Franco Zonta

Comparative studies of the postjunctional activities of some very potent muscarinic agonists

SummaryThis study was undertaken to determine the potenties of seven muscarinic agonists (methylfurtrethonium, dioxolane, oxathiolane, carbachol, muscarine, muscarone and oxotremorine) on the postjunctional muscarinic receptors of seven isolated preparations (guinea pig taenia-coli, ileum, jejunum, trachea and atria and rat jejunum and urinary bladder).The results indicate that the rank order of sensitivity of the preparations varies independently of the potency of the agonist used and it is almost the same for all the compounds with the exception of oxotremorine.Muscarone was the most potent compound in all the tissues. Intergroup comparisons in each preparation and the evaluation of the equieffective molar ratios relative to muscarone revealed that carbachol possesses a certain degree of cardioselectivity and oxathiolane, on the other hand, is much less active on the cardiac tissue than on the others.Oxotremorine is a peculiar compound endowed with cardioselectivity.

Synthesis and pharmacological investigation of cholinergic ligands structurally related to muscarone

Abstract Five new analogs of muscarone were synthesized in order to evaluate the influence of the carbonyl group on muscarinic activity. We chose to introduce structural variations at the C-2 and C-3 positions of the tetrahydrofuran ring. The muscarinic activity was evaluated in vitro on guinea pig atria and ileum as well as on rat jejunum and urinary bladder. All the new derivatives are less potent than muscarone and three of them displayed a potency very close to that previously reported for muscarine. The tissue selectivity observed for the 3-methylene derivative which is eight times more potent on guinea pig ileum than atria is worth noting. The present data show the lack of a simple relationship between the polarity of the group located in the 3-position of the ring and the muscarinic activity.

Insulin-like growth factor-I ameliorates delayed kidney graft function and the acute nephrotoxic effects of cyclosporine.

BACKGROUND Delayed graft function (DGF) is a relatively common complication after cadaveric renal transplantation. The adverse effect of DGF on long-term graft survival has lead to intensive efforts to reduce ischemic graft injury. In this study we examined the effects of a new protective treatment based on insulin growth factor (IGF)-I. We evaluated the impact of the treatment on renal recovery and on the nephrotoxicity that is a common side effect of mainstream immunosuppressants. Because therapy with IGF-I or the analog des(1-3)IGF-I is effective in treating experimental ischemic renal failure, these peptides may be useful as perspective clinical treatments. METHODS We have addressed three areas relating to the potential use of IGF-I and its analog des(1-3)IGF-I. First, because of the immunogenic properties of IGF-I, we assessed the effect of des(1-3)IGF-I on the rejection of skin allografts in Lewis rats. Next we determined whether treatment with des(1-3)IGF-I influences the early function of transplanted kidneys in a model of DGF induced by a combination of warm and cold ischemia. Finally we tested whether IGF-I protects against acute cyclosporine nephrotoxicity. RESULTS Des(1-3)IGF-I did not accelerate the rejection of the skin grafts (P=0.57). The administration of this peptide in a model of syngenic renal transplant improved the early function of the graft. Postoperative values of creatinine and blood urea nitrogen were significantly better (P<0.05) in treated animals. IGF-I also ameliorated the nephrotoxicity of cyclosporine, with better values of creatinine and blood urea nitrogen (P<0.05). CONCLUSIONS In evaluating this study it should be recognized that the animal models studied, although widely used, differ from the human condition. However, IGF-I and des(1-3)IGF-I exhibit properties that strongly suggest their value in preventing clinical DGF, and they deserve further studies.

