Biomedical subjects

E Grana

Publications and source records attributed to E Grana.

At least 19 recordsLinked to original sources

PubMed2000

Differential effects of drugs interacting with autonomic transmitters on responses of rat vas deferens to field stimulation.

1. Frequency-response curves (0.1-30 Hz) were obtained in the epididymal portion of rat vas deferens. At low frequencies (0.1-1 Hz), the parameters evaluated were the first twitch and the fourth twitch at each frequency. The responses to trains of stimuli at intermediate (2-5 Hz) and high (10-30 Hz) frequencies were biphasic consisting of phase I (the first rapid phase of tetanus) and of phase II (the secondary slowly developing one). 2. Prazosin inhibited the first and the fourth twitch but not when the frequency was < 1 Hz. Suramin inhibited the first twitch while substantially depressing the fourth one. The combination of prazosin and suramin almost completely abolished all the twitches evoked by a train of stimuli at low frequencies. Nifedipine left almost unaltered the first twitch while markedly depressing the fourth one, especially at relatively high frequency (1 Hz). Verapamil was devoid of any inhibitory action. Papaverine depressed the first twitch while only at the highest concentration used (1 x 10(-4) M) markedly inhibited the fourth one. Chloroethylclonidine (CEC) depressed the first twitch and increased the fourth. 3. When intermediate (2-5 Hz) and high (10-30 Hz) frequencies are considered, prazosin and suramin partially inhibited both phase I and phase II, while in combination they almost completely abolished both phases. Nifedipine and verapamil selectively suppressed phase II, leaving phase I unaffected. Papaverine completely abolished both phase I and phase II. CEC was able to completely abolish phase I but increased phase II. 4. These results suggest that the response to the first twitch of a train at low frequency is prevailingly noradrenergic, prazosin-sensitive, while when the twitches are close enough (i.e. at 1 Hz) a summation of stimuli takes place and a predominant purinergic component, both suramin- and nifedipine-sensitive, becomes evident. 5. At high frequencies, both phases are due to the concomitant release of noradrenaline and adenosine triphosphate (ATP). The noradrenergic component of phase I is nifedipine-insensitive and CEC-sensitive, resembling the pharmacological profile of the endogenously released noradrenaline by single pulse, while that of phase II, nifedipine-sensitive and CEC-insensitive, is similar to that produced by exogenously applied noradrenaline.

Adrenergic alpha-Antagonists↗

PubMed1999

Differential blockade of alpha,beta-methylene-ATP, noradrenaline and electrically evoked contractions of the rat vas deferens.

The present study was carried out compare the effects of nifedipine, verapamil, papaverine and chloroethylclonidine (CEC) on the electrically-induced contractions and on the contractions produced by exogenous noradrenaline and alpha, beta methylene-ATP in the epidydimal portion of rat vas deferens. Nifedipine inhibited in a concentration-dependent fashion the purinergic component (phase I) of the electrically evoked response. Verapamil (10(-7) M-10(-5) M) did not inhibit significantly or even potentiated phase I of the contractile response to single-pulse field stimulation but inhibited the response to alpha,beta methylene-ATP. Papaverine left unaffected phase I but depressed the response to alpha,beta methylene-ATP. CEC significantly potentiated the purinergic component of the electrically-evoked response and the response induced by alpha,beta methylene-ATP. Nifedipine was devoid of any inhibitory action on the noradrenergic component (phase II) of the response to single shock. Verapamil at the highest concentration used (10(-5) M) was able to partially inhibit phase II. Papaverine abolished in a concentration-dependent manner the noradrenergic component of the response to single shock. CEC abolished the phase II of single shock while was devoid of any inhibitory action on exogenous noradrenaline. The implications of these differences among the compounds studied in the present work are discussed in relation to roles of calcium channels.

Adenosine Triphosphate↗

PubMed1998

Effect of calcium antagonists on the response to noradrenaline in the whole and bisected rat vas deferens.

