibition of ChAT activity by bromoacetylcholine (10 μM) reduced the growth of cultured human bronchial
epithelial cells. Inhibition of cell growth occurred also in the presence of atropine (1 μM) together with (±)-tubocurarine
In conclusion, the present experiments demonstrate a widespread existence of non-neuronal acetylcholine in surface cells of
man. Non-neuronal acetylcholine may act as a local signalling molecule.
Archiv für Experimentelle Pathologie und Pharmakologie 02/1997; 355(4):515-523. · 2.65 Impact Factor