ISSN 00124966, Doklady Biological Sciences, 2015, Vol. 460, pp. 12–16. © Pleiades Publishing, Ltd., 2015. Original Russian Text © V.I. Tsirkin, A.D. Nozdrachev, Yu.V. Korotaeva, 2015, published in Doklady Akademii Nauk, 2015, Vol. 460, No. 4, pp. 480–485.
PHYSIOLOGY
The Effect of Histidine on the Contractility and Adrenoreactivity of the Myocardium of Nonpregnant and Pregnant Rats V. I. Tsirkina, Academician A. D. Nozdrachevb, and Yu. V. Korotaevac Received August 11, 2014
DOI: 10.1134/S0012496615010123
Earlier we demonstrated that histidine had a posi tive inotropic effect (IE) in experiments with myocar dial strips of the right rat ventricle (at concentrations of 10–4 and 10–3 g/mL) [5] and with biopsy material of the right atrial auricle of a human heart (at a concen tration of 10–5 g/mL) [3]. However, this effect of histi dine (10–5 g/mL) was not observed in experiments with frog myocardium [7]. It was also found that histi dine (10–5 g/mL) increased the effectiveness of activa tion of βadrenergic receptors (ARs) of an intact frog myocardium [5, 7], but this effect was not observed in experiments with an intact rat myocardium using his tidine at a concentration of 10–4 g/mL [7], though at the same concentration it had a βadrenosensitizing activity in experiments with rat myocardium, and the effectiveness of activation of β ARs by it was decreased by lysophosphatidylcholine [7]. In experiments with strips of the right atrial auricle of a heart subjected to aortocoronary bypass, histidine (10–5 g/mL) did not increase the positive IE of adrenaline, though 100, 500, and 1000fold dilutions of the blood serum of nonpregnant women (as a source of endogenous sen sitizer of βadrenergic receptors, ESBAR) revealed βadrenosensitizing activity [3]. It was also demon strated that histidine (10–5 and 10–4 g/mL) restored the effectiveness of activation of β2 ARs of rat myo metrium decreased by propranolol [6].
Therefore, the purpose of this study was assessing the effect of histidine on the contractility of the myo cardium of the right ventricle of the heart of nonpreg nant and pregnant rats and that on the effectiveness of activation of β1 and β2 ARs, including that in the pres ence of the adrenoceptor antagonists propranolol, atenolol, or nicergoline. Sixty strips of the myocardium (length, 8–13 mm; width, 1–2 mm) excised parallel to the longitudinal axis of the right ventricle of rats and consisting of tra becula and papillary muscles were studied; 8 rats being at proestrus; 11, at estrus; 13, at metaestrus; 8, at diestrus; 4, at the 5th to 9th day of pregnancy; 6, at the 12th to 14th day, and 10, at the 16th to 20th day of pregnancy. The contractile activity induced by single rectan gular stimuli (5 ms, 20 V, 1 Hz) was recorded using an ESL1 stimulator (Fig. 1) according to the method [5] at 37°C in a dish (1 mL) of a Myocytograph (Noris, Russia) of a vertical type under the conditions of con tinuous perfusion at a rate of 1.1 mL/min with an oxy genated (100% oxygen) Krebs solution using a syringe dispenser (Noris), isometric force meter (Honeywell, United States), and an LA70 analogtodigital con verter (Rudnev–Shilyaev, Russia). One end of the strip was attached to an anchor (a silverplated metal rod) serving as an active electrode, and its other end was attached to the force meter. A passive electrode was a silverplated metal rod immersed in a bath. In each experiment, the strip was stretched up to the optimal length at which the contraction force was maximal. Histidine (SigmaAldrich, Japan), adrenaline hydrochloride (Moskovskii Endokrinnyi Zavod, Rus sia), propranolol (Uralbiofarm, Russia), atenolol (Sintez, Russia), and nicergoline (FP Obolenskoe, Russia) were used. Krebs solution (pH 7.4) contained 136 mM NaCl, 4.7 mM KCl, 2.52 mM CaCl2, 1.2 mM MgCl2, 0.6 mM KH2PO4, 4.7 mM NaHCO3, and 11 mM C6H12O6.
