Apparent capacity of cardiac muscarinic receptors for different radiolabeled antagonists.

Muscarinic receptors in sarcolemmal membranes, digitonin-solubilized extracts, and purified preparations from porcine atria have revealed a shortfall in the apparent capacity for N-[3H]methylscopolamine, which was only about 75% of that for [3H]quinuclidinylbenzilate. Since binding at near-saturating concentrations of [3H]quinuclidinylbenzilate was inhibited fully at comparatively low concentrations of unlabeled N-methylscopolamine, the data are inconsistent with the notion that [3H]quinuclidinylbenzilate binds selectively to a subclass of distinct, non-interconverting, and mutually independent sites. The discrepancy is resolved by adjusting the specific activity of N-[3H]methylscopolamine to account for unlabeled scopolamine that was identified in some batches of the radioligand. Also, there was no shortfall in capacity when N-[3H]methylscopolamine was devoid of scopolamine, and the predicted effect was obtained when pure N-[3H]methylscopolamine was supplemented with known amounts of scopolamine. A small discrepancy in the levels of scopolamine estimated pharmacologically and by mass spectrometry can be attributed largely to a difference in the efficiency of ionization between scopolamine and N-methylscopolamine. Different capacities for different radioligands are not uncommon with muscarinic and other G protein-coupled receptors, and in some cases the effect may have been due wholly or in part to an unlabeled impurity. Binding data can be mechanistically ambiguous, particularly when acquired only at graded concentrations of the radioligand. The predicted effects of an unlabeled impurity mimic or resemble those of alternative scenarios such as sequestration behind a hydrophobic barrier, a nucleotide-regulated interconversion from one state of affinity to another, and cooperativity between interacting sites.

Nature of the oligomers formed by muscarinic m2 acetylcholine receptors in Sf9 cells.

Wild-type, FLAG-tagged, and c-myc-tagged muscarinic m2 receptors extracted in digitonin-cholate from singly and co-infected Sf9 (Spodoptera frugiperda) cells were indistinguishable in their binding of [3H]quinuclidinylbenzilate, either before or after purification. The FLAG epitope was found to coimmunoprecipitate with the c-myc epitope when co-infected cells were solubilised in digitonin-cholate, n-dodecyl-beta-D-maltoside or Lubrol-PX. The degree of coprecipitation in digitonin-cholate was unaffected by preincubation of the extract for up to 60 min at 30 degrees C, with or without muscarinic receptor ligands; no coimmunoprecipitation occurred in mixed extracts from singly infected cells. As measured by [3H]quinuclidinylbenzilate, the efficiency of immunoprecipitation from co-infected cells was 87% of that from singly infected cells. The amount of receptor immunoprecipitated from the latter, as determined by densitometry, was 2.3-fold that expected from the loss of binding from the extract. The data suggest that at least some of the receptors were trimeric or larger and that oligomers neither formed nor dissociated under the conditions of the experiments. Also, some receptors appear to be non-functional or latent in digitonin-solubilised extracts.

Potentiation of purinergic transmission by angiotensin in prostatic rat vas deferens.

1. Angiotensin II (AII) elicited only a minute, if any, direct contractile response in smooth muscle cells of prostatic rat vas deferens, but it potentiated contractile responses to field stimulation. 2. Angiotensin-potentiated contractile response to field stimulation was concentration-dependent, and the order of potency was AII > AIII approximately AI. The EC50 of AII was 8.11 +/- 2.79 nM. 3. AII did not modify the contractile response of exogenous noradrenaline (NA) on non-stimulated prostatic vas deferens. Furthermore, the concentration-response curve for AII-potentiated contractile responses to field stimulation in reserpine-treated rats did not significantly differ from the control group. 4. Desensitization of purinoceptors with 30 microM alpha, beta-methylene-ATP almost completely abolished the potentiation of the contractile response to field stimulation by AII. 5. The response to AII in the prostatic rat vas deferens was blocked by the AT1 selective antagonist losartan, but not by the AT2 selective antagonist CGP 42112. Losartan acted as a competitive antagonist with a pA2 value of 8.75. 6. In conclusion, AII potentiated purinergic transmission in the prostatic rat vas deferens via the AT1 receptor.

Characterization of contractile response to angiotensin in epididymal rat vas deferens.

Angiotensin had a dual action on the epididymal half of rat vas deferens. It potentiated electrical stimulated contraction and exerted a direct contractile effect on the muscle. The potentiation of electrically stimulated response may be mediated by presynaptic facilitation of neurotransmitter release. Muscular contractile response to angiotensin is concentration dependent. Angiotensin II was found to be much more potent than angiotensin III, and the order of potencies was angiotensin II > angiotensin I > angiotensin III. The presence of a mixture of protease inhibitors (10 microM chymostatin, 50 microM bacitracin, 10 microM leupeptin and 10 microM pepstatin) did not alter the contractile activity of angiotensin II. In contrast, angiotensin I (10 nM)-induced contraction was significantly reduced in the presence of ACE inhibitor SQ 20881 (500 nM). The angiotensin II induced contraction was not reduced by CGP 42112, a specific AT2 receptor antagonist, but was significantly inhibited by losartan, a specific AT1 receptor antagonist. Losartan shifted the dose-response curve of angiotensin II to the right with a pA2 value of 8.68. In addition, p-aminophenylalanine6 angiotensin II, which is proposed as an AT2 receptor agonist, did not induce contraction. It is concluded that the AT1 receptor predominantly mediates angiotensin-induced contraction in epididymal rat vas deferens.