Guinea Pig Left Atria Research Articles

A mathematical model for analysis of pharmacologically induced changes in the kinetics of cardiac muscle

A mathematical model of the isometric contraction of cardiac muscle is developed and utilized to characterize the inotropic and lusitropic effects of cardioactive compounds in isolated guinea pig left atria. In contrast to metrics that are based on minima and maxima of an isometric twitch and its derivative function, the entire time course of the twitch is used to quantify the kinetics of the contraction-relaxation cycle. The model relates observed tension to a time-dependent activation function that describes generation of internal force and a coupling function that determines mechanical response to the activation function. The model is structured so that it is suitable for nonlinear curve fitting to observed data. Results obtained using the model for fitting experimental data from tissues treated with different classes of cardioactive compounds agree with more qualitative results presented by other authors. Experiments using the model to fit data over an extended (90 min) time course revealed differences in the kinetic profiles of milrinone and forskolin. Computer simulations that demonstrate the effect of each model parameter on twitch kinetics are presented, and the relationships between the model and other theoretical and empirical models of cardiac muscle are discussed. The mathematical model is useful to enable a more quantitative understanding of the kinetics of cardiac muscle contraction and relaxation and identify compounds that may be selective for inotropic or lusitropic effects.

Histamine H 3 -Receptor–Mediated Inhibition of Calcitonin Gene-Related Peptide Release From Cardiac C Fibers

Antidromic stimulation of cardiac sensory C fibers releases calcitonin gene-related peptide (CGRP), which increases heart rate, contractility, and coronary flow. C-fiber endings are closely associated with mast cells, and CGRP may release mast-cell histamine. Because prejunctional histamine H3-receptors inhibit transmitter release from autonomic nerves, we tested the hypothesis that H3-receptors modulate CGRP release in the heart. CGRP released by bradykinin in the electrically paced guinea pig left atrium and by capsaicin in the spontaneously beating isolated heart caused marked positive inotropic and chronotropic effects, respectively. Capsaicin significantly enhanced the overflow of CGRP (fivefold) and histamine (twofold) into the coronary effluent. All of these effects were prevented by prior chemical destruction of C fibers in vivo. The H3-receptor agonist imetit attenuated the inotropic response to bradykinin by 50%. Imetit also decreased the capsaicin-induced tachycardia and the increase in CGRP overflow by 50%. Imetit, however, did not modify the response to exogenous CGRP. The effects of imetit were blocked by the H3-receptor antagonist thioperamide. Notably, thioperamide by itself potentiated the capsaicin-evoked increases in heart rate and CGRP overflow (by 25% and 50%, respectively). Thus, our findings identify a negative-feedback loop, whereby CGRP releases histamine from cardiac mast cells and histamine in turn inhibits CGRP releases by activating H3-receptors on C-fiber terminals. Because CGRP release is augmented in pathophysiological conditions, such as septic shock, heart failure, and acute myocardial infarction, modulation of CGRP release may be clinically relevant.

Syntheses of (14β,17α)-14-Hydroxy- and (14β,17α)-2,14-Dihydroxyestradiols and Their Activities.

Structure 1 is proposed for the Inagami-Tamura endogenous digitalis-like factor (EDLF), and (14β,17α)-14-hydroxy- and (14β, 17α)-2,14-dihydroxyestradiols (2 and 3) were synthesized as models for studies on 1. The latter compound was remarkably potent in inducing a contractile response in isolated rat aorta and guinea pig left atrium.

Syntheses of (14 beta,15 beta,16 beta,17 alpha)- and (14 beta,15 alpha,16 alpha,17 alpha)-1,3,5(10)-estratriene-2,3,14,15,16,17- hexaols, possible candidates for the Inagami--Tamura endogenous digitalis-like factor, and their activity.

Structures are proposed for the Inagami-Tamura endogenous digitalis-like factor (EDLF), and two possible candidates, (14 beta,15 beta,16 beta,17 alpha)- and (14 beta,15 alpha,16 alpha,17 alpha)-2,3,14,15,16,17-hexahydroxy-1,3,5(10)-estratrienes, were synthesized. Both compounds were potent in inducing a contractile response in isolated rat aorta and guinea pig left atrium.

Pharmacological characteristics of indoline derivatives in muscarinic receptor subtypes.

