Anton D. Michel

Direct labeling of rat M3-muscarinic receptors by [3H]4DAMP.

The muscarinic receptors of rat submaxillary gland, rat heart and rat cortex were directly labeled using the ligand [3H]4-diphenylacetoxy-N-methyl-piperidine methiodide [( 3H]4DAMP). In the rat submaxillary gland, [3H]4DAMP predominantly bound with high affinity (Kd = 0.2 nM) to a population of binding sites that displayed the pharmacology of the M3 muscarinic receptor subtype. In rat heart, [3H]4DAMP labeled the M2 muscarinic receptor with low affinity (Kd = 4 nM). In rat cortex [3H]4DAMP predominantly bound to a population of sites with high affinity (Kd = 0.2 nM). The pharmacology of these sites was consistent with [3H]4DAMP labeling both M1 and M3 muscarinic receptors present in rat cortex with high affinity. These data indicate that [3H]4DAMP represents a useful ligand for selectively labeling the M1 and M3 muscarinic receptor subtypes.

Induction of the angiotensin AT2 receptor subtype expression by differentiation of the neuroblastoma × glioma hybrid, NG-108-15

In vitro differentiation of the mouse neuroblastoma-rat glioma hybrid cell line, NG-108-15, with dimethyl sulphoxide (1.5%) and low serum (0.5%), produced a marked increase in the number of angiotensin II receptors, from a level at the limit of sensitivity using labelled angiotensin II with a high specific activity ([125I]angiotensin II), in undifferentiated cells, to a Bmax of 1077 (1070-1268) fmol/mg in 5-day-differentiated cells. The affinity (Kd) of radiolabelled angiotensin II for the receptors in differentiated cells was 8.1 (7.5-10) nM. The recently available selective non-peptide antagonists, DuP 753 and PD 123177 and the peptide analogues of angiotensin II, CGP 42112A and p-aminophenylalanine6 angiotensin II, were used to characterize the angiotensin II receptors by competing for 125I-[Sar1-Ile8]angiotensin II binding to membranes prepared from undifferentiated and differentiated cells. The predominant angiotensin II receptor subtype expressed by undifferentiated cells was AT1 and after differentiation AT2. This change in receptor expression was evident 2 days after initiation of differentiation, was maximal at 4-5 days and was stable for at least 8 days. Administration of angiotensin II induced intracellular Ca2+ mobilization in both undifferentiated and differentiated cells. This was antagonised by the selective AT1 antagonist, DuP 753, indicating an action at the AT1 receptor subtype in both undifferentiated and differentiated cells. The selective AT2 antagonist, PD 123177 was without effect on the angiotensin II induced increase in intracellular Ca2+. This effect of DuP 753 on Ca2+ was specific for angiotensin II since the drug had no effect on bradykinin induced increases in intracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

1 Pharmacology and Structure-Activity Relationships of α2-Adrenoceptor Antagonists

Publisher Summary This chapter discusses the pharmacology and structure–activity relationships of α 2 -adrenoceptor antagonists. The α-adrenoceptor was originally subdivided on an anatomical basis into pre- and postsynaptic subtypes. It has subsequently been pointed out that the terms pre- and postjunctional are more encompassing and therefore these will be used in the chapter. This original classification followed the demonstration of pre-junctional α-adrenoceptors that inhibited neurotransmitter release in a wide range of tissues from several species. The terms α 1 - and α 2 -adrenoceptors are subsequently adopted to describe post- and prejunctional adrenoceptors, respectively. However, the demonstration that α 2 -adrenoceptors occur postjunctionally in human platelets, human adipocytes, and vascular smooth muscle has led to the formulation of alternative methods of subclassification. The α 2 -adrenoceptor has been examined in a wide range of experimental systems using a diversity of methods. Direct-binding studies in membrane preparations have enabled an accurate assessment of the affinity of both agonists and antagonists for the α 2 -adrenoceptor, while the ability of α 2 -adrenoceptor agonists to attenuate the activity of adenylate cyclase has provided a more functional and biochemical means of examining these receptors.

