Dependency on Mg-and Ca-concentration of cycle length in spontaneously beating guinea-pig atria

Summary In the isolated, spontaneously beating guinea-pig atrium, Mg exerts a negative, and Ca a positive chronotropic effect. Contrary to Ca, for Mg a linear relationship between cycle length and concentration was observed in the range from 0.4 to 12.8 mM. The chronotropic effect produced per mmole Mg exceeded that of Ca by factors between 1.2 and greater than 5. The half-time for the development of the maximal effect on cycle length was about 1 min for Mg and of the order of 2 min for Ca. It is assumed that the extracellular, not the intracellular concentrations of Mg and Ca determine the change in cycle length. In atria incubated at different Ca-levels, the Mg-effect decreased with rising Ca, whereas, in atria incubated at different Mg-levels, the Ca-effect was remarkably augmented by increasing Mg-concentrations. It is discussed, that these paradoxical effects might be the result of the interaction of Mg and Ca at different sites of the isolated heart preparation, only one of which is responsible for the change in cycle length.I wish to thank Mrs.H. Wilbert for her valuable help when carrying out the experiments.     

Stimulatory effect of lymphocytes from Chagas'' patients on spontaneously beating rat atria.

The aim of this work was to study the effect of lymphocytes from individuals infected with Trypanosoma cruzi (Chagas' patients) on the contractile behaviour of living heart tissue. Chagas' lymphocytes (ChL) reacted with isolated rat atria preparations increasing the isometric development tension (IDT) and frequency of contractions (FC) in a dose-dependent manner. The maximal stimulatory effect was reached after 30-40 min of contract. In contrast, normal lymphocytes (NL) did not alter the basal IDT and FC values. beta-adrenergic antagonists, anti-histamine agents and inhibitors of the synthesis and action of arachidonic acid (AA) products were used to study the mechanisms of the reaction. (-)-propranolol (10(-7)M) and pyrilamine (10(-6)M) had no effect ruling out the participation of beta-adrenergic agonists or histamine. However, indomethacin (10(-6)M) and acetylsalicylic acid (1.8 X 10(-4)M) enhanced the effect of ChL. Inhibitors of the lipoxygenase pathway (5,8,11,14-eicosatetraynoic acid, 10(-7)M; nordihydroguairetic acid, 10(-5)M) and FPL55712, an antagonist of one of its terminal products: the slow reacting substance of anaphilaxis (SRS-A), abolished the reaction. Therefore, a fundamental role for SRS-A in the production of the stimulatory effect is postulated. Lymphocytes of the T cell lineage (E rosette forming cells, ERFC) are the effector cells involved in this reaction, whereas non-rosetting ChL depressed IDT. T ascertain if effector cells could be replaced by soluble factors, ChL were reacted with homogenates of rat atria and the cell free supernatants were added to beating rat atria. Positive ino- and chronotropic effects were obtained, indicating that soluble factors generated during the reaction can substitute for the intact effector cells. On the other hand if the effector cells were purified from Chagas' patients that had been treated 1 month to 6 years before the assay with trypanocidal drugs (3-methyl-4-(5'-nitrofurfurylidene-amino)-tetrahydro-4H-1, 4-tiazine-1, 1-dioxide, nifurtimox or N-benzyl-2-nitro-imidazolacetamide, benznidazole) only depressor effects were found. The depressor inotropic action of lymphocytes from treated patients (tr-ChL) was abolished with indomethacin and acetyl salicylic acid indicating that products of the cyclooxygenase pathway of AA were involved. While this work provides additional evidence for the hypothesis that lymphocytes from T. cruzi infected patients may react with heart tissue and alter its contractile behaviour, the results should not be extrapolated to the in vivo situation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increase in passive tension of spontaneously beating atria in hyperosmolar media.

Spontaneously beating rabbit atria were used for study of increases in passive tension induced by hyperosmolar Tyrode bathing media. The time course and degree of passive tension change was dependent on the level of osmolality and the osmotic agent used. The effectiveness of various agents added to Tyrode solution in increasing resting tension was in the order sucrose greater than mannitol greater than or equal to NaCl greater than glucose greater than = no change. The hyperosmotically induced "contracture" was similar to contracture induced by high Ca2+ medium, but the maximum hyperosmotic effect was greater than that of high Ca2+. In contrast to active atria, inactive atria showed no increase in passive tension in hyperosmotic solutions or in Tyrode solution containing high Ca2+. After depolarization with Tyrode medium containing high K+ or zero K+, increased Ca2+ caused contracture in inactive left atria greater than that caused by hyperosmotic media (+200 mM sucrose) in active right atria or atrial pairs. Results of the study indicate that although other factors may be involved, increased Ca2+ flux inward could largely account for the passive tension response.     