PHARMACOLOGICAL CHARACTERIZATION OF ALPHA -ADRENOCEPTORS THAT MEDIATE CONTRACTION IN SPLENIC ARTERY STRIPS FROM THE PIG

The α-adrenoceptors that mediate contractions in strips of splenic artery from the pig were characterized by the use of selective agonists and subtype-selective antagonists. Noradrenaline, the α1-selective agonist phenylephrine and the α1-/α2-agonist oxymetazoline caused the preparations to contract with potency (pD2) values of 6.94, 6.14 and 7.27, respectively. Compared to noradrenaline, phenylephrine and oxymetazoline induced 93% and 78% of noradrenaline maximum effect. Conversely, the two α2-selective agonists clonidine and B-HT 920 induced only 31% and 13% of noradrenaline maximum effect. B-HT 920 only marginally contracted the tissue even when it was precontracted with phenylephrine. The α2-selective antagonist yohimbine antagonized oxymetazoline- and phenyleprine-induced contractions with affinity (pA2) values (6.80 and 6.74, respectively) consistent with α1-adrenoceptor interaction. This suggests that the pig splenic artery possesses only functional α1-adrenoceptors. The α1-adrenoceptor antagonists of varying subtype selectivities like WB-4101, 5-methylurapidil, benoxathian and BMY 7378 competitively antagonized phenylephrine-induced contractions with affinity values of 9.46, 8.26, 9.06 and 6.91, respectively. These values correlated highly with published affinity values for functional α1A-adrenoceptors (r=0.92) and α1a-clones (r=0.94) and less well with affinity values for functional α1B-adrenoceptors (r=0.84) and α1b-clones (r=0.87). Conversely, correlation with functional α1D-adrenoceptors (r=0.26) and α1d-clones (r=0.33) was poor. In addition the α1D-selective antagonist BMY 7378 had a low affinity value compared to that reported for α1D-adrenoceptors. Therefore, based on correlation studies, the plot that resembled the line of equal values most closely was that for the α1A-subtype. The α1A-selective antagonist RS-17053 antagonized phenylephrine-induced contractions in an apparently non-competitive manner and gave an apparent pA2 value of 7.06 which is similar to the “low” affinity values reported in other α1A-containing tissues. Exposure to the irreversible α1B/D-antagonist chloroethylclonidine slightly decreased maximum response to phenylephrine without significantly affecting its potency value, indicating that the phenylephrine response is substantially chloroethylclonidine-insensitive.It is concluded that splenic artery strips from the pig contract in response to phenylephrine through activation of α1-adrenoceptors which display the pharmacological profile of the α1A-subtype for which the recently reported α1A-selective antagonist RS-17053 shows low affinity. Evidence for contribution of the α1B-subtype in the overall contractile response is elusive while no evidence was obtained for the involvement of the α1D-subtype. The contribution of functional α2-adrenoceptors to the contractile response was ruled out.

Determination of dissociation constants and relative efficacies of some potent muscarinic agonists at postjunctional muscarinic receptors

SummaryThis study was undertaken to determine dissociation constants (KA) and relative efficacies (er) of seven muscarinic agonists (methylfurtrethonium; dioxolane, oxathiolane, carbachol, muscarine, muscarone and oxotremorine) in three isolated tissues (guinea-pig ileum and atria and rat urinary bladder).The rank order of affinities (-log KA) of the various compounds varied depending on the tissue used. er values for the different agonists did not differ significantly from each other in any of the three tissues, except that the er of muscarine in the guinea-pig ileum was higher than those of the other compounds and that of oxotremorine in the rat urinary bladder was lower than those of the other agonists.Comparisons among tissues show that KA and er values were the same in different tissues for some compounds (muscarone, muscarine and methylfurtrethonium), while significant differences were found for the other compounds. This suggests the existence of a discrete receptor population recognized by some but not all agonists.For oxotremorine er as well as -log KA, is greater in atria than in smooth muscle: these factors combine to determine the cardioselectivity of this compound which can now ascribed to receptor selectivity.