Binding of angiotensin antagonists to rat liver and brain membranes measured ex vivo.

1 The effects of the angiotensin antagonists GR117289, losartan and Sar1Ala8‐angiotensin II on the ex vivo binding of [125I]‐Sar1Ile8‐angiotensin II to rat liver and cortex/hippocampus (Cx/H) membranes have been investigated. 2 GR117289 (0.1–30 mg kg−1, s.c., 2 h pretreatment) caused a dose‐dependent reduction in [125I]‐Sar1Ile8‐angiotensin II binding to both liver and cortex/hippocampus membranes. 3 Administration of a submaximal dose of GR117289 (1 mg kg−1, s.c.) indicated that the peak inhibition of binding in the liver occurred within 0.5 h, whereas the peak inhibition of binding in the Cx/H occurred 2 h after drug treatment. 4 The effect of GR117289 was long lasting. Binding was still reduced in the Cx/H 48 h after drug treatment (10 mg kg−1, s.c.) but had returned to normal 72 h after drug treatment. In the liver binding was still reduced 72 h after treatment with the same dose. 5 Losartan (1 − 30 mg kg−1, s.c.) was equipotent with GR117289 in its ability to reduce liver binding, but was less effective at inhibiting binding to central receptors. 6 The non‐peptide antagonist Sar1Ala8‐angiotensin II (3 and 10 mg kg−1) reduced binding in the liver but not in the Cx/H membranes. 7 These results suggest that, unlike the peptide antagonist Sar1Ala8‐angiotensin II, the non‐peptide angiotensin antagonists, GR117289 and losartan, are able to cross the blood brain barrier and occupy central angiotensin II receptors.

Pharmacological characterization of angiotensin-induced depolarizations of rat superior cervical ganglion in vitro.

1 The depolarizing responses to angiotensin II and angiotensin III of the rat superior cervical ganglion have been characterized in vitro, by the use of peptidase inhibitors, peptide and non‐peptide antagonists and dithiothreitol (DTT). 2 Angiotensin II and III depolarized the ganglion in a concentration‐related manner. Angiotensin II was approximately 30 fold more potent than angiotensin III. 3 The endopeptidase inhibitor, bacitracin, increased the potency of angiotensin II and III by approximately 4 and 20 fold respectively. The aminopeptidase inhibitor, amastatin, further increased the potency of angiotensin III (but not angiotensin II) by approximately 4 fold. In the presence of bacitracin and amastatin, angiotensin II and III were equipotent. 4 The peptide antagonist [Il7]angiotensin III (0.01–0.3 μm) produced a non‐parallel rightward displacement of the angiotensin II concentration‐response curve, with a suppression of the maximum response. The potency of [Ile7]angiotensin III was increased by bacitracin and amastatin. 5 The AT1‐selective non‐peptide antagonist losartan (DuP 753; 0.03 and 0.1 μm) produced a parallel rightward displacement of the angiotensin II concentration‐response curve, with an apparent pKB of 8.3 ± 0.1. A higher concentration of losartan (0.3 μm) depressed the maximum agonist response by 32 ± 6.5%, possibly reflecting non‐competitive behaviour of the antagonist. The potency of losartan was not influenced by bacitracin. 6 The AT2‐selective non‐peptide antagonist, PD123177 (3 μm) failed to antagonize the angiotensin II‐induced depolarizations. 7 DTT (1 mm) produced a 22% reduction of the maximum response to angiotensin II. 8 We conclude that the angiotensin II‐induced depolarizations of the rat superior cervical ganglion are mediated by angiotensin II receptors of the AT1 subclass. The ability of peptidase inhibitors to modify the potency of peptide agonists and antagonists highlights the difficulties associated with the use of peptide agents to characterize angiotensin II receptors in this preparation.