An effect of the electrogenic sodium pump on the membrane potential in beating guinea-pig atria

Summary The membrane potential of guinea-pig atria was measured in different media before, during and after electrical stimulation of various durations. The stimulus duration and frequency (3 pulses/sec) were kept constant.During short stimulation (<1 min) in Tyrode solution at 35°C the membrane potential depolarized first quickly and later on more slowly. Following cessation of the stimulus train the membrane potential repolarized to the resting value within 1–2 min.Prolonged stimulation (1–7 min) in Tyrode solution at 35°C likewise caused an initial depolarization of membrane potential. Thereafter, the membrane potential repolarized although stimulation continued. After the end of the stimulation period the membrane potential hyperpolarized beyond the resting potential for several minutes. Maximum hyperpolarization was achieved ca. 1 min after the end of the stimulus train.In Tyrode solution at 25°C both the repolarization of the membrane potential during long stimulation and the hyperpolarization after it were diminished. The same effect was observed in Na poor fluids, which contained only 50 or 33% of the Na concentrations in Tyrode solution.Digitoxigenine (3·10^–6 g/ml), when added to Tyrode solution at 35°C, also reduced the repolarization of membrane potential during, and the hyperpolarization after, a long stimulus train.Compared to the resting value the membrane resistance was essentially unchanged 1–3 min after a 2 min stimulation period.From these results it is concluded, that an active, electrogenic Na pump contributes to the membrane potential of beating guinea-pig auricles.     

Cyclic AMP-dependent and independent effects of prostaglandins on the contraction-relaxation cycle of spontaneously beating isolated rat atria

The effects of prostaglandin (PG) F_2α, E₂, E₁ and I₂ on the amplitude, duration of the contraction-relaxation cycle (CRC), the second derivative of developed tension and the cyclic adenosine-3′,5′-monophosphate (cAMP) level and on ⁴⁵Ca uptake were studied in isolated spontaneously beating rat atria. The order of capacity to generate positive inotropic effects was PGF_2α>PGE₂>PGE₁?PGI₂. Only PGI₂ and PGE₁ decreased the duration of CRC. PGF_2α produced an increase during the first 2.5min, whereafter the duration returned to the initial level. PGE₂ had no significant effect on the shape of the CRC. The ratios of the maximum and minimum of the second derivative of the developed tension were reduced by PGI₂ and PGF_2α 2.5 and 5 min after administration, respectively. The ⁴⁵Ca uptake was stimulated equally by all of the tested PGs, but only PGI₂ and PGE₁ could significantly increase the cAMP level. The results do not support the conception that cAMP could mediate the positive inotropic effect of PGs. Rather the contrary, cAMP, increased by PGE₁ or PGI₂, could be responsible for increased relaxation, which might prevent the full development of tension.     

Cyclic AMP-dependent and -independent effects of prostaglandins on the contraction-relaxation cycle of spontaneously beating isolated rat atria

The effects of prostaglandin (PG) F2 alpha, E2, E1 and I2 on the amplitude, duration of the contraction-relaxation cycle (CRC), the second derivative of developed tension and the cyclic adenosine-3', 5' monophosphate (cAMP) level and on 45Ca uptake were studied in isolated spontaneously beating rat atria. The order of capacity to generate positive inotropic effects was PGF2 alpha greater than PGE2 greater than PGE1 approximately PGI2. Only PGI2 and PGE1 decreased the duration of CRC. PGF2 alpha produced an increase during the first 2.5 min, whereafter the duration returned to the initial level, PGE2 had no significant effect on the shape of the CRC. The ratios of the maximum and minimum of the second derivative of the developed tension were reduced by PGI2 and PGF2 alpha 2.5 and 5 min after administration, respectively. The 45Ca uptake was stimulated equally by all of the tested PGs, but only PGI2 and PGE1 could significantly increase the cAMP level. The results do not support the conception that cAMP could mediate the positive inotropic effect of PGs. Rather the contrary, cAMP, increased by PGE1 or PGI2, could be responsible for increased relaxation, which might prevent the full development of tension.     