The present study was designed to investigate which muscarinic receptors the indoline derivatives interact with and also their pharmacological properties. Compounds I and II contracted guinea-pig ileum in a concentration-dependent manner, whereas compounds III-IX behaved as antagonists and Schild plots gave straight lines. 4-DAMP antagonized the contractile responses to compounds I and II, and the pA2 values for 4-DAMP were 8.96 +/- 0.23 and 9.09 +/- 0.06, respectively. In guinea-pig left atrium, compound I partly inhibited twitch responses, whereas compound II did not have any effect. In rabbit vas deferens, compounds I and II produced inhibitory effects on twitch responses evoked by field stimulation. Pirenzepine antagonized the inhibitory responses of compounds I and II, and the pA2 values for pirenzepine were 7.90 +/- 0.13 and 8.12 +/- 0.06, respectively. Compound I has about 150-fold higher affinity to M1 receptors than to M3 receptors, while compound II has about 360-fold higher affinity to M1 receptors. Our results indicate that compounds I and II show 7- and 16-fold higher M1 receptor selectivity than McN-A-343, respectively. Therefore, compound II is selective for M1 receptors over M2 or M3 receptors.

Synthesis and smooth muscle calcium channel antagonist effects of alkyl 1,4-dihydro-2,6-dimethyl-4-(pyridinyl)-5-[2-(4,5-dihydro- 4,4-dimethyloxazolin-2-yl)]-3-pyridinecarboxylates.

A group of racemic alkyl 1,4-dihydro-2,6-dimethyl-4-(3- or 4-pyridinyl)-5-[2-(4,5-dihydro-4,4-dimethyloxazolin-2-yl)-3- pyridinecarboxylates 11a-e were prepared by using the Hantzsch reaction involving condensation of the Knoevenagel adducts 9a-e with 1-[2-(4,5-dihydro-4,4-dimethyloxazolin-2-yl)]-1-propen-2-ami ne (10). In contrast, the 4-(2-pyridinyl) analogue 11f was prepared by thionyl chloride mediated cyclization of the 5-¿N-(1,1-dimethyl-2-hydroxyethyl)aminocarbonyl¿ moiety of 16 to the 5-[2-(4,5-dihydro-4,4-dimethyloxazolin-2-yl)] ring system (11f). In vitro calcium channel antagonist activity was determined by using the guinea pig ileum longitudinal smooth muscle (GPILSM) assay. Compared to the reference drug nifedipine (IC50 = 1.43 x 10(-8) M), the title compounds 11 exhibited weak calcium channel antagonist activity (10(-5) to 10(-6) M range). A comparison of compounds 11 having a C-4 3-pyridinyl substituent showed that with respect to the alkyl ester R2-substituent, the relative potency order was i-Bu (11c) > or = i-Pr (11e) > Me (11a). The point of attachment of the C-4 pyridinyl substituent in the isopropyl ester isomeric series of compounds was a determinant of activity where the potency profile was 4-py (11d) > or = 3-py (11e) > 2-py (11f). Although less effective, the 4,5-dihydro-4,4-dimethyloxazolin-2-yl moiety acts as a bioisostere of the alkyl ester substituent present in classical 1,4-dihydropyridine calcium channel antagonists. The 4,5-dihydro-4,4-dimethyl-oxaxolin-2-yl ring system is not an effective bioisostere of the 3-nitro group present in 1,4-dihydropyridine calcium channel agonists since isopropyl 1,4-dihydro-2,6-dimethyl-4-(2-pyridinyl)-5- [2-(4,5-dihydro-4,4-dimethyloxazolin-2-yl)]-3-pyridinecarboxyla te (11f) produced a modest 10% increase in the in vitro contractile force of guinea pig left atrium at a concentration of 1.64 x 10(-7) M, relative to the reference 3-nitro analogue 1 (EC50 = 9.6 x 10(-6) M).

Interaction of class III antiarrhythmic drugs with muscarinic M2 and M3 receptors: radioligand binding and functional studies.