Cholinergic agents structurally related to furtrethonium. 1

Abstract A series of 5-substituted-2-(dimethylaminomethyl)-furyl derivatives 4 was prepared, with the aim of discovering novel antimuscarinic agents which are selective for smooth muscle as opposed to cardiac tissue. Both non-quaternary and quaternary ammonium compounds were synthesised. The agonist starting point, furtrethonium 3 , was gradually transformed into antagonist by introduction of lipophilic and bulky groups in position 5 of this molecule. In particular, the introduction of α-hydroxy-α-cyclohexylbenzyl moiety (compound 9b ), a lipophilic group characteristic of antimuscarinic agents, caused an appreciable increase of the antagonists potency, and the lengthening of the distance between this lipophilic group and the furan ring, obtained by introduction of an ester, ether or amide group, led to some selectivity towards smooth muscle (compounds 19, 21, 25 ). Interestingly, compound 19 , with an ester moiety as a spacer group, proved to be at least 20 times more potent in rat ileum (p K B = 7.3) and rat bladder (p K B = 7.2) than guinea-pig atria (p K B = 5.9).

Effects of cromakalim (BRL 34915) on mechanical responses of rat vas deferens to noradrenaline and naphazoline

The contractile responses produced by noradrenaline were compared to those produced by naphazoline in the rat isolated vas deferens, a preparation which is usually quiescent. The responses to noradrenaline were biphasic: prominent spontaneous contractile activity was superimposed on contractions produced by naphazoline. After removal of naphazoline the rat vas deferens displayed periodic spontaneous activity. The effects of cromakalim and verapamil were compared on contractions induced by noradrenaline and naphazoline and on spontaneous activity induced by exposure to naphazoline. Cromakalim (1-10 x 10(-7) M) shifted to the right the cumulative concentration-response curve of naphazoline but not that of noradrenaline; at the same range of concentrations, cromakalim inhibited naphazoline-induced periodic spontaneous activity. Verapamil (3-30 x 10(-8) M) flattened in a dose-dependent manner the concentration-response curves of noradrenaline and naphazoline; higher concentrations of verapamil (3-30 x 10(-6) M) were required to modify periodic spontaneous activity. It was concluded that naphazoline but not noradrenaline can block K+ channel(s) and that this property can represent an important difference between imidazolines and phenethylamines.

Isolated porcine bronchi provide a reliable model for development of bronchodilator anti-muscarinic agents for human use

Background and purpose: In human airways, muscarinic acetylcholine receptors (mAChRs) exert a predominant role in the control of airways resistance and anti‐muscarinic agents are currently included in the pharmacological treatment of chronic obstructive pulmonary disease (COPD). However, the development of more effective mAChR antagonists is hampered by considerable species variability in the ultrastrucural and functional control of airway smooth muscle, making extrapolation of any particular animal model questionable. This study was designed to characterize the mAChRs in a bronchial preparation from pigs, animals considered to provide close models of human biology.

Inhibition by glibenclamide of the effects of cromakalim on responses of rat vas deferens to naphazoline.

Cromakalim has been shown to inhibit naphazoline-induced contractions and spontaneous activity induced by exposure to naphazoline in the rat isolated vas deferens. Glibenclamide 10(-6) M blocked both these effects of cromakalim. Our data add to the list of data derived mainly from experiments on vascular smooth muscle; they suggest that the same glibenclamide-sensitive K+ channel is present in vascular and non-vascular smooth muscle and that it may be involved in the relaxant actions of cromakalim.

The influence of verapamil and papaverine on the calcium- and epinephrine-induced responses of isolated guinea-pig atria☆

The effects of papaverine and verapamil on Ca2+ - and epinephrine evoked responses were investigated on spontaneously beating atria of the guinea-pig. Papaverine showed a non competitive antagonism against epinephrine but was ineffective against Ca2+; verapamil antagonized both the action of epinephrine and Ca2+ in a different manner but at the same concentrations. The pharmacological prerequisite to classify an anti-Ca2+ substance as a blocker of Ca2+ entry (that is the ability to selectively antagonize the Ca2+ -induced contraction relative to those of norepinephrine in vascular smooth muscle) was not met by the results obtained in the cardiac tissue. The behaviors of verapamil and papaverine with regard to Ca2+ response suggest another criterion for classifying a substance as a Ca2+ entry blocker based on the ability or inability to inhibit the response of the atrial preparation to Ca2+.