1. The present study was carried out to look at the effect of different calcium antagonists on the response to noradrenaline in the whole and bisected rat vas deferens considering that the response consisted of three components (I) the phasic response (II) the tonic response and (III) the spikes (rhythmic contractions). 2. Nifedipine (3 x 10(-9)-1 x 10(-7) M) inhibited all the components at the same concentration range, verapamil (1 x 10(-7)-1 x 10(-5) M) inhibited the phasic and tonic response but not the rhythmic activity. This latter component, at a certain concentration range and especially in the prostatic portion was markedly potentiated. Diltiazem and flunarizine lay in an intermediate position. 3. Papaverine, a Ca2+ antagonist that acts mainly intracellularly, inhibited preferentially the tonic component; ryanodine was practically inactive. 4. Cromakalim inhibited only partially the phasic and tonic components but totally inhibited the rhythmic contractions. 5. These results can be explained by postulating two types of calcium channels opened by alpha-adrenoceptor stimulation. The first one is verapamil- and nifedipine-sensitive and allows the entry of Ca2+ directly available for the contraction and responsible for the phasic and partially responsible for the tonic component. The second channel is merely nifedipine-sensitive and allows the entry of Ca2+ trigger which can release Ca2+ from intracellular sites: the mobilized Ca2+ is able to sustain the tonic component and is the main one responsible for the rhythmic activity. There is the possibility that this second channel is associated with ATP-sensitive K+ channels.

Animals↗

PubMed1997

Effect of cromakalim on the purinergic and cholinergic transmission in the rat detrusor muscle.

Contraction of the rat detrusor muscle is mediated by cholinergic and purinergic mechanisms. The present study was carried out to look at the influence of cromakalim, compared with atropine, suramin and nifedipine on the contractile response evoked by single shock and exogenous agonists (carbachol and ATP) in rat urinary bladder. Cromakalim was able to inhibit only partially the response to carbachol and profoundly affected the response to exogenous ATP. Atropine suppressed the response to carbachol and was inactive versus ATP. Suramin was inactive versus carbachol and was able to antagonize the response to ATP. Nifedipine proved to be a non-competitive antagonist versus carbachol (pD2 = 7.66 +/- 0.05) and deeply affected the response to ATP. Cromakalim inhibited only partially the first, purinergic, phase of the electrically evoked response but was able to inhibit in a concentration-dependent manner the second, cholinergic, phase (logIC50 = 6.87 +/- 0.05). Nifedipine blocked both the phases. Atropine blocked partially only the second phase. Suramin inhibited the first phase but, at least partially, also the second one. The combination of atropine and suramin enhanced the inhibition of the second phase. The antagonistic effect of suramin on the second phase could indicate an overlap of the purinergic and cholinergic components. The comparison between pre- and postjunctional effects indicates that cromakalim acts on purinergic transmission predominantly postjunctionally. On the contrary, the action on cholinergic transmission seems to occur mainly at prejunctional level. This conclusion can be relevant in view of the claimed importance of K+ channel openers in the treatment of urinary disorders.

Adenosine Triphosphate↗

PubMed1997

Cromakalim blocks the purinergic response evoked in rat vas deferens by single-pulse electrical stimulation.

The present study was carried out to look at the influence of the K+ channel opener cromakalim, compared with suramin and prazosin, on the contractile response evoked by single-pulse field stimulation and exogenous agonists in epididymal and prostatic portions of rat vas deferens. In the epididymal portion suramin abolished the first phase of the response to single shock, prazosin deeply affected the second phase and a combination of both antagonists almost completely abolished both phases. Cromakalim was able to inhibit in a concentration-dependent manner the first purinergic phase (pD2 = 5.90 +/- 0.11), leaving practically unaffected the second, adrenergic phase. This inhibitory effect of cromakalim on the electrically evoked response was counteracted by glibenclamide. Cromakalim and prazosin, but not suramin, affected the response to exogenous noradrenaline. Suramin but not cromakalim was able to antagonize responses to alpha, beta-methylene-ATP. In the prostatic portion because of a less clear discrimination between adrenergic and purinergic phases of the electrically evoked response, the picture was less clear although the trend was identical. Cromakalim was not able to antagonize the response to ATP. It is concluded that in rat vas deferens cromakalim inhibits purinergic transmission by acting prejunctionally.

Adenosine Triphosphate↗

PubMed1997

Profiles of the response to noradrenaline in the whole and bisected rat vas deferens.