We have recently reported that class III antiarrhythmic drugs inhibit the muscarinic acetylcholine (ACh) receptor-operated K+ current (IK,ACh) in guinea-pig atrial cells by different molecular mechanisms. The data obtained from the patch-clamp study suggest that D,L-sotalol inhibits IK,ACh by blocking the muscarinic receptors, whereas MS-551 inhibits the K+ current by blocking the muscarinic receptors and depressing the function of the K+ channel itself and/or the guanine nucleotide-binding protein (G protein). This study was undertaken to determine whether the class III antiarrhythmic drugs D,L-sotalol and MS-551 interact with the muscarinic receptors of cardiac and peripheral tissues. Both drugs inhibited concentration dependently the specific [3H]N-methylscopolamine ([3H]-NMS) binding to membrane preparations obtained from guinea-pig atria and submandibular glands. The competition curves of these drugs for [3H]-NMS binding to glandular membranes were monophasic, suggesting competition with [3H]-NMS at a single site. Although the competition curve of D,L-sotalol for [3H]-NMS binding to atrial membranes was monophasic, that of MS-551 was biphasic and showed high- and low-affinity states of binding. D,L-Sotalol showed slightly, but significantly, higher affinity for cardiac-type muscarinic receptors (M2) than for glandular-type muscarinic receptors (M3). The inhibition constant (Ki) for MS-551 in glandular membranes was also slightly greater than the high-affinity Ki value for the drug in atrial membranes. In guinea-pig left atria and ilea, D,L-sotalol shifted the concentration-response curves for the negative inotropic effect and the contracting effect of carbachol in a parallel manner. The slopes of Schild plot were not significantly different from unity, suggesting competitive antagonism, and the pA2 for D,L-sotalol in left atria was slightly greater than that in ilea. MS-551 also shifted the concentration response curve for the negative inotropic effect of carbachol in atrial preparations to a greater extent than that for the contracting effect in ileal preparations, although MS-551 failed to show a pure competitive antagonism. These results suggest that both D,L-sotalol and MS-551 interact with cardiac M2 and peripheral M3 receptors, and that at high concentrations they exert anticholinergic activity in cardiac and peripheral tissues.

The ( S)-(+)-enantiomer of dimethindene: a novel M 2-selective muscarinic receptor antagonist

The present study was designed to determine the in vitro affinity profile of ( R)-(−)-dimethindene and ( S)-(+)-dimethindene at muscarinic receptor subtypes using both functional and binding assays. In addition, the racemate was investigated in functional studies. The functional muscarinic receptors studied were putative M 1 receptors in rabbit vas deferens and rat duodenum, M 2 receptors in guinea-pig left atria and rabbit vas deferens, as well as M 3 receptors in guinea-pig ileum and trachea. Furthermore, the histamine H 1 antagonism by ( R)-(−)- and ( S)-(+)-dimethindene has been examined in guinea-pig ileum. Muscarinic binding selectivity was assessed in homogenates from human neuroblastoma NB-OK 1 cells (M 1), rat heart (M 2), pancreas (M 3) and striatum (M 4). The results demonstrate that ( S)-(+)-dimethindene is a potent M 2-selective muscarinic receptor antagonist ( pA 2 = 7.86 7.74 ; p K i = 7.78) with lower affinities for the muscarinic M 1 ( pA 2 = 6.83 6.36 ; p K i = 7.08), the M 3 ( pA 2 = 6.92 6.96 ; p K i = 6.70) and the M 4 receptors (p K i = 7.00), respectively. The ( S)-(+)-enantiomer was more potent (up to 41-fold) than the ( R)-(−)-enantiomer in all muscarinic assays. In contrast, the stereoselectivity was inverse at histamine H 1 receptors, the ( R)-(−)-enantiomer being the eutomer (pA 2 = 9.42; pA 2 (S)- isomer = 7.48 ). In conclusion, ( S)-(+)-dimethindene is a useful tool to investigate muscarinic receptor heterogeneity. In addition, this lipophilic compound might become the starting point for the development of M 2-selective muscarinic receptor antagonists useful as diagnostic tools for quantifying muscarinic M 2 receptors in the central nervous system with positron emission tomography imaging, and to test the hypothesis that muscarinic M 2 receptor antagonists show beneficial effects in the treatment of cognitive disorders.