Mechanism of action of H2-antagonists on histamine-or dimaprit-stimulated H2-receptors of spontaneously beating guinea-pig atrium

Cimetidine, ranitidine and famotidine are antagonists of the histamine H₂-receptors on the spontaneously beating right atrium of the guinea pig. When analyzed by the classical Schild method theirpA ₂-values are respectively: 6.3, 6.8 and 7.7 with dimaprit as agonist and 5.8, 6.5 and 7.7 with histamine as agonist. Radiolignad-displacement studies with [³H]-tiotidine as radioligand resulted inpK _d values for cimetidine, ranitidine and famotidine of 6.3; 6.9 and 8.2 respectively.In dimaprit-stimulated atria all antagonists added at concentrations above theirK _d values depressed the maximal increase in frequency. In the presence of histamine this effect was much less pronounced and only visible at concentrations of ranitidine and famotidine around 10 ·K _d.The rightward shift of the curves as well as the decrease inE _max are reversible but the dissociation constants of the antagonists are small (less than 10^–3 s^–1).The spontaneously beating right atrium showed receptor reserve for histamine and virtually no receptor reserve for dimaprit.The results have been interpreted in a model in which H₂-antagonists act mainly by competing with the agonist for the histamine receptor site but have in addition a distinct affinity for a secondary site on the receptor. Occupation of this site by the antagonist prevents building up of the stimulus elicited by the agonist and thus decreases theE _max. In systems with receptor reserve (histamine) the effect of antagonist binding to the secondary binding site is seen only at high concentration of antagonist while in absence of receptor reserve (dimaprit) the depression ofE _max is directly visible.Simulations of the model show that the affinity of this secondary binding site is 50- (famotidine) to 100-(cimetidine and ranitidine) fold lower than for the agonist binding site.     

Effect of lowering of the temperature on the tissue levels of cAMP, cGMP, PGE and PGF2 alpha in spontaneously beating rat atria preparation.

Tissue levels of cAMP, cGMP, PGE, and PGF2 alpha were measured in spontaneously beating rat atria preparation at 20C and 37C. At 20C the formation of cAMP increased in correlation with the markedly increased amplitude and reduced frequency. The other biochemical parameters were not significantly affected by the low temperature.     

Effect of lowering of the temperature on the tissue levels of cAMP,cGMP, PGE and PGF2β, in spontaneously beating rat atria preparation

Tissue levels of cAMP, cGMP, PGE, and PGF_2β were measured in spontaneously beating rat atria preparation at 20°C and 37°C. At 20°C the formation of cAMP increased in correlation with the markedly increased amplitude and reduced frequency. The other biochemical parameters were not significantly affected by the low temperature.     

Temporal dissociation between the negative inotropism and the increase in cyclic GMP level induced by choline esters in spontaneously beating rat atria preparations

The effects of acetylcholine and carbacholine on cyclic GMP levels and contraction amplitude were investigated simultaneously in spontaneously beating rat atria preparations. Both drugs at the concentrations of 5 x 10(-7) M or higher produced a very rapid decline of amplitude. However, the cyclic GMP levels was not elevated earlier than 5 s later, when the amplitude was already reduced to 50% of the initial value. Concentrations of acetylcholine below 5 x 10(-7) M were not able to affect the cyclic GMP level, although they reduced the contractility significantly. Acetylcholine had no effect on the cyclic AMP level. The data demonstrate that cyclic GMP does not mediate the negative inotropic effect of choline esters in rat atria preparations.     

Temporal dissociation between the negative inotropism and the increase in cyclic GMP level induced by choline esters in spontaneously beating rat atria preparations1

The effects of acetylcholine and carbacholine on cyclic GMP levels and contraction amplitude were investigated simultaneously in spontaneously beating rat atria preparations. Both drugs at the concentrations of 5times10^--⁷ M or higher produced a very rapid decline of amplitude. However, the cyclic GMP level was not elevated earlier than 5 s later, when the amplitude was already reduced to 50% of the initial value. Concentrations of acetylcholine below 5times 10^--⁷ M were not able to affect the cyclic GMP level, although they reduced the contractility significantly. Acetylcholine had no effect on the cyclic AMP level. The data demonstrate that cyclic GMP does not mediate the negative inotropic effect of choline esters in rat atria preparations.     