1. The present study was carried out to look at the differences between the whole and bisected rat vas deferens in response to cumulative and non-cumulative administration of noradrenaline in terms of sensitivity and maximum response. An effort was made to quantify the parameters characterizing the various components of the mechanical response to single concentrations of the agonist. 2. The sensitivity, as measured by the -log EC50 value for noradrenaline, was significantly lower in the prostatic portion than in the whole vas deferens and epididymal portion. The maximum contractions reached by the epididymal and the prostatic half were 65.03 +/- 10.43% and 13.84 +/- 5.28% of the maximal contraction evoked by noradrenaline in the whole rat vas deferens. 3. Various concentrations of noradrenaline were tested as single concentrations in the three preparations. These components of the response to noradrenaline were individualized, i.e. an early rapid phasic component followed by a second slower tonic component with spikes superimposed. The time-course of these components, as well as their ratios, were different depending on the concentration of agonist tested and on which half of the vas deferens was considered. 4. The importance of oxidation of exogenous noradrenaline as well of the neuronal and extraneuronal uptake mechanisms was considered. EDTA was devoid of any action. Cocaine enhanced potency and the maximum contraction of noradrenaline and all the parameters related to the different phases of the contraction were increased. Hydrocortisone at the highest concentration employed (2 X 10-5 M) was able to reduce significantly the -log EC50 value of the cumulative concentration-response curve to noradrenaline and both the phasic and tonic components of the contradiction to noradrenaline (3 X 10-6 M). 5. In conclusion, our evaluation of the parameters of the mechanical response of the whole and bisected vas deferens to noradrenaline rationalize the, often contradictory, findings of the current literature. Chiefly, the attainment of a careful description of the features of the response to noradrenaline can offer a dependable approach to the study of the underlying mechanisms of the three components.

Adrenergic alpha-Agonists↗

PubMed1995

Mode of antagonism of methoctramine, AF-DX 116 and hexahydrosiladifenidol in guinea-pig left atrium and ileum: comparison of Schild and resultant analysis.

1. Methoctramine, AF-DX 116 and hexahydrosiladifenidol (HHSiD) are the muscarinic antagonists most widely used to study muscarinic receptor subtypes. 2. The present study was undertaken to examine the mode of antagonism of these compounds in guinea-pig left atrium and ileum by comparison of the Schild and resultant analysis. With this method the effect of various concentrations of the test antagonist on the antagonism produced by specific concentrations of a reference antagonist was measured and the equilibrium dissociation constant of the test antagonist-receptor complex estimated. Atropine was used for comparative purposes and scopolamine as the reference antagonist. 3. At the cardiac level the affinity values obtained by Schild and resultant analysis for methoctramine and AF-DX 116, as for atropine, are very similar: these results indicate that the two cardio-selective antagonists and the non-selective antagonist, atropine, bind at a common site with the reference antagonist scopolamine. The resultant plot for the ileo-selective HHSiD has a slope considerably less than unity: this finding might indicate that this antagonist binds to a site different from that of scopolamine and it should be considered like an allosteric antagonist. 4. At the ileal level the affinity values obtained by Schild and resultant analysis are identical for the ileo-selective antagonist HHSiD as for atropine but not for methoctramine and AF-DX 116. This indicates a mutual binding site with scopolamine for HHSiD and atropine but not for the two cardio-selective antagonists. However, it is worth emphasizing that the difference between affinity values obtained by Schild and resultant analysis is seen when relatively high concentrations are required: a dual mode of interaction (both competitive and allosteric) could be involved.

Animals↗

PubMed1995

Synthesis and pharmacological investigation of new chiral muscarinic antagonists.

The two pairs of enantiomers of isoxazolidin-3-ones 3 and 4 were synthesized by means of Lipase PS-catalyzed hydrolyses of suitable racemic butyrates. The same butyrates were also employed as key intermediates in the preparation of racemic 3 and 4. The antimuscarinic potency of the new compounds was assayed in two in vitro functional tests. The eutomers (-)-3 and (-)-4 share the same stereochemistry (5R) of the most potent enantiomer of "azamuscarone" 2, a structurally related muscarinic agonist. Such a spatial arrangement around the chiral center of 2-4, coupled with the low values of eudismic ratio, represents an anomaly among the chiral muscarinic ligands. This anomaly was accounted for by the absence of a chiral center at C-2, a position whose configuration is crucial in determining the high enantioselectivity of muscarinic agonists and antagonists.

Animals↗

PubMed1995

Cholinergic agents structurally related to furtrethonium. 3. Synthesis and antimuscarinic activity of a series of N-(5-methyl-2-furfuryl and-tiophenyl)-dialkyl amines and of 2-dimethylamino methyl-4-(1'-substituted-acetoxy-methyl)-1,3-dioxolanes.