Characterization of the interaction of zamifenacin at muscarinic receptors in vitro

The interaction of zamifenacin ((3 R)-(+)-diphenylmethoxy-1-(3,4)-methylenedioxyphenethyl)piperidine) at muscarinic receptor subtypes was studied using radioligand binding and functional techniques, in vitro. In radioligand binding studies, zamifenacin acted as a competitive antagonist, with the following p K i values; rat cerebral cortex (M 1) 7.90 ± 0.08, myocardium (M 2) 7.93 ± 0.13, submaxillary gland (M 3) 8.52 ± 0.01 and rabbit lung (M 4) 7.78 ± 0.04. In functional studies zamifenacin acted as a surmountable antagonist, exhibiting the following apparent affinity values; canine saphenous vein (putative M 1) 7.93 ± 0.09, guinea-pig left atria (M 2) 6.60 ± 0.04, guinea-pig ileum (M 3) 9.31 ± 0.06, guinea-pig oesophageal muscularis mucosae (M 3) 8.84 ± 0.04, guinea-pig trachea (M 3) 8.16 ± 0.04, and guinea-pig urinary bladder (M 3) 7.57 ± 0.15. Therefore, zamifenacin is selective for muscarinic M 3 receptors in guinea-pig ileum, oesophageal muscularis mucosae, trachea and bladder over muscarinic M 2 receptors in atria. The degree of muscarinic M 3 M 2 receptor selectivity depends upon the muscarinic M 3 receptor preparation studied.

Pyrrolo[2,1-c][1,4]benzothiazines: synthesis, structure-activity relationships, molecular modeling studies, and cardiovascular activity.

The synthesis and pharmacological evaluation of a series of pyrrolo[1,4]benzothiazine derivatives are described. These compounds, related to diltiazem, have been shown to be representative of a novel series of calcium channel antagonists. The IC50S for inhibition of [3H]nitrendipine binding calculated by radioreceptor assay on rat cortex and rat heart homogenates showed that some of the described compounds possess an affinity equal to or higher than those of the reference calcium antagonists verapamil and cis-(+)-diltiazem. Furthermore, the alteration of the benzothiazepinone system of diltiazem to the pyrrolo[1,4]benzothiazine system of the title compounds resulted in a clear-cut selectivity for cardiac over vascular tissue, as shown in functional studies. In fact comparison of calcium antagonist activity on guinea pig aorta strips with the negative inotropic activity, determined by using an isolated guinea pig left atrium, revealed that the compounds examined displayed higher selectivity than the reference standard, within a wide variation of data. A number of structure-activity relationship trends have been identified, and possible explanation is advanced in order to account for the observed differences in selectivity. Prerequisite for in vitro calcium channel-blocking activity is the presence of two pharmacophores, namely, the substitution at C-4 and the substitution on the pyrrole ring. Two of the tested compounds, 8b and 28a, were identified as potent calcium antagonists selective for cardiac over vascular tissue.

Use of resampling techniques to estimate the variance of parameters in pharmacological assays when experimental protocols preclude independent replication: An example using schild regressions

Estimates of variance in pharmacological assays are usually made by repeating the experiment with different tissues. Biological factors, such as the inability to wash a drug from tissue, may preclude the type of replication that is appropriate for the statistics of interest. For example, in Schild regressions, replication is usually done at each concentration of antagonist. In some test systems, replication of dose-response curves is not possible. For example, some persistent agonists cannot be removed from tissues after exposure, while in other systems, rapid desensitization severely alters tissue sensitivity to repeated challenge with agonist. In this paper, we demonstrate how a statistical resampling method, bootstrapping, can be used to derive estimates of the confidence intervals for pA 2, pK B, and slope from Schild plots. This method utilizes the speed of the computer to estimate variance by repeatedly resampling the data. The advantage to this method is that it can be used for many different experimental designs. For a data set obtained from a Schild regression of atenolol antagonism of isoproterenol in the guinea pig left atrium, bootstrap estimates of confidence limits were calculated for cases where dose ratios were derived from the same tissue and randomly paired tissues. These estimates showed good agreement with estimates obtained using conventional analytical methods, thus suggesting that this method may be useful in practice.

Cooliatoxin, the first toxin from Coolia monotis (Dinophyceae).