Effects of methoxamine and alpha-adrenoceptor antagonists, prazosin and yohimbine, on the sleep-wake cycle of the rat

A study was carried out on the effects of methoxamine, prazosin, and yohimbine on the sleep-wake cycle in rats prepared for chronic sleep recordings. Methoxamine (4-8 mg/kg), an alpha 1-adrenoceptor agonist, induced a dose-related increase in wakefulness (W) and a decrease in slow-wave sleep (SWS) and REM sleep (REMS). Prazosin (0.125-1 mg/kg), which selectively blocks alpha 1-adrenoceptors, modified only slightly the amount of time spent in W and SWS, and consistently decreased REMS values. Prazosin (0.5 mg/kg) reversed the effects of methoxamine, decreasing W and increasing sleep. Yohimbine (3 mg/kg), which blocks alpha 2-adrenoceptors, augmented W and diminished sleep. Methoxamine (4 mg/kg) in animals pretreated with yohimbine (3 mg/kg) induced a further decrease of SWS and REMS and an increase of W. Thus, pharmacological activation of alpha 1- or blocking of alpha 2-adrenoceptors appears to decrease sleep and increase W. Further, blocking of alpha 1-adrenoceptors decreases REMS. Rapid eye movement sleep depression by the alpha 1-agonist or the alpha 1-antagonist is tentatively ascribed to a critical change in noradrenergic transmission in the brain.     

Effects of a selective alpha 1-adrenoceptor agonist, methoxamine, on sexual behavior and penile reflexes

Methoxamine, an adrenergic agonist with selectivity for the alpha₁-adrenoceptor, when administered intraperitoneally 10 minutes prior to mating tests (1 to 5 mg/kg), effected reductions in the ejaculatory threshold, evidenced by a decrease in the number of intromissions preceding ejaculation. In mounting tests after penile anesthetization, a test which specifically assesses sexual motivation, 3 mg/kg methoxamine was without a stimulatory effect. Further, in penile reflex tests (ex copula) 1 mg/kg methoxamine was without effect, whereas 5 mg/kg decreased the number of erections, cups and flips per test, and increased the incidence of seminal emission. These data indicate a facilitation of the ejaculatory mechanism, both in and ex copula, coupled with an inhibition of erectile responses for moderate doses of methoxamine. Treatment of male rats with the alpha₂-adrenoceptor agonist clonidine (0.25 mg/kg, IP, five minutes pretest) drastically reduced the number of animals exhibiting intromissive and ejaculatory behavior in mating tests. This suppressive effect of clonidine was not prevented by prior treatment with methoxamine (3 mg/kg, 10 minutes pretest and five minutes preclonidine). Further, ST-91, a polar analog of clonidine which does not readily enter the central nervous system, was without effect on male sexual behavior. Since (1) the effects of methoxamine administration are not of similar quality or magnitude to those reported earlier after yohimbine, an alpha₂-adrenoceptor antagonist, (2) since concurrent stimulation of alpha₁- (by methoxamine) and alpha₂- (by clonidine) adrenoceptors is followed by a suppression of sexual behavior similar to that seen after clonidine alone, and (3) since a peripherally acting alpha₂-adrenoceptor agonist is without effect on sexual behavior, we suggest that one of the substrates underlying the clonidine-induced suppression of male rats' sexual behavior are postsynaptic alpha₂-adrenoceptors located within the central nervous system. Further, alpha₁-adrenoceptors appear to play a role in the regulation of the ejaculatory threshold in and ex copula, and in the regulation of erection ex copula.     