Given a decrease in the effectiveness of activation of β ARs of the myocardium during hypertension [14] and cardiac failure [12] observed in pregnant women [8], a question arises of cardiologic applications of his tidine as a substance increasing contractility and as an exogenous sensitizer of β ARs of the myocardium. a
Kazan State Medical University, ul. Derendyaeva 50, Kirov, 610017 Russia; email: [email protected] b St. Petersburg State University, Moskovskii pr. 193, 196066 Russia c Vyatka State University of Humanities, ul. Svobody 122, Kirov, 610002 Russia
We performed eight series of experiments. In series I (nonpregnant rats, NPR) and II (pregnant rats, PR) consisting of 32 stages, the effect of histidine (in a con 12
THE EFFECT OF HISTIDINE ON THE CONTRACTILITY AND ADRENOREACTIVITY
centration range from 10–10 to 10–4 g/mL) on the amplitude of evoked contractions of the strips was studied, including that in the presence of adrenaline at a concentration of 10–9 g/mL (a fragment of the experiments is given in Fig. 2). In series III and V (NPR) and in series IV and VI (PR) consisting of eight stages, the effect of adrenaline (10–5 g/mL) on the con traction amplitude and that of histidine (10–4 g/mL) on the effect of adrenaline in the presence of propranolol (10–8 g/mL, series III and IV) or atenolol (10–6 g/mL in series V, 10–8 g/mL in series VI) were studied. In series VII and VIII (NPR), nicergoline (10–8 g/mL, series VII, eight stages) or a mixture of nicergoline and propranolol (the concentration of the both drugs was 10–8 g/mL, series VIII, four stages) were used as adrenoceptor antagonists. Since the distribution of the data obtained did not fit the normal law, the results of the study were given in the form of a median and interquartile range (the 25th and 75th percentiles) [1]. The differences between two dependent samples (between stages of series) and those between independent samples (between series) were determined using the Wilcoxon T test and the Wilcoxon–Mann–Whitney U test, respectively. The differences were considered significant at p < 0.05. On the whole (series I–VIII), it was found that in nonpregnant rats (not taking the phase of the cycle into account) the wet weight of 40 strips was 88 mg (the 25th and 75th percentiles were 72 and 103 mg, respec tively) and the dry weight was 22 mg (20 and 35 mg). In pregnant rats (the 5th to 20th days of pregnancy, 20 strips), they were 86 mg (70 and 124 mg) and 21 mg (18 and 28 mg), respectively. Herewith, the differences between groups were statistically nonsignificant for each index (p > 0.05, the Mann–Whitney U test). This made it possible to compare nonpregnant and preg nant rats with each other studying the myocardium contractility. It was found that the amplitude of contractions of strips of the right ventricle of rats induced by electrical stimuli did not depend on the phase of the estrous cycle and the presence of pregnancy. Thus, nonpreg nant rats with estrogen prevailing (proestrus, estrus) had the amplitude of 3.4 mN (2.0 and 4.6 mN) and 0.03 mN (0.02 and 0.04 mN) per 1 mg wet weight of the strip and 0.12 mN (0.08 and 0.18 mN) per 1 mg dry weight. When progesterone prevailed (metaestrus, diestrus), it was 2.5 mN (2.2 and 4.0 mN) and 0.03 mN (0.02 and 0.04 mN) per 1 mg wet weight, respectively, and 0.11 mN/mg dry weight (p > 0.05 according to the Mann–Whitney U test). On the whole, not taking the phase of the cycle into account for nonpregnant rats, these indices were 2.8 mN (2.0 and 3.1 mN) and 0.03 mN (0.02 and 0.04 mN) per 1 mg wet weight and 0.12 mN (0.08 and 0.15 mN) per 1 mg dry weight. The pregnant rats had the same DOKLADY BIOLOGICAL SCIENCES
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4 5 3 1 9 2
8
7
6 Fig. 1. The scheme of transmural electrostimulation of a strip of the right ventricle of the heart of nonpregnant and pregnant rats upon continuous electrostimulation (5 ms, 1 Hz) during perfusion with an oxygenated (100% O2) Krebs solution: 1, the working chamber of a Myocyto graph; 2, a strip anchor (a silverplated metal rod) serving as an active electrode; 3, a silverplated metal rod (a pas sive electrode); 4, an electrostimulator; 5, a force meter; 6, Krebs solution; 7, a strip of the right ventricle of the heart; 8, a syringe dispenser; and 9, a reservoir for the per fusate.