Coolia monotis is a benthic dinoflagellate previously thought to be non-toxic. We describe a new toxin, named cooliatoxin, purified from cultures of a strain of C. monotis isolated from Australia. Cooliatoxin is likely a mono-sulphated, polyether toxin (M = 1,062; i.p. LD50 = 1 mg/kg in mice) that induces hypothermia and respiratory failure in mice after a pronounced delay period during which there are no obvious signs of intoxication. These signs in mice are similar to those reported for the shellfish toxin named yessotoxin and the molecular weight of cooliatoxin corresponds to the mono-sulphated form of yessotoxin, suggesting that cooliatoxin may be an analogue of yessotoxin. Cooliatoxin has no effect on the mouse phrenic nerve or diaphragm musculature in vitro but causes initial stimulation and subsequent block of unmylenated nerves in vitro. In isolated guinea pig left atria, cooliatoxin (above 20 nm) induced a slow developing concentration dependent sustained inotropic response. Propranolol or tetrodotoxin reversed the positive inotropic effects, indicating that the majority of the cooliatoxin induced response was mediated by stimulation of nerves associated with the atrial musculature, resulting in the release of noradrenaline. Cooliatoxin induced transient contractions in isolated guinea pig vas deferens preparations. Atria and vas deferens preparations were tachyphylactic to a second equivalent dose of cooliatoxin applied after the effects of the first dose had diminished. The observed in vitro effects of cooliatoxin on peripheral nerves are unlikely to account for the lethal effects in mice and a central action of this toxin is suspected.

Enhancement of adenosine A1 receptor functions by benzoylthiophenes in guinea pig tissues in vitro.

Previous reports on a series of benzoylthiophenes, including PD 81,723 [2-amino-4,5-dimethyl-3-(3-trifluoromethyl-benzoyl) thiophene], have shown specific enhancement of agonist binding at the adenosine A1 receptor. We have studied the effects of two substituted benzoylthiophenes, PD 78,416 (thieno[2,3-c]pyridine-6(5H)-carboxylic acid, 2-amino-3-benzoyl-4,7-dihydro-ethyl ester) and RS-74513-000 [2-amino-4-ethyl-5-methyl-3-(3-trifluoro-methyl-benzoyl) thiophene] on response elicited by adenosine A1 receptors in isolated guinea pig left atrium and ileum. In the electrically paced left atrium, PD 78,416 antagonized negative inotropic effect elicited by the agonist CPA [N6-cyclopentyladenosine] with a pKB value of 6.2 +/- 0.2 (n = 4). At a low concentration which had no antagonistic effect (0.1 microM), PD 78,416 enhanced the effect of CPA. The concentration-response curve to CPA was shifted leftward by 5.1 fold (95% confidence limits 2.4-11.2). In field stimulated isolated ileum, PD 78,416 (0.1, 0.3, 1 microM) did not enhance or antagonize effects of CPA. At concentrations above 1 microM, PD 78,416 decreased electrically induced contraction. This effect was not sensitive to adenosine deaminase and was not antagonized by the A1 antagonist CPX [8-cyclopentyl-1,3-dipropyl-xanthine] (1 microM). Unlike PD 78,416, RS-74513-000 (0.01, 0.1, 1, 3, 10 microM) did not antagonize or enhance effects of CPA in the left atrium. However, effects of CPA in ileum were enhanced by RS-74513-000 (1 and 3 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Quinidine-induced potentiation of cardiovascular effects of nitrendipine: functional aspects and possible molecular mechanisms.

The functional interaction between the dihydropyridine calcium channel blocker nitrendipine and quinidine was studied in isolated preparations from guinea-pig cardiac ventricle and in mesenteric arterial segments under a variety of experimental conditions. The negative inotropic potency of nitrendipine is clearly enhanced by quinidine (3 x 10(-6)-10(-4) mol/l) by up to two orders of magnitude, i.e. cardiac nitrendipine effects are potentiated. Vasorelaxant effects, however, remain largely unaffected (nitrendipine potency is increased by half an order of magnitude maximally). To elucidate the mechanism of this interaction, the ability of quinidine to potentiate the negative inotropic effect of a series of 12 dihydropyridines was compared with their voltage-dependence of action in guinea-pig left atria. No significant correlation is found (r = 0.18). Furthermore, quinidine inhibits rather than stimulates binding of tritiated nitrendipine, nimodipine or (S)-isradipine to isolated cardiac membranes. Therefore, the mechanism of the quinidine-nitrendipine interaction differs from those previously proposed for modulation of dihydropyridine binding by other drugs. We hypothesize that quinidine-occupied calcium channels adopt an intermediate affinity for nitrendipine, higher than in resting channels, but lower than the high affinity present with inactivated channels. Model calculations which are based on this assumption are able to reproduce all experimental findings of this study.

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.