Comparison of the response of mast cells in guinea-pig cardiac atria and ventricles

A comparison was made between collagenase-dispersed guinea-pig atrial and ventricular tissues. Heparin containing cells were stained with alcian blue at pH 2.2, and counted by an automated technique (Technicon H6000). The cells were challenged with the specific antigen (ovalbumin), with antisera to guinea-pig IgG (non-subclass specific), IgG₁ and IgG₂, and the calcium ionophore A23187. Histamine release was measured by an automated spectrofluorometric technique, and leukotriene C₄ was measured by radioimmunoassay.All of the following parameters were higher in the atrial than in ventricular cells (mean ratio and SEM of atrial: ventricular mast cell parameters in parenthesis): 1) Histamine content/g wet tissues (3.32±0.71:1) (p<0.05), 2) Absolute mast cell number as a proportion of total cell count (3.75±1.64:1), 3) Histamine release induced by antigen (significant in one out of four experiments), anti-IgG (significant in three out of four experiments), anti-IgG₁ (significant in two out of four experiments), and anti-IgG₂ (higher but not statistically significant).Ionophore A23187 gave an inconsistent histamine release pattern: significantly higher release from atria in five treatments (different concentrations in different experiments), and higher ventricular release in three.Significantly more leukotriene C₄ was released by antigen and the ionophore A23187 (mean of 3–5 treatments), but not with anti-IgG.     

甜菜碱对戊四氮致痫大鼠海马GFAP、甘氨酸和甘氨酸受体表达的影响

 目的：研究甜菜碱对癫痫大鼠海马胶质细胞原纤维酸性蛋白（GFAP）、甘氨酸(Gly)及甘氨酸受体(GlyR)表达的影响。方法：将健康雄性Wistar大鼠随机分为：正常对照组（腹腔注射生理盐水,1.0 mL生理盐水灌胃);癫痫组（腹腔注射戊四氮,1.0 mL生理盐水灌胃）;甜菜碱高、中、低浓度组（腹腔注射戊四氮,甜菜碱灌胃）;丙戊酸钠组（腹腔注射戊四氮,丙戊酸钠灌胃）。实验结束后大鼠眼眶取血检测血清中同型半胱氨酸的含量;在低温条件下迅速取脑组织,分析Gly含量的变化,免疫荧光检测GFAP的水平,兔疫荧光和Western bloting检测海马区GlyR表达的变化。结果：各组大鼠大发作潜伏期无显著差异(P＞0.05),但是甜菜碱治疗组较癫痫组的首次大发作持续时间显著缩短(P<0.01)。癫痫组同型半胱氨酸的含量与正常组比较显著降低（P<0.01）,甜菜碱高、低浓度组同型半胱氨酸含量与癫痫组比较明显降低（P<0.05）。癫痫组甘氨酸的含量与正常对照组相比显著下降（P<0.01）。甜菜碱高、中、低浓度组甘氨酸的含量与癫痫组比较显著增高（P<0.01）。免疫荧光检测GFAP结果,癫痫组与正常组比较显著增高（P<0.01）,而甜菜碱高中低浓度与癫痫组相比显著降低（P<0.01）。免疫荧光与Western bloting检测GlyR结果,癫痫组GluR的表达与正常对照组相比显著降低（P<0.01）,甜菜碱高、中、浓度组较癫痫组显著升高（P<0.01）。结论：甜菜碱具有较好的抗癫痫作用。     

Pharmacological interactions between ranitidine,cimetidine, metiamide and tiotidine at histamine H2-receptor sites in guinea-pig atria

Cumulative concentration-response curves to histamine or dimaprit were constructed on guinea-pig isolated right atria and agonist dose-ratios were determined following addition of ranitidine, cimetidine, metiamide or tiotidine alone or a combination of any two of these H₂-receptor blocking drugs. The observed histamine or dimaprit dose-ratios for combinations of any two of the H₂-antagonists tested were consistent with results predicted from the equation, DR₁₊₂=DR₁+DR₂–1, for two antagonists competing for the same receptor sites. Therefore we conclude that all four H₂-antagonists compete for the same histamine H₂-receptor.     

H2-receptor antagonist activity of N-methylthiourea,N-cyano-N′-methylguanidine,N-cyanoamidine,N-carbamoylamidine and N-sulfamoylamidine 5-substituted thiazole derivatives on guinea-pig atria

The H₂-antagonist activity of thiazole derivatives, substituted on position 5 with urea-equivalent groups, has been tested on guinea-pig isolated atria stimulated by dimaprit. By comparing the activities of the 2,5-disubstituted thiazole derivatives with those of the correseponding 2,4-disubstituted derivatives it can be seen that the side-chain position is critical to activity and differently influences activity in the various series. The heteroaromatic ring atom sequence N-C-S-C-side chain is always associated with a low antagonist activity.