The introduction of an homologous series of cyclic amines at position 2 of 5-methylfurane and its isoster 5-methylthiophene induced a weak antagonist behaviour, probably depending on the steric hindrance of the substituents at the nitrogen, in the case of the cardiac tissue. Surprisingly, when evaluated on guinea-pig ileum preparatons, these compounds showed non-muscarinic effects, not-related either to nicotinic or istaminergic effects, the nature of which awaits to be explained. Substitution of the furane ring on the structure of the lead 2a, b, obtained in a previous study, with the bioisoster 1,3-dioxolane moiety gave potent but not selective analogues (17 and 18).

Animals↗

PubMed1994

Cholinergic agents structurally related to furtrethonium. 2. Synthesis and antimuscarinic activity of a series of N-[5-[(1'-substituted-acetoxy) methyl]-2-furfuryl]dialkylamines.

In the first part of this study, devoted to the discovery of selective antimuscarinic agents, (+/-)- N-[5-[(1'-phenyl-1'-cyclohexylacetoxy)methyl]-2-furfuryl]dimeth yla mine (5a) proved to be at least 20 times more potent in the rat ileum and bladder than in guinea pig atria. Several (+/-)-N- [5-[(1'-substituted-acetoxy)methyl]-2-furfuryl]dialkylamine analogs of 5a were subsequently prepared. This involved exploration of the tertiary nitrogen substituents and modulation of the lipophilic side chain at position 5 of the furan ring, using the Hansch approach. A QSAR study was conducted to correlate activity with physicochemical properties of substituents. The possibility of describing all compounds in a single model indicates that variations of nitrogen and the lipophilic side chain contribute independently to activity. Compounds 5b, c,j, with bulky lipophilic substituents at the tertiary nitrogen, showed unprecedented selectivity between the two smooth muscle tissues, their antimuscarinic potency being from 10 to 90 times higher in the ileum than in the bladder. It is suggested that their interesting tissue selectivity is probably related to nonspecific phenomena involving the receptor environment, rather than real differences between the muscarinic receptors in the two tissues.

Animals↗

PubMed1993

Synthesis and muscarinic activity of some muscarone analogs.

The synthesis of some muscarone analogs has been accomplished and their muscarinic activity has been assayed in three in vitro functional tests. The results of such an investigation put in evidence the active role played by the methylene moiety, placed exocyclic to the pentaatomic ring, on the interaction of the ligands with the different muscarinic receptor subtypes. Worth noting is the ileum-atria selectivity displayed by methylene derivative 7.

Animals↗

PubMed1993

Synthesis and muscarinic activity of the chiral forms of methylenemuscarones.

The synthesis of the two pairs of enantiomers of methylenemuscarones 3 and 4 has been accomplished by using (R)- and (S)-lactic esters as starting materials. Due to the existence of different muscarinic receptor subtypes, the compounds were examined for their ability to bind membranes from cerebral cortex (M1), heart (M2), and salivary glands (M3) and were assayed in "in vitro" functional tests as well. The results of such an investigation put in evidence that, in both binding and functional tests, (-)-3 (2S,5S) and (-)-4 (2R,5S) were the eutomers and shared the stereochemistry of the eutomer of muscarone and allomuscarone respectively. It is worth noting that the distomer of 3 and 4 behaves as a partial agonist in the cardiac tissue and as a full agonist in the other preparations. This peculiarity of the chiral forms of 3 and 4 could account for the remarkable selectivity for the ileum observed in the corresponding racemates.

Animals↗

PubMed1992

Synthesis and pharmacological investigation of the enantiomers of muscarone and allomuscarone.

A strategy based on the use of (R)- and (S)-lactic ester as starting materials allowed the synthesis of the two enantiomers of muscarone [(-)-1 and (+)-1] and allomuscarone [(-)-5 and (+)-5] in greater than 98% enantiomeric excess. The compounds were examined for their ability to bind to membranes from cerebral cortex (M1), heart (M2), and salivary glands (M3) and to recognize affinity agonist states of the muscarinic receptors. The two pairs of enantiomers were also tested in five functional assays, and their muscarinic potency was determined. In both binding and functional tests, (-)-1 (2S,5S) and (-)-5 (2R,5S) were the eutomers of muscarone and allomuscarone, respectively. The eudismic ratio of muscarone, evaluated in the functional tests, spanned a range of 280-440. These values are substantially different from ones (2.4-10.1) reported in the literature. From a stereochemical point of view, muscarone behaves as muscarine and all other major muscarinic agonists; as a consequence, the hypotheses advanced to account for the anomalies of muscarone no longer have reason to exist.

Animals↗

PubMed1992

Synthesis and pharmacological investigation of stereoisomeric muscarines.