Effects of ω-toxins on noradrenergic neurotransmission in beating guinea pig atria

The effects of four ω-toxins, known to block various subtypes of neuronal voltage-activated Ca 2+ channels, on the beating guinea pig left atrium have been analyzed. Atria were suspended in oxygenated Krebs-bicarbonate solution at 32°C and driven with electrical pulses delivered by a stimulator at 1 Hz, 1 ms, 4 V. A 10-fold increase of voltage caused a potent and rapid enhancement of the size of contractions (about 3- to 4-fold above basal), which reflects the release of endogenous noradrenaline from sympathetic nerve terminals. ω-Conotoxin MVIIC, ω-conotoxin MVIIA and ω-conotoxin GVIA inhibited the inotropic responses to 10 × V stimulation with IC 50 values of 191, 44 and 20.4 nM, respectively. ω-Agatoxin IVA did not affect the contractile responses. The inotropic responses to exogenous noradrenaline were unaffected by the toxins. The potent blocking effects of ω-conotoxin GVIA were present even in conditions in which the release of noradrenaline was strongly facilitated by presynaptic α 2-adrenoceptor blockade by phenoxybenzamine. These effects were not reversed upon repeated washing of the tissue with toxin-free medium. In contrast, the blockade induced by ω-conotoxin MVIIC and ω-conotoxin MVIIA were fully reversed, with t 1 2 of 13.5 and 31.2 min, respectively. ω-Conotoxin MVIIC (1 μM) protected against the irreversibility of the blockade induced by ω-conotoxin GVIA (100 nM). The results are compatible with the following conclusions: (i) at cardiac sympathetic neuromuscular junctions, the release of noradrenaline and the noradrenergic transmission is maintained and regulated by N-type Ca 2+ channels; (ii) the modulation by presynaptic α 2-adrenoceptors of noradrenaline release is exerted via N-type Ca 2+ channels; (iii) ω-conotoxin MVIIC and ω-conotoxin MVIIA reversibly block N channels and the noradrenergic neurotransmission; (iv) ω-conotoxin GVIA and ω-conotoxin MVIIC seem to recognize the same binding site on the N-type Ca 2+ channels; however, their binding kinetics might considerably differ; (v) P-type Ca 2+ channels are unlikely involved in the regulation of transmitter release at cardiac sympathetic nerve terminals.

Synthesis and Calcium Channel Modulating Effects of Alkyl 1,4‐Dihydro‐2,6‐dimethyl‐4‐(pyridinyl or 2‐trifluoromethylphenyl)‐5‐(1H‐tetrazol‐5‐yl)‐3‐pyridinecarboxylates

AbstractA group of racemic alkyl 1,4‐dihydro‐2,6‐dimethyl‐4‐(pyridinyl or 2‐trifluoromethylphenyl)‐5‐(1H‐tetrazol‐5‐yl)‐3‐pyridinecarboxylates 11–14 were prepared using the Hantzsch reaction that involved the condensation of 2‐, 3‐ or 4‐pyridinecarboxaldehyde, or 2‐trifluoromethylbenzaldehyde, 8a–d with isopropyl or methyl 3‐aminocrotonate (9a–b) and 5‐(2‐oxopropyl)‐1H‐tetrazole (10a) or 5‐(2‐oxopropyl)‐1‐methyl‐1H‐tetrazole (10b). In vitro calcium channel (CC) antagonist and agonist activities were determined using guinea pig ileum longitudinal smooth muscle (GPILSM) and guinea pig left atrium (GPLA) assays, respectively. In the series of compounds 11–14, only compounds 14a and 14b exhibited some weak CC antagonist activity (10−6 M range) relative to the reference drug nifedipine (IC50 = 1.43 × 10−8 M). Compounds 11b‐14b having a 1‐methyl‐1H‐tetrazol‐5‐yl substituent were inactive CC agonists on GPLA. In contrast, compounds 12a‐14a having a 1H‐tetrazol‐5‐yl substituent exhibited CC agonist activity on GPLA, but the C‐4 2‐pyridinyl analog 11a exhibited a mild negative inotropic effect. The approximately equipotent CC agonist activity (GPLA) exhibited by the C‐4 pyridinyl 13a and 4‐(2‐trifluoromethylphenyl) 14a compounds compared favorably with that of the reference drug (±)‐Bay K 8644 (EC50 = 7.7 × 10−7 M) indicating that the 1H‐tetrazol‐5‐yl moiety is a suitable replacement for the nitro group present in Bay K 8644 with respect to cardiac CC agonist activity. These latter compounds 13a and 14a should serve as useful probes to study the structure‐function relationships of calcium channels.

