Offset rate of action of muscarinic antagonists depends on their structural flexibility.

Time course measurements of the action of muscarinic antagonists were performed in the spontaneously beating carp atrium. Several high affinity drugs, which embody the quinuclidine structure were examined. The structural flexibility of these molecules was reflected in the dissociation of the drugs from the muscarinic receptor. The dissociation of rigid drugs was very much prolonged as compared to flexible drugs of the same affinity.     

Dissociation constants of 4-acetoxy-piperidines and -thiacyclohexanes at the muscarinic receptor.

The dissociation constants (KA) and the relative intrinsic efficacies (e) of 4-acetoxy-piperidines and -thiacyclohexanes were determined on the guinea-pig isolated left atrium. The differences in the muscarinic potencies are associated with differences in affinities and efficacies, respectively.     

Dissociation constants of 4-acetoxy-piperidines and-thiacyclohexanes at the muscarinic receptor

Summary The dissociation constants (K_A) and the relative intrinsic efficacies (e) of 4-acetoxy-piperidines and-thiacyclohexanes were determined on the guinea-pig isolated left atrium. The differences in the muscarinic potencies are associated with differences in affinities and efficacies, respectively.Structure and conformation activity relationships of heterocyclic acetylcholine analogues, VIII.We thank the German Research Association for supporting this work, and Mrs Ch. Röttger for her valuable assistance in carrying out the investigations.     

The kinetics of estrogen binding to rat alpha-fetoprotein

Fluid obtained from rat fetuses was utilized to characterize the affinity, number of binding sites, and the association and dissociation rate kinetics of the binding of estradiol and estrone to AFP. Statistical analysis demonstrated no differences when the values for the AFP-estradiol interaction were compared with those obtained for the ATP-estrone interaction. These data demonstrate that rat AFP specifically binds estradiol and estrone with a high capacity, high affinity, and similar binding kinetics.     

The kinetics of estrogen binding to rat α-fetoprotein

Summary Fluid obtained from rat fetuses was utilized to characterize the affinity, number of binding sites, and the association and dissociation rate kinetics of the binding of estradiol and estrone to AFP. Statistical analysis demonstrated no differences when the values for the AFP-estradiol interaction were compared with those obtained for the AFP-estrone interaction. These data demonstrate that rat AFP specifically binds estradiol and estrone with a high capacity, high affinity, and similar binding kinetics.This work was supported by NSF grant PCM-8109847 and by a Grant-in-Aid of Research from Sigma Xi, The Scientific Research Society.     

Determination of odour affinities based on the dose-response relationships of the frog's electro-olfactogram

Summary Electro-olfactograms (EOG's) recorded from the frog's olfactory epithelium for 11 substances were used to calculate dissociation constants which in turn serve as an index for the affinity between odorant and receptor site. These constants were calculated with and without a correction for the odour partition between water and air. For a homologous series of 7 n-alcohols these values decrease up to 1-heptanol. The dose-response relationships were based on the peaks of the EOG's since the peak/plateau-ratio was concentration-dependent for some of the substances.This work was supported by Z.W.O. grant number 12-24-12 of the Dutch Government.     

Action of D-penicillamine on immunocomplexes containing rheumatoid factor

Summary The affinity between purified rheumatoid factors (RF) and native or heat aggregated human IgG has been studied in vitro by polarization florescence in the presence and in the absence of D-penicillamine. The value of the dissociation constant was the same using native and heat aggregated IgG suggesting that binding to the aggregated protein is not dependent on the exposure of a new determinant lacking in the native molecule. The results obtained in the presence of D-penicillamine suggest that the concentration of the drug necessary to get a pronounced effect on the apparent dissociation constant of the immunocomplex between IgG and RF is not reached in vivo, in clinical situations.     

Specific cleavage of insulin-like growth factor-binding protein-1 by a novel protease activity

Insulin-like growth factor-binding protein-1 (IGFBP-1) is secreted in a highly phosphorylated form that binds IGF-I with high affinity and is resistant to proteolysis. We have purified IGFBP-1-specific protease activity from the urine of an individual with multiple myeloma. This protease efficiently cleaves both phosphorylated and non-phosphorylated IGFBP-1 at Ile130-Ser131, generating fragments that together have higher association and dissociation rates for IGFs compared with intact IGFBP-1. The proteolytic fraction contained azurocidin, a protease homologue hitherto considered inactive. After cleavage of IGFBP-1, there was a lower affinity, but higher capacity for IGF-I binding, suggesting both N- and C-terminal fragments may interact with ligand independently. There was decreased inhibition of IGF-II-stimulated cell growth and glucose uptake. Alone, proteolysed IGFBP-1 stimulated glucose uptake in muscle. We conclude that specific cleavage of IGFBP-1 at target tissues is important in cellular growth and metabolism and opens novel strategies for targeting IGFBP-1 in treatment of disease.     

