Endothelins and sarafotoxins: effects on motility, binding properties and phosphoinositide hydrolysis during the estrous cycle of the rat uterus.

The effects of four peptides of the endothelin/sarafotoxin (ET/SRTX) family on the motility of the rat uterus were examined during the different stages of the estrous cycle. ET-1, ET-3, SRTX-b and SRTX-c showed similar effects on the contraction of the uterus: a slight increase in the maximum tension of the spontaneous rhythmic contractions, a suppression of the relaxation phase of these contractions and an increase in their rate. All three effects were concentration dependent. Of the four peptides, ET-1 and SRTX-b showed the highest potency and efficacy, suggesting that among the various peptides of this family so far studied, ET-1 and SRTX-b are the two full agonists. The rank order of susceptibility of the different stages was, in most cases: proestrus greater than estrus greater than metestrus. Freshly excised diestrus uteri showed no spontaneous contractions and did not respond to any of the peptides. The binding potency of ET-1 and SRTX-b to uterine membranes was similar at the various estrous stages, but their maximal binding decreased gradually from proestrus to diestrus. All four peptides induced phosphoinositide (PI) hydrolysis in uterine slices at all four different stages, with ET-1 and SRTX-b again being more potent than ET-3 or SRTX-c. The maximal PI hydrolysis correlated with the increased rate of the rhythmic contractions. It is suggested that the reaction of the rat uterus to the ET/SRTX peptides depends on its hormonal status and that ET may act in concert with steroid hormones in the modulation of the estrous cycle.     

Reversible and irreversible inhibition of rat brain muscarinic receptors is related to different substitutions on bisquaternary pyridinium oximes

The role of the functional substituents on the pyridinium ring of bisquaternary pyridinium compounds, mostly oximes, in exerting reversible and irreversible inhibition of binding of [³H]-N-methyl-4-piperidyl benzilate ([³H]-4NMPB) to rat brain stem muscarinic receptors was studied. The drugs tested, i.e. HGG-42, HGG-12, HGG-52, HI-6, obidoxim, SAD-128 and TMB-4, could reversibly inhibit binding of [³H]-4NMPB, with the highest potency (K_I=1.7–6 M) exhibited by analogs possessing hydrophobic substituents at position 3 or 4 of the pyridinium ring. Bisquaternary drugs possessing an oxime moiety at position 2, but not at position 4 of the pyridinium ring, could also induce about 30% reduction of maximal binding capacity (B_max) (loss of muscarinic receptors) in addition to their reversible effect. Thus the structural correlates of the reversible and the irreversible effects of these drugs are different.     

Recognition of the muscarinic receptor by its endogenous neurotransmitter: binding of [3H]acetylcholine and its modulation by transition metal ions and guanine nucleotides.

Agonist binding to the muscarinic receptor in rat cerebral cortex membranes was studied by using the neurotransmitter itself, [3H]acetylcholine [( 3H]AcCho). By using 10 microM atropine or oxotremorine to define specific binding, it was possible to demonstrate specific binding of [3H]AcCho that was sensitive to muscarinic but not to nicotinic ligands. Equilibrium binding experiments with 5-240 nM [3H]AcCho indicated specific binding of the ligand to a saturable population of muscarinic receptors (361 +/- 29 fmol/mg of protein; Kd = 76 +/- 17 nM). This value represented 25% of the available binding sites for a labeled antagonist in the same preparation and corresponds to the proportion of high-affinity agonist binding sites observed previously in competition experiments with labeled antagonists. Inclusion of transition metal ions (e.g., 2 mM Ni2+) in the assay increased the equilibrium binding of [3H]AcCho (628 +/- 38 fmol/mg of protein, Kd = 86 +/- 21 nM) but did not affect equilibrium binding of 3H-labeled antagonists, indicating conversion of low- into high-affinity muscarinic agonist binding sites. The increase developed slowly over 30 min of incubation at 25 degrees C but could be reversed rapidly (approximately equal to 2 min) by the chelating agent EDTA or by guanine nucleotides. These data directly reveal a slow though quickly reversible interconversion of low- into high-affinity muscarinic agonist binding sites.     

Mechanism of acetylcholine release: possible involvement of presynaptic muscarinic receptors in regulation of acetylcholine release and protein phosphorylation.