indices as the nonpregnant rats (pPR–NPR > 0.05) 2.8 mN (2.2 and 4.1 mN) and 0.02 mN (0.02 and 0.04 mN) per 1 mg wet weight and 0.13 mN (0.08 and 0.20 mN) per 1 mg dry weight, respectively. Our data, on one hand, indicate stability of the spe cific contractility of the myocardium at different stages of the reproductive process, which, however, does not agree with some reports on an increase in the contractility of the rat myocardium during pregnancy [13], and, on the other hand, make it possible to com pare pregnant and nonpregnant rats with each other in regards to the effect of adrenergic agents and histidine on the myocardium contractility. We have also demonstrated (series I and II) that histidine (10–10 to 10–4 g/mL) does not affect the amplitude of contractions of the myocardium of non pregnant rats (for all the concentrations (p > 0.05 according to the Wilcoxon T test), but increases it (though independently on the concentration) in preg nant rats (for all the concentrations, p < 0.05 accord ing to the Wilcoxon T test). Thus, the amplitude of contractions in the nonpregnant rats (series I) under the action of histidine at a concentration range of 10–10 to 10–4 g/mL was 99 (97 and 114), 97 (86 and 107), 99 (91 and 128), 102 (94 and 115), 109 (72 and
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TSIRKIN et al. (a)
4 mN 3 min
KS
Ad9
KS
His10
KS
His10 + Ad9
KS
His10 + Ad9
KS
(b)
4 mN 3 min
KS
Ad9
KS
His10
KS
Fig. 2. Mechanograms of strips of the right ventricle of the heart of (a) nonpregnant (estrogen phase) and (b) pregnant (19.5 days) rats upon application of adrenaline (10–9 g/mL; Ad9), histidine (10–10 g/mL, His10), or their mixture under continuous elec trostimulation (1 Hz, 5 ms, 20 V). These fragments of series I and II (stages 1–7) demonstrate the presence of a positive inotropic effect of histidine in experiments with the myocardium of nonpregnant rats and the absence of β adrenosensitizing activity of his tidine in the intact myocardium. KS is Krebs solution. Calibration: 4 mN, 3 min.
124), 104 (76 and 134), and 103 (84 and 120)% of the amplitude observed in the case of perfusion with the Krebs solution (hereinafter, PKS). In the pregnant rats (series II), it was 113 (105 and 152), 110 (104 and 126), 118 (105 and 151), 115 (107 and 125), 119 (119 and 165), and 130 (114 and 148)% of the amplitude observed during PKS. Figure 2 gives a fragment of the experiments with the myocardium strips of nonpreg nant and pregnant rats studying histidine at concen tration of 10–10 g/mL. Thus, we have confirmed the data [5] on the ability of histidine to increase the contractility of the myocar dium of the right ventricle of nonpregnant rats but demonstrated that this ability is also observed in the case of pregnant rats. This indicates that histidine is promising for the use in clinical practice to increase myocardium contractility in pregnant women. We have also found (experiments of series I and II) that adrenaline alone used at a relatively low concen tration (10–9 g/mL) does not affect the amplitude of contraction of the strips of the myocardium of non pregnant (irrespective of the phase of the cycle) and pregnant rats (p > 0.05 according the Wilcoxon T test). Indeed, in the presence of adrenaline, the amplitude of contractions was 104 (102 and 115) and 99 (92 and 127)% of the initial level. Histidine (10–10 to 10–4 g/mL) in both groups did not enhance the effect of adrenaline (Fig. 2). In the experiments with histidine at a concentration of 10–4 g/mL, the ampli tude of contractions of the strips of nonpregnant rats
(in percent of the amplitude of contractions recorded at the previous stage in the case of PKS) upon applica tion of adrenaline was 104 (102 and 115)%, upon that of adrenaline combined with histidine, it was 83 (75 and 114)%, and, upon repeated application of adren aline without histidine, it was 104 (94 and 118)%. Herewith, the differences between the effects of the three tests with adrenaline were statistically nonsignif icant (р1,2,3 > 0.05 according to the Mann–Whitney U test). For the strips of the pregnant rats, these values were 99 (92 and 127), 125 (93 and 150), and 112 (105 and 126)% (р1,2,3 > 0.05 according to the Mann–Whit ney U test). These data prove the notion [5] that histi dine acting on the intact rat myocardium has no β1 or β2 adrenosensitizing activity. At the same time, our results demonstrate that this conclusion is correct for a wide range of histidine concentrations not only in regards to the myocardium of male rats [5], but also in regards to the myocardium of pregnant and nonpreg nant rats. We suggest that the β adrenosensitizing activity of histidine is prevented by the presence in car diac hystiocytes of rats of β3 ARs, their activation being known [10] to have a negative inotropic effect. The other series of experiments (series III–VIII) have revealed that adrenaline used at a concentration of 10–5 g/mL in the experiments with 40 myocardium strips of nonpregnant rats and 20 myocardium strips of pregnant rats has a positive inotropic effect. Its inten sity did not depend on the phase of the cycle and the presence of pregnancy. In particular, adrenaline statis DOKLADY BIOLOGICAL SCIENCES