Inhibition of in vitro RNA synthesis by hycanthone,oxamniquine and praziquantel

Summary The schistosomicides, hycanthone, oxamniquine and praziquantel, were found to inhibit the in vitro RNA synthesis using isolated hamster liver nuclei. Preincubation of the nuclei with these drugs revealed that the inhibitory effect of oxamniquine was irreversible and progressed with time, whereas that of hycanthone and parziquantel was reversible. On the other hand, hycanthone and praziquantel have a high affinity for DNA but oxamniquine does not. The data indicate that the mechanism of inhibition by oxamniquine is different from that of hycanthone and praziquantel.     

Inhibition of in vitro RNA synthesis by hycanthone, oxamniquine and praziquantel.

The schistosomicides, hycanthone, oxamniquine and praziquantel, were found to inhibit the in vitro RNA synthesis using isolated hamster liver nuclei. Preincubation of the nuclei with these drugs revealed that the inhibitory effect of oxamniquine was irreversible and progressed with time, whereas that of hycanthone and praziquantel was reversible. On the other hand, hycanthone and praziquantel have a high affinity for DNA but oxamniquine does not. The data indicate that the mechanism of inhibition by oxamniquine is different from that of hycanthone and praziquantel.     

Genetic and pharmacological models of cholinergic supersensitivity and affective disorders

Increased muscarinic sensitivity has been associated with altered hormonal states (hypothyroidism and hyperadrenocorticism), chronic administration of muscarinic antagonists or antidepressants with muscarinic actions, selective breeding for anticholinesterase sensitivity, and certain inbred strains of rats and mice. Thus, both genetic and environmental factors may influence muscarinic receptor sensitivity. The reasonably detailed studies on the selectively-bred rats have revealed that the Flinders Sensitive Line (FSL) rats weigh less, are less active, are more sensitive to muscarinic agonists and to stressors, and have higher concentrations of hippocampal and striatal muscarinic receptors than 'normal', or the selectively-bred, Flinders Resistant Line (FRL) rats. Thus, there are a number of parallels between FSL rats and depressed humans. The FSL rats may be the first animal model of depression to mimic the actual trait of depression, and not just the state.     

Characterization of apoptosis induced by marine natural products in non small cell lung cancer A549 cells

The effects of different marine derived agents were studied in A549 cell growth. These drugs induced cell cycle arrest at the G2-M phase associated with the up-regulation of GADD45alpha-gamma and down-regulation of c-Myc. In treated cells, GADD45alpha-gamma and c-Myc were up- and down-regulated, respectively. A cascade of events leading to apoptotic mitochondrial 'intrinsic' pathway was observed in treated cells: (1) dephosphorylation of BAD serine136; (2) BAD dissociation from 14-3-3 followed by its association with BCL-XL; (3) cytochrome c release; (4) caspase-3 activation, and (5) cleavage of vimentin. Caspase(s) inhibitor prevented the formation of cleavage products and, in turn, apoptosis was inhibited through a p53-independent mechanism. Moreover, these compounds did not activate NF-kappaB. Our findings may offer new insights into the mechanisms of action of these agents in A549 cells. The better understanding of their effects might be important to fully exploit the potential of these new drugs.     

Interaction of smooth muscle relaxant drugs with calmodulin and cyclic nucleotide phosphodiesterase

Some smooth muscle relaxant drugs with an unknown mechanism of action have been tested for their interaction with calmodulin and with calmodulin-induced cyclic nucleotide phosphodiesterase (PDE) activity. The affinity of these drugs for calmodulin does not parallel their inhibitory effect on the calmodulin activation of PDE. The lack of parallelism could be due to a binding of the drugs to different sites on calmodulin; furthermore a binding of papaverine, octylonium bromide and felodipine to PDE molecule might also be considered to explain their inhibitory effect on PDE basal activity. The myolytic effect of octylonium bromide and pinaverium bromide may be due to their interaction with calmodulin-dependent systems.     