The synthesis of the eight stereoisomers of muscarine has been efficiently accomplished by utilizing the two enantiomers of lactic esters as starting material. The synthetic strategy is based on a SnCl4-catalyzed addition of allyltrimethylsilane to O-protected lactic aldehydes followed by an iodocyclization process. All the final derivatives possess an enantiomeric excess higher than 98%. The four pairs of enantiomers bound to M1, M2, and M3 muscarinic receptor subtypes in membranes from cerebral cortex, heart, and salivary glands, respectively, and recognized heterogeneous states of the receptors. Of the eight isomers, only natural muscarine (+)-1 recognized three affinity states of the M2 receptor. The compound was also the only one to show selectivity in the binding study, demonstrating 37- to 44-fold higher affinity for the M2 than for the M1 or M3 receptors. In addition, the compounds were tested in functional assays on isolated guinea pig atria (M2 receptors) and ileum (mixed population of M2 and M3 receptors) and their muscarinic potencies were determined. Among the eight isomers, again only (+)-1 enantiomer was found to be very active on both tissues. Its potency was more than two orders of magnitude higher than that of its enantiomer (-)-1 as well as the other six isomers. The eudismic ratios (E.R.) deduced from the two functional tests were 324 and 331.

Animals↗

PubMed1992

Influence of lithium pretreatment and of cooling on the responsiveness of the rat isolated jejunum and urinary bladder to muscarinic agonists.

1. The aim of the present study was to see whether contractile responses induced by muscarinic agonists in the rat jejunum and urinary bladder were differently affected by procedures that mainly influence the steps following agonist-receptor interaction. Thus, the effects of ex vivo lithium pretreatment (6.8 mmol kg-1 i.p. for 3 days) and in vitro cooling from 37 degrees C to 20 degrees C) on the contractile responses to full and partial agonists, carbachol, oxotremorine, muscarine and pilocarpine were studied. 2. Lithium pretreatment did not affect muscarinic responses on the urinary bladder. It significantly reduced responses to carbachol and oxotremorine but not to muscarine and pilocarpine on the rat jejunum. 3. A decrease of the bath temperature from 37 degrees C to 20 degrees C potentiated responses to carbachol, muscarine and oxotremorine and abolished those to pilocarpine in the urinary bladder. The same lowering of the bath temperature potentiated responses to carbachol, did not affect those to muscarine and to oxotremorine and reduced those to pilocarpine in the jejunum. 4. Together the findings indicate that differences exist in the stimulus-response coupling induced by muscarinic agonists between the two tissues and that, in a given tissue, differences exist among agonists in their ability to activate the agonist-receptor-transducer complex.

Animals↗

PubMed1992

Effects of cromakalim (BRL 34915) on resting and evoked activity in frog semicircular canals.

The effects of endolymphatic administration of cromakalim and glibenclamide were tested in isolated semicircular canals of the frog. The actions of the drugs were evaluated by recording: (1) the transepithelial potential between the endolymphatic and the perilymphatic sides of the crista ampullaris; (2) the slow nerve potential from the ampullar nerve; (3) the action potential discharge in afferent ampullar nerve fibres; and (4) the perilympathatic potassium concentration in the fluid bathing the outer surface of the crista ampullaris. The above mentioned parameters were recorded both at rest and during mechanical stimulation of the sensory organ. The results demonstrated that the endolymphatic administration of cromakalim (10(-4) M) produced an increase in both ampullar receptor resting activity and in perilymphatic K+ resting levels. By contrast all the parameters related to the mechanically evoked responses were practically unaffected. Glibenclamide (10(-4) M) proved able to cancel or to prevent cromakalim effects. These data suggest that the membrane of the hair cells is endowed with K+ channels regulated by internal ATP whose activation is mainly involved in the processes sustaining ampullar receptors' resting firing rate.

Adenosine Triphosphate↗

PubMed1992

A novel class of cholinergic agents structurally related to 2-morpholinol.

A series of esters and ethers of N-alkylmorpholin-2-ols, and their methiodides, which can be considered cyclic analogues of acetylcholine, were synthesized. The amines were obtained by acylation or etherification of morpholinols with the appropriate acyl chlorides and alcohols. All compounds were tested for their ability to interact with the muscarinic receptor M2 (guinea-pig atria) or M3 (rat ileum and urinary bladder) subtype. Some compounds, although endowed with relatively low potency, proved interesting for their organ selectivity. Some considerations on the structure-activity relationship are made and the results obtained with reference agonists and antagonists are also shown.

Animals↗

PubMed1991

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.

Animals↗