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THE EFFECT OF HISTIDINE ON THE CONTRACTILITY AND ADRENOREACTIVITY
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An amplitude of induced contractions (in mN and % to an experimental stage indicated, median and 25th and 75th percentiles) of myocardium strips of the right ventricle of the heart of nonpregnant (with the phase of the cycle not taken into account) and pregnant (5th to 20th day) rats upon application of adrenaline (10–5 g/mL) including that against application of an adrenoceptor antagonist (propranolol, atenolol, nicergoline) or an adrenoceptor antagonist combined with histidine (10–4 g/mL) Propranolol, 10–8 g/mL
Atenolol, 10–6 (series V) and 10–8 g/mL (series VI)
Nicergoline, 10–8 g/mL
Nicergoline + propra nolol, 10–8 g/mL
series III
series IV
series V
series VI
series VII
series VIII
nonpregnant rats (n = 10)
pregnant rats (n = 10)
nonpregnant rats (n = 10)
pregnant rats (n = 10)
nonpregnant rats (n = 10)
nonpregnant rats (n = 8)
Stage 1. Background amplitude under the conditions of perfusion with Krebs solution, mN 3.64 2.45 2.89 2.21 2.80 3.35 (1.85; 5.42) (2.06; 3.80) (2.03; 4.29) (1.80; 3.57)# (2.10; 3.65) (1.90; 5.70) Stage 2. Contraction amplitude upon application of adrenaline (10–5 g/mL), in % to the amplitude at stage 1 119 121 117 116 126 128 (111; 142)* (113; 126)* (109; 124)* (107; 129) (116; 170)* (119; 138)* Stage 4. Contraction amplitude upon application of an inhibitor, in % to the amplitude at stage 3 92 105 86 110 97 102 (89; 101) (99; 117)# (76; 101) (94; 135)# (85; 108) (88; 128) Stage 5. Contraction amplitude upon application of adrenaline (10–5 g/mL) and inhibitor, in % to the amplitude at stage 4 70 82 85 76 88 90 (65; 85)* (76; 100)* (75; 99)* (66; 98)* (77; 104) (82; 101) Stage 6. Contraction amplitude upon application of adrenaline (10–5 g/mL), inhibitor, and histidine (10–4 g/mL), in % to the amplitude at stage 4 96 91 98 105 114 103 (87; 103) (77; 110) (87; 107) (90; 142) (110; 125)* (88; 127) Stage 8. Contraction amplitude upon application of adrenaline (10–5 g/mL), in % to the amplitude at stage 1 128 119 108 118 128 Not studied (119; 144)* (99; 143)# (106; 113) (111; 155)* (119; 138)* p < 0.05 according to the Wilcoxon T test; # p < 0.05 according to the Mann–Whitney U test. Stages 3 and 7 are perfusion of the myocardium strips with Krebs solution.
tically significantly (p < 0.05 according to the Wil coxon T test) increased the amplitude of contractions of the myocardium strips of the nonpregnant rats to 117 (108 and 127)% of the initial level and that of the pregnant rats to 121 (113 and 126)% (pPR–NPR > 0.05 according to the Mann–Whitney U test). Propranolol and atenolol alone did not affect (p > 0.05 according to the Wilcoxon T test) the amplitude of contractions of the strips of the pregnant and nonpregnant rats; the same was observed for a mixture of nicergoline and propranolol and nonpregnant rats (table). At further stages of series III–VIII, it was found that, under the action of each of the adrenoceptor antagonists, adrenaline (10–5 g/mL) had a negative inotropic effect instead of a positive one, and addi tional introduction of histidine (10–4 g/mL) to the medium inhibited the negative inotropic effect of adrenaline, though did not restore its ability to have a positive inotropic effect. Thus, in the experiments with propranolol (10–8 g/mL, series III, table) adrenaline first (stage 2) DOKLADY BIOLOGICAL SCIENCES
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statistically significantly (p < 0.05 according to the Wilcoxon T test) revealed a positive inotropic effect increasing the amplitude of contractions to 119 (111 and 142)% of the initial level. Propranolol (stage 4) alone did not affect the contraction amplitude: it was 92 (89 and 101)% of the level under the PKS condi tions (stage 3). However, adrenaline applied against the background of propranolol (stage 5) had a negative inotropic effect instead of positive one: it statistically significantly (p < 0.05 according to the Wilcoxon T test) decreased the contraction amplitude to 70 (65 and 85)% of the level at stage 4. The addition of histidine (10–4 g/mL) to the mixture of adrenaline and propranolol (stage 6) prevented the negative inotropic effect of adrenaline but did not restore its ability to exert a positive inotropic effect. Under these condi tions, the contraction amplitude was 96 (87 and 103)% of the amplitude observed against the background of propranolol at stage 4. The washing of the strip with Krebs solution from propranolol and histidine was accompanied by restoration of the positive inotropic effect of adrenaline (stage 8): it statistically signifi