Interaction of smooth muscle relaxant drugs with calmodulin and cyclic nucleotide phosphodiesterase

Summary Some smooth muscle relaxant drugs with an unknown mechanism of action have been tested for their interaction with calmodulin and with calmodulin-induced cyclic nucleotide phosphodiesterase (PDE) activity. The affinity of these drugs for calmodulin does not parallel their inhibitory effect on the calmodulin activation of PDE. The lack of parallelism could be due to a binding of the drugs to different sites on calmodulin; furthermore a binding of papaverine, octylonium bromide and felodipine to PDE molecule, might also be considered to explain their inhibitory effect on PDE basal activity. The myolytic effect of octylonium bromide and pinaverium bromide may be due to their interaction with calmodulin-dependent systems.     

The response to potassium of the Na-K pump ATPase in low-K red blood cells from cattle at birth and in later life

It is shown that in low-K red blood cells of cattle the apparent affinity for K(1/Kapp K) at an inhibitory site of the Na-K ATPase increases markedly during the first 3 months of life. This site probably is the Na accepting site at the internal membrane surface and the change in Kapp K reflects an increase in KNa/KK, the ratio of the true dissociation constants. This effect may explain the concomitant fall in cellular K concentration.     

Genetic and pharmacological models of cholinergic supersensitivity and affective disorders

Summary Increased muscarinic sensitivity has been associated with altered hormonal states (hypothyroidism and hyperadrenocorticism), chronic administration of muscarinic antagonists or antidepressants with muscarinic actions, selective breeding for anticholinesterase sensitivity, and certain inbred strains of rats and mice. Thus, both genetic and environmental factors may influence muscarinic receptor sensitivity. The reasonably detailed studies on the selectively-bred rats have revealed that the Flinders Sensitive Line (FSL) rats weigh less, are less active, are more sensitive to muscarinic agonists and to stressors, and have higher concentrations of hippocampal and striatal muscarinic receptors than normal, or the selectively-bred, Flinders Resistant Line (FRL) rats. Thus, there are a number of parallels between FSL rats and depressed humans. The FSL rats may be the first animal model of depression to mimic the actual trait of depression, and not just the state.     

Desensitization of the muscarinic receptor of bullfrog atrial muscle

Summary The muscarinic ACh receptors, which hyperpolarize the resting membrane and also depress the action potential of bullfrog atrial muscle, show desensitization to the action of ACh. This suggests that the molecular mechanism of these muscarinic ACh receptor-ionic channel (voltage-dependent) complexes is comparable to that of the nicotinic ACh receptor-ionic channel (voltage-independent) complex of the end-plate.Acknowledgment. This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.     

Serotonergic and cholinergic interaction in the regulation of pituitary-adrenal function in rats

It has been proposed that the central serotonergic inputs which modulate pituitary-adrenal secretion are mediated by cholinergic neurons. We have tested this hypothesis in intact rats. Male Sprague-Dawley rats were injected with cholinergic and serotonergic agents which enhanced transmitter function and with receptor blocking agents. Agents were injected, singly and in combination, into both unstressed and stressed animals. Since the response to cholinergic agents might be due to changes to vasopressin release, Brattleboro (vasopressin deficient) rats were also injected with cholinergic agents. The level of plasma corticosterone at 1-h post-injection was determined. Results indicate that the serotonin receptor blockade decreased the stimulatory, cholinergic effect of physostigmine. Cholinergic receptor blockers did not significantly reduce the corticosterone rise induced by 5-hydroxytryptophan. These results do not support the hypothesis of cholinergic mediation of serotonergic input. Nicotinic and muscarinic receptors appeared to exert opposing influences on the system. The nicotinic receptor antagonist was able to block the stimulatory effect of physostigmine. The muscarinic receptor antagonist significantly elevated plasma corticosterone levels. No differences were found in the effect of physostigmine on Brattleboro rats as compared to controls. These data are interpreted as suggesting that 1) the acetylcholine-induced stimulation of pituitary-adrenal function is mediated, in part, by serotonergic neurons; and 2) stimulation of nicotinic receptors is facilitatory whereas stimulation of muscarinic receptors is inhibitory to pituitary-adrenal function.