AF-DX 116 discriminates heart from gland M2-cholinoceptors in man.

The M2-cholinoceptor subtype selective antagonist AF-DX 116 was compared with atropine with respect to effects on heart rate and salivary flow in healthy volunteers. These effects were related with in vitro occupancy of M-cholinoceptor subtypes in radioreceptor assays of plasma samples. Radioreceptor assays comprised M1-cholinoceptors in bovine cerebral cortex and M2-cholinoceptors in pig heart and rat salivary gland membranes. 3H-pirenzepine served as a label in the cerebral cortex 3H-N-methyl-scopolamine in the heart and gland preparations. Oral administration of 240 mg AF-DX 116 led to a time dependent increase in heart rate with a maximum effect comparable to atropine 40 micrograms/kg i.v. The effects of both drugs on heart rate were matched by a greater than 80% occupancy of heart M2-cholinoceptors in the radioreceptor assay of plasma samples. In contrast to the complete inhibition of salivary flow after atropine, AF-DX 116 induced an increase of salivation. The effects on salivary flow coincided with a greater than 80% occupancy of glandular M2-cholinoceptors after atropine but no detectable occupancy after AF-DX 116. Occupancy of the M1-subtype amounted to 61.7% after AF-DX 116 and a blockade of inhibitory, presynaptic M1-autoreceptors at missing postsynaptic blockade of glandular M2-cholinoceptors might explain the hypersalivation induced by AF-DX 116.

Secretory antibodies against Giardia intestinalis in lactating Nicaraguan women.

Secretory IgA (sIgA) antibodies are important in the host defence against the intestinal protozoan parasite Giardia intestinalis. However, few antigens have been identified. In this study 100 milk and saliva samples from lactating women, living in an endemic region (León, Nicaragua), were screened for the presence of antibodies against G. intestinalis. Most milk and saliva samples contained anti-Giardia antibodies (59% and 52%, respectively), with a mean sIgA content 50 times higher in milk than in saliva. The positive samples reacted with trophozoite membrane, flagella and cytoplasmic antigens. Western blot analysis showed that milk and saliva anti-Giardia sIgA recognized up to 16 different Giardia proteins in the molecular weight region 20-165 kDa. Two-dimensional Western blotting showed that the major immunoreactive proteins were the same as the immunoreactive proteins identified by serum from acute giardiasis patients in a non-endemic country. The major difference was a stronger reactivity against the variant surface proteins (VSPs) in the milk samples. Milk sIgAs also recognized recombinant Giardia proteins such as alpha-1 giardin, ornithine carbamoyl transferase, VSP-4EX, arginine deaminase and alpha-enolase. These antigens will be important targets in the development of new immunodiagnostic tools and vaccines.

Conformation of a beta-adrenoceptor-derived signal transducing peptide as inferred by circular dichroism and 1H NMR spectroscopy.

The peptide T345-359 representing the fourth intracellular loop of the avian beta-adrenoceptor has been shown to strongly inhibit receptor-mediated adenylate cyclase activity [Münch, G., Dees, C., Hekman, M., & Palm, D. (1991) Eur. J. Biochem. 198, 357-364]. Circular dichroism and two-dimensional 1H NMR techniques were used to investigate the three-dimensional structure of the peptide in trifluoroethanol, phospholipid micelles, and small unilamellar phospholipid vesicles. The prepared vesicles were tested for size distribution and stability by using electron microscopy, photon correlation spectroscopy, and 31P NMR spectroscopy. The peptide T345-359 adopted a predominantly alpha-helical conformation in either trifluoroethanol or phospholipid micelles and vesicles. No structural differences were found for the conformation of the peptide in the presence of phospholipid micelles or vesicles, respectively, using 2D 1H NMR techniques, suggesting a unique conformation of T345-359 when associated with model membranes. A computer-aided model of the micelle-associated peptide was derived. The relevance of the 3D structure of the intracellular loops of receptors to communicate with the G protein in the signal transduction cascade is discussed.

Comparison of the effects of atropine in vivo and ex vivo (radioreceptor assay) after oral and intramuscular administration to man.

The effects of an oral dose of atropine (0.03 mg/kg body weight) and an IM (0.02 mg/kg) dose on the heart rate and salivary flow in seven healthy adult volunteers were compared to see whether the oral dose was sufficient to inhibit vagal reflexes of the heart. Atropine concentrations in plasma were determined by an M2-selective radioreceptor assay, and the in vitro occupancy of porcine cardiac M2-cholinoceptors was measured in parallel. In ligand-binding studies, atropine has been shown to have a comparable affinity for human and porcine cardiac M2-cholinoceptors (Ki 4.0 and 5.9, respectively). Slight changes in heart rate after oral administration were not significant. After IM administration, however, the heart rate increased significantly, by a maximum of 22 beats.min-1 after 45 min. The slight increase in heart rate after the oral dose corresponded to a receptor occupancy in vitro near the lower limit of detection, whereas the significant increase in heart rate after the IM dose corresponded to a receptor occupancy of up to 47%. The maximum reduction in salivary flow was similar after the oral and IM doses (84.3 and 87.5%, respectively). The almost complete inhibition of salivary flow could be explained by the lower vagal tone in the salivary glands compared with to the heart. The difference in the effect on heart rate was probably due to lower absorption of the oral dose. Thus, an oral dose greater than 0.03 mg atropine/kilogram body weight is required to compensate for low gastrointestinal absorption and to overcome the high vagal tone of the heart.