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cantly (p < 0.05 according to the Wilcoxon T test) increased the contraction amplitude to 128 (119 and 144)% of the level observed during PKS at stage 7. Similar data were obtained in the experiments with the myocardium strips of pregnant rats (series IV). Ini tially, adrenaline had a positive inotropic effect; pro pranolol alone did not change the contraction ampli tude; adrenaline in the presence of propranolol induced a negative inotropic effect, and histidine inhibited it. However, restoration of the positive ino tropic effect occurred after propranolol and histidine were removed at the final stage 8 (table). Similar results were observed in the experiments with atenolol (table) acting on the myocardium strips of nonpreg nant rats (series V) and pregnant rats (series VI). In the experiments with nicergoline (10–8 g/mL, series VII, table) performed on the myocardium strips of nonpregnant rats, we demonstrated that, in the presence of nicergoline, which itself had no effects on the contraction amplitude, adrenaline lost its ability to exert a positive inotropic effect. Herewith, in contrast to the series with propranolol and atenolol, nicer goline did not induce a negative inotropic effect. Addi tion of histidine (10–4 g/mL) restored the ability of adrenaline to exert a positive inotropic effect even in the presence of nicergoline. In the experiments with the myocardium of nonpregnant rats where a mixture of nicergoline and propranolol was used (10–8 g/mL, series VIII, table), it was also demonstrated that the antagonists themselves did not affect the contraction amplitude but prevented a positive inotropic effect of adrenaline, which, however, was not restored com pletely upon addition of histidine. The results of series III–VIII confirm our earlier data [4] that, upon inhibition of β1 ARs by atenolol and that of β1 and β2 ARs by propranolol, adrenaline in the myocardium strips of nonpregnant rats had a negative inotropic effect instead of positive one. In this study, we demonstrated that this reversion of the adrenaline effect was also observed for the myocar dium strips of pregnant rats. Taking into account liter ature data on a decrease in the contraction amplitude of the myocardium during activation of β3 ARs [10] and α1 ARs [9], we suggest that the negative inotropic effect upon application of propranolol, atenolol, and nicergoline (as a nonselective antagonist of β1 and β2 ARs) is caused by the activation of β3 ARs and α1 ARs. Removal of the negative inotropic effect of adrenaline and even restoration of the ability of adrenaline to exert a positive inotropic effect upon application of histidine including those observed in the myocardium of pregnant rats were caused by an increase in the effectiveness of activation of β1 ARs and β2 ARs upon application of histidine due to β1 and β2 adrenosensi tizing activity of histidine. It cannot be excluded that the presence of β3 ARs in this case prevents the com
plete restoration of the ability of adrenaline to exert a positive inotropic effect. The results of the experiments with histidine and data obtained by us earlier [4] make it possible to for mulate three assumptions. (1) Patients taking antago nists of β ARs upon stress could have, instead of an increase in the heart functioning, its decrease and, as a result, develop acute heart failure, since catechola mines against the background of inhibition of β1 ARs and β2 ARs could activate β3 ARs and α1 ARs. Thus, in these cases, it could be reasonable to use analogs of ESBAR (e.g., mildronat or histidine). This is also proved by clinical experience of using mildronat (as a metabolitic drug) together with antagonists of β1 ARs in myocardial infarction [2]. (2) It is reasonable to use histidine, mildronat, and other analogs of ESBAR in chronic heart failure, which is known [11] to be char acterized by a decreased effectiveness of activation of β ARs and in the cases of threatened preterm birth to increase the effectiveness of the socalled β AR inhibi tion mechanism [6]. (3) It is necessary to create exog enous sensitizers of β1 ARs and β2 ARs, including those based on histidine, tryptophan, or tyrosine. REFERENCES 1. Glantz, S., Primer of Biostatistics, New York: McGraw Hills, 2012, 7th edition. 2. Gordeev, I.G., Lyusov, V.A., Il’ina, E.E., et al., Kardi ologiya, 2007, vol. 47, no. 2, pp. 22–24. 3. Korotaeva, K.N., Nozdrachev, A.D., Vyaznikov, V.A., et al., Vestn. S.Peterb. Gos. Univ. Ser. 3 Biol., 2011, no. 2, pp. 45–57. 4. Korotaeva, Yu.V. and Tsirkin, V.I., Vyat. Med. Vestn., 2013, no. 3, pp. 24–30. 