Acetylcholine (AcCho) release from purely cholinergic Torpedo synaptosomes was evoked by K+ depolarization in the presence of Ca2+. Activation of muscarinic receptors, present in the synaptosomal fraction, by the agonist oxotremorine resulted in the inhibition of AcCho liberation. This inhibition was abolished by the muscarinic antagonist atropine, which by itself has no effect. These findings suggest that the muscarinic receptor, present in the electric organ of Torpedo is presynaptic and that its physiological function is to regulate AcCho release by negative feedback. The mechanism of presynaptic muscarinic inhibition was investigated by examining the effect of muscarinic ligands on synaptosomal 45Ca2+ uptake and on the level of phosphorylation of specific synaptosomal proteins. Ca2+-dependent K+ depolarization-induced synaptosomal AcCho release was accompanied by 45Ca2+ uptake and by a marked increase in the phosphorylation of a specific synaptosomal protein (band alpha) of approximately 100,000 daltons. Activation of the muscarinic receptor by the agonist oxotremorine had no detectable effect on synaptosomal 45Ca2+ uptake but resulted in the concomitant inhibition of AcCho release and of phosphorylation of band alpha. The muscarinic antagonist atropine abolished the inhibitory effect of oxotremorine both on AcCho liberation and on phosphorylation of band alpha. These findings suggest that phosphorylation of band alpha may be involved in regulation of the presynaptic processes that underly AcCho release and that activation of the muscarinic receptor by agonists may inhibit AcCho release by blocking the phosphorylation of band alpha.     

Endothelins and sarafotoxins: physiological regulation, receptor subtypes and transmembrane signaling.

The endothelins and sarafotoxins are two structurally related families of potent vasoactive peptides. Although the physiological functions of these peptides are not entirely clear, the endothelins are probably involved in pathophysiological conditions such as hypertension and heart failure. This review summarizes the state of the art in some areas of this intensively studied subject, including: (1) structure-function relationships of ET/SRTX, (2) ET concentrations in plasma, (3) ET/SRTX receptor subtypes and (4) signaling events mediated by the activation of ET/SRTX receptors.     

Psychotomimetics as anticholinergic agents. I. 1-Cyclohexylpiperidine derivatives: anticholinesterase activity and antagonistic activity to acetylcholine

Phencyclidine (sernyl^®, 1-[(1 -phenylcyclohexyl)piperidinel]) and ten of its derivatives, known for their psychotomimetic activity, are potent competitive inhibitors of butyry-lcholinesterase and acetylcholinesterase. The drugs also protect the enzyme against inactivation by an organophosphate (sarin), presumably by their direct interaction with the active site. In addition to the acetylcholine-like structural factors identifiable in these molecules, the cyclohexyl moiety is considered necessary for the interaction. The drugs are also competitive antagonists of acetylcholine in perfused organs (guinea-pig ileum, frog rectus abdominis) and in the eye of three mammals. This peripheral activity is three orders of magnitudes less potent than that of atropine. Antiacetylcholine activity in the central nervous system was studied through the antidotal effect of THA (tacrine), before and after injection of the drugs. The correlation between the anticholinesterase activity and the CNS activity as well as the structural relation of the drugs to agonists and antagonists of the cholinergic system is discussed     

Disopyramide and quinidine bind with inverse selectivity to muscarinic receptors in cardiac and extracardiac rat tissues

We investigated the interactions of disopyramide and quinidine with the muscarinic receptor in tissue homogenates from rat atrium, ventricle, cortex, submandibular gland, and urinary bladder by means of competition binding experiments, using the tritium-labeled antagonist N-methyl-4-piperidyl benzilate. The drugs displayed heterogeneous characteristics of binding to the muscarinic receptors in the different tissues. The binding affinity of quinidine to the muscarinic receptor in atrial tissue was five to 10 times greater than in the other tissues studied, whereas the affinity of disopyramide to the muscarinic receptor in the heart was five times lower than in the other tissues. This inverse selectivity shown by the two drugs in their binding to cardiac and to noncardiac tissues may explain the extracardiac antimuscarinic side effects of treatment with disopyramide and their absence with quinidine.     

In vivo and in vitro studies on the antimuscarinic activity of some amino esters of benzilic acid

The cholinergic antimuscarinic properties of some aliphatic and heterocyclic aminoesters of benzilic acid were evaluated in the CNS as well as in peripheral organs. Antagonism to hypothermia and to forced motor activity disturbances, both centrally mediated and induced in mice by oxotremorine, was kinetically determined in vivo. The antiacetylcholine activity of these compounds on the isolated guinea pig ileum, and their affinity for muscarinic high-affinity binding-sites in mouse brain homogenate were determined and correlated in vitro. 3-Quinuclidinyl benzilate (QNB) was found to be the most potent drug in vivo as well as in vitro, while dimethylaminoethyl benzilate was the least active. Atropine was as potent as scopolamine in the competition experiments in vitro but ten times less active in the two in vivo tests. Rate constants for the onset (k_on) and offset (k_off) of the antimuscarinic activity determined on the isolateed ileum, were found to increase and decrease, respectively, with the increase in the affinity of the drugs for the muscarinic binding-sites. The relationship between the molecular structure and the properties of these compounds is discussed in terms of the factors contributing to the observed rate constants of antimuscarinic activity and of the reasons for the decrease in the apparent potency of atropine in vivo.