Potentiation by alcuronium of the antimuscarinic effect of N-methylscopolamine in guinea pig left atria

Alcuronium is known to stabilize allosterically the binding of the muscarinic antagonist N-methylscopolamine to muscarinic M 2 receptors and thus to elevate the equilibrium binding of N-methylscopolamine in homogenized cardiac tissue. In order to check for a functional consequence of this effect, the action of alcuronium alone and in combination with N-methylscopolamine was determined in contracting guinea pig left auricles with oxotremorine-M as the negative inotropic agonist. For sake of comparison, the allosteric modulator W84 = hexane-1,6-bis(dimethyl-3′-phthalimidopropyl-ammonium bromide) was included. Alcuronium displayed a weak antimuscarinic action (pA 2 = 5.7). In conjunction with 10 −7 M N-methylscopolamine, alcuronium (≥ 10 −6 M) induced a more pronounced antimuscarinic effect than expected for a combination of competitive antagonists. The extent of overadditivity with combinations of W84 and 10 −7 M N-methylscopolamine was smaller. In conclusion, alcuronium potentiates the antimuscarinic effect of N-methylscopolamine in contracting cardiac preparations with high effectivity.

Effects of Sematilide, a Novel Class III Antiarrhythmic Agent, on Action Potential in Guinea Pig Atrium

Electrophysiological effects of sematilide, a novel class III antiarrhythmic agent, were examined and compared with those of (±)sotalol in guinea pig left atrium by a conventional microelectrode technique. Application of 0.1-1000 μM sematilide or 1-1000 μM (±)sotalol concentration-dependently prolonged the duration of action potentials (APD) that were elicited by electrical stimulation at 1 Hz. Other parameters of action potentials such as the maximum upstroke velocity of phase 0 depolarization, action potential amplitude and resting membrane potential were not affected significantly by these drugs in the concentration ranges employed. The prolongation of APD by sematilide or (±)sotalol was accompanied by a corresponding increase in the effective refractory period (ERP). Approximately a 30% increase in ERP was obtained by the treatment with 5 μM sematilide or 100 μM (±)sotalol, suggesting that sematilide as a class III antiarrhythmic agent is approximately 20 times more potent than (±)sotalol on a molecular basis. When the stimulation rate was increased stepwise from 0.2 to 2 Hz, the relative increase in APD at 90% repolarization by the treatment with sematilide and (±)sotalol was slightly larger at 2 Hz than at 0.2 Hz, indicating that “reverse rate-dependence” was not observed under these conditions. These results may suggest a possibility that sematilide effectively blocks atrial arrhythmia.

Effect of amiloride on inotropic and toxic actions of ouabain in guinea-pig left atria

The effect of amiloride on the positive inotropic and toxic effects of ouabain in guinea-pig left atria has been studied. In atria driven at 1 Hz, amiloride (0.3 and 0.5 mM) decreased the EC 50 but did not affect the maximal tension developed by ouabain. At 0.1 Hz, amiloride did not change either the EC 50 or the maximal tension developed by ouabain. Ouabain toxicity (onset of arrhythmias) was not changed by amiloride at either frequency of stimulation. Therefore, amiloride did not antagonize either the positive inotropic or the toxic effect of ouabain. The positive inotropic effect of amiloride has been ascribed to the inhibition of the Na +/Ca 2+ exchanger. Since amiloride inhibits also the Na +/H + exchanger, 5-( N-ethyl- N-isopropyl)amiloride (EIPA), an amiloride derivative which selectively inhibits the Na +/H + exchange, has been tested to evaluate the role of the Na +/H + exchange in the amiloride-ouabain interaction. EIPA increased the EC 50 values of ouabain and decreased the maximal developed tension by the glycoside in atria driven at 0.1 and 1 Hz, but did not antagonize the toxic response (arrhythmias) of atria to ouabain. It is suggested that the inhibition of Ca 2+ exit through the Na +/Ca 2+ exchange by amiloride and ouabain may explain the observation that the positive inotropic effects of amiloride and ouabain are additive.