5. Penkina, Yu.A., Nozdrachev, A.D., and Tsirkin, V.I., Vestn. S.Peterb. Gos. Univ. Ser. 3 Biol., 2008, no. 1, p. 551. 6. Toropov, A.L., Nozdrachev, A.D., and Tsirkin, V.I., Vestn. S.Peterb. Gos. Univ. Ser. 3 Biol., 2011, no. 1, pp. 27–42. 7. Tsirkin, V.I., Sizova, E.N., Kaisina, I.G., et al., Ros. Vestn. Akush.Ginek., 2004, no. 2, pp. 4–9. 8. Shekhtman, M.M., Rukovodstvo po ekstragenital’noi patologii u beremennykh (Handbook on Extragenital Pathology in Pregnancy), Moscow: Triada, 2011. 9. Chu, C., Thai, K., Park, K., et al., Am. J. Physiol. Heart Circ. Physiol., 2013, vol. 304, no. 7, pp. 946–953. 10. Gill, R., Cheung, P., Yu, X., et al., Clin. Med. Insights. Cardiol., 2012, vol. 6, pp. 45–51. 11. Guggilam, A., Hutchinson, K., West, T., et al., J. Mol. Cell. Cardiol., 2013, vol. 57, pp. 47–58. 12. SirykBathgate, A., Dabul, S., and Lymperopoulos, A., Drug Des. Devel. Ther., 2013, vol. 7, pp. 1209–1222. 13. VirgenOrtiz, A., Marin, J., Elizalde, A., et al., J. Phys iol. Sci., 2009, vol. 59, no. 5, pp. 391–396. 14. Wehling, M., Herz, 2002, vol. 27, suppl. 1, pp. 16–25.
Translated by E. Berezhnaya DOKLADY BIOLOGICAL SCIENCES
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2015
PHYSIOLOGY
The Effect of Histidine on the Contractility and Adrenoreactivity of the Myocardium of Nonpregnant and Pregnant Rats V. I. Tsirkina, Academician A. D. Nozdrachevb, and Yu. V. Korotaevac Received August 11, 2014
DOI: 10.1134/S0012496615010123
Earlier we demonstrated that histidine had a posi tive inotropic effect (IE) in experiments with myocar dial strips of the right rat ventricle (at concentrations of 10–4 and 10–3 g/mL) [5] and with biopsy material of the right atrial auricle of a human heart (at a concen tration of 10–5 g/mL) [3]. However, this effect of histi dine (10–5 g/mL) was not observed in experiments with frog myocardium [7]. It was also found that histi dine (10–5 g/mL) increased the effectiveness of activa tion of βadrenergic receptors (ARs) of an intact frog myocardium [5, 7], but this effect was not observed in experiments with an intact rat myocardium using his tidine at a concentration of 10–4 g/mL [7], though at the same concentration it had a βadrenosensitizing activity in experiments with rat myocardium, and the effectiveness of activation of β ARs by it was decreased by lysophosphatidylcholine [7]. In experiments with strips of the right atrial auricle of a heart subjected to aortocoronary bypass, histidine (10–5 g/mL) did not increase the positive IE of adrenaline, though 100, 500, and 1000fold dilutions of the blood serum of nonpregnant women (as a source of endogenous sen sitizer of βadrenergic receptors, ESBAR) revealed βadrenosensitizing activity [3]. It was also demon strated that histidine (10–5 and 10–4 g/mL) restored the effectiveness of activation of β2 ARs of rat myo metrium decreased by propranolol [6].
Therefore, the purpose of this study was assessing the effect of histidine on the contractility of the myo cardium of the right ventricle of the heart of nonpreg nant and pregnant rats and that on the effectiveness of activation of β1 and β2 ARs, including that in the pres ence of the adrenoceptor antagonists propranolol, atenolol, or nicergoline. Sixty strips of the myocardium (length, 8–13 mm; width, 1–2 mm) excised parallel to the longitudinal axis of the right ventricle of rats and consisting of tra becula and papillary muscles were studied; 8 rats being at proestrus; 11, at estrus; 13, at metaestrus; 8, at diestrus; 4, at the 5th to 9th day of pregnancy; 6, at the 12th to 14th day, and 10, at the 16th to 20th day of pregnancy. The contractile activity induced by single rectan gular stimuli (5 ms, 20 V, 1 Hz) was recorded using an ESL1 stimulator (Fig. 1) according to the method [5] at 37°C in a dish (1 mL) of a Myocytograph (Noris, Russia) of a vertical type under the conditions of con tinuous perfusion at a rate of 1.1 mL/min with an oxy genated (100% oxygen) Krebs solution using a syringe dispenser (Noris), isometric force meter (Honeywell, United States), and an LA70 analogtodigital con verter (Rudnev–Shilyaev, Russia). One end of the strip was attached to an anchor (a silverplated metal rod) serving as an active electrode, and its other end was attached to the force meter. A passive electrode was a silverplated metal rod immersed in a bath. In each experiment, the strip was stretched up to the optimal length at which the contraction force was maximal. Histidine (SigmaAldrich, Japan), adrenaline hydrochloride (Moskovskii Endokrinnyi Zavod, Rus sia), propranolol (Uralbiofarm, Russia), atenolol (Sintez, Russia), and nicergoline (FP Obolenskoe, Russia) were used. Krebs solution (pH 7.4) contained 136 mM NaCl, 4.7 mM KCl, 2.52 mM CaCl2, 1.2 mM MgCl2, 0.6 mM KH2PO4, 4.7 mM NaHCO3, and 11 mM C6H12O6.
Given a decrease in the effectiveness of activation of β ARs of the myocardium during hypertension [14] and cardiac failure [12] observed in pregnant women [8], a question arises of cardiologic applications of his tidine as a substance increasing contractility and as an exogenous sensitizer of β ARs of the myocardium. a
Kazan State Medical University, ul. Derendyaeva 50, Kirov, 610017 Russia; email: [email protected] b St. Petersburg State University, Moskovskii pr. 193, 196066 Russia c Vyatka State University of Humanities, ul. Svobody 122, Kirov, 610002 Russia
We performed eight series of experiments. In series I (nonpregnant rats, NPR) and II (pregnant rats, PR) consisting of 32 stages, the effect of histidine (in a con 12
THE EFFECT OF HISTIDINE ON THE CONTRACTILITY AND ADRENOREACTIVITY
centration range from 10–10 to 10–4 g/mL) on the amplitude of evoked contractions of the strips was studied, including that in the presence of adrenaline at a concentration of 10–9 g/mL (a fragment of the experiments is given in Fig. 2). In series III and V (NPR) and in series IV and VI (PR) consisting of eight stages, the effect of adrenaline (10–5 g/mL) on the con traction amplitude and that of histidine (10–4 g/mL) on the effect of adrenaline in the presence of propranolol (10–8 g/mL, series III and IV) or atenolol (10–6 g/mL in series V, 10–8 g/mL in series VI) were studied. In series VII and VIII (NPR), nicergoline (10–8 g/mL, series VII, eight stages) or a mixture of nicergoline and propranolol (the concentration of the both drugs was 10–8 g/mL, series VIII, four stages) were used as adrenoceptor antagonists. Since the distribution of the data obtained did not fit the normal law, the results of the study were given in the form of a median and interquartile range (the 25th and 75th percentiles) [1]. The differences between two dependent samples (between stages of series) and those between independent samples (between series) were determined using the Wilcoxon T test and the Wilcoxon–Mann–Whitney U test, respectively. The differences were considered significant at p < 0.05. On the whole (series I–VIII), it was found that in nonpregnant rats (not taking the phase of the cycle into account) the wet weight of 40 strips was 88 mg (the 25th and 75th percentiles were 72 and 103 mg, respec tively) and the dry weight was 22 mg (20 and 35 mg). In pregnant rats (the 5th to 20th days of pregnancy, 20 strips), they were 86 mg (70 and 124 mg) and 21 mg (18 and 28 mg), respectively. Herewith, the differences between groups were statistically nonsignificant for each index (p > 0.05, the Mann–Whitney U test). This made it possible to compare nonpregnant and preg nant rats with each other studying the myocardium contractility. It was found that the amplitude of contractions of strips of the right ventricle of rats induced by electrical stimuli did not depend on the phase of the estrous cycle and the presence of pregnancy. Thus, nonpreg nant rats with estrogen prevailing (proestrus, estrus) had the amplitude of 3.4 mN (2.0 and 4.6 mN) and 0.03 mN (0.02 and 0.04 mN) per 1 mg wet weight of the strip and 0.12 mN (0.08 and 0.18 mN) per 1 mg dry weight. When progesterone prevailed (metaestrus, diestrus), it was 2.5 mN (2.2 and 4.0 mN) and 0.03 mN (0.02 and 0.04 mN) per 1 mg wet weight, respectively, and 0.11 mN/mg dry weight (p > 0.05 according to the Mann–Whitney U test). On the whole, not taking the phase of the cycle into account for nonpregnant rats, these indices were 2.8 mN (2.0 and 3.1 mN) and 0.03 mN (0.02 and 0.04 mN) per 1 mg wet weight and 0.12 mN (0.08 and 0.15 mN) per 1 mg dry weight. The pregnant rats had the same DOKLADY BIOLOGICAL SCIENCES
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4 5 3 1 9 2
8
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6 Fig. 1. The scheme of transmural electrostimulation of a strip of the right ventricle of the heart of nonpregnant and pregnant rats upon continuous electrostimulation (5 ms, 1 Hz) during perfusion with an oxygenated (100% O2) Krebs solution: 1, the working chamber of a Myocyto graph; 2, a strip anchor (a silverplated metal rod) serving as an active electrode; 3, a silverplated metal rod (a pas sive electrode); 4, an electrostimulator; 5, a force meter; 6, Krebs solution; 7, a strip of the right ventricle of the heart; 8, a syringe dispenser; and 9, a reservoir for the per fusate.
indices as the nonpregnant rats (pPR–NPR > 0.05) 2.8 mN (2.2 and 4.1 mN) and 0.02 mN (0.02 and 0.04 mN) per 1 mg wet weight and 0.13 mN (0.08 and 0.20 mN) per 1 mg dry weight, respectively. Our data, on one hand, indicate stability of the spe cific contractility of the myocardium at different stages of the reproductive process, which, however, does not agree with some reports on an increase in the contractility of the rat myocardium during pregnancy [13], and, on the other hand, make it possible to com pare pregnant and nonpregnant rats with each other in regards to the effect of adrenergic agents and histidine on the myocardium contractility. We have also demonstrated (series I and II) that histidine (10–10 to 10–4 g/mL) does not affect the amplitude of contractions of the myocardium of non pregnant rats (for all the concentrations (p > 0.05 according to the Wilcoxon T test), but increases it (though independently on the concentration) in preg nant rats (for all the concentrations, p < 0.05 accord ing to the Wilcoxon T test). Thus, the amplitude of contractions in the nonpregnant rats (series I) under the action of histidine at a concentration range of 10–10 to 10–4 g/mL was 99 (97 and 114), 97 (86 and 107), 99 (91 and 128), 102 (94 and 115), 109 (72 and
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TSIRKIN et al. (a)
4 mN 3 min
KS
Ad9
KS
His10
KS
His10 + Ad9
KS
His10 + Ad9
KS
(b)
4 mN 3 min
KS
Ad9
KS
His10
KS
Fig. 2. Mechanograms of strips of the right ventricle of the heart of (a) nonpregnant (estrogen phase) and (b) pregnant (19.5 days) rats upon application of adrenaline (10–9 g/mL; Ad9), histidine (10–10 g/mL, His10), or their mixture under continuous elec trostimulation (1 Hz, 5 ms, 20 V). These fragments of series I and II (stages 1–7) demonstrate the presence of a positive inotropic effect of histidine in experiments with the myocardium of nonpregnant rats and the absence of β adrenosensitizing activity of his tidine in the intact myocardium. KS is Krebs solution. Calibration: 4 mN, 3 min.
124), 104 (76 and 134), and 103 (84 and 120)% of the amplitude observed in the case of perfusion with the Krebs solution (hereinafter, PKS). In the pregnant rats (series II), it was 113 (105 and 152), 110 (104 and 126), 118 (105 and 151), 115 (107 and 125), 119 (119 and 165), and 130 (114 and 148)% of the amplitude observed during PKS. Figure 2 gives a fragment of the experiments with the myocardium strips of nonpreg nant and pregnant rats studying histidine at concen tration of 10–10 g/mL. Thus, we have confirmed the data [5] on the ability of histidine to increase the contractility of the myocar dium of the right ventricle of nonpregnant rats but demonstrated that this ability is also observed in the case of pregnant rats. This indicates that histidine is promising for the use in clinical practice to increase myocardium contractility in pregnant women. We have also found (experiments of series I and II) that adrenaline alone used at a relatively low concen tration (10–9 g/mL) does not affect the amplitude of contraction of the strips of the myocardium of non pregnant (irrespective of the phase of the cycle) and pregnant rats (p > 0.05 according the Wilcoxon T test). Indeed, in the presence of adrenaline, the amplitude of contractions was 104 (102 and 115) and 99 (92 and 127)% of the initial level. Histidine (10–10 to 10–4 g/mL) in both groups did not enhance the effect of adrenaline (Fig. 2). In the experiments with histidine at a concentration of 10–4 g/mL, the ampli tude of contractions of the strips of nonpregnant rats
(in percent of the amplitude of contractions recorded at the previous stage in the case of PKS) upon applica tion of adrenaline was 104 (102 and 115)%, upon that of adrenaline combined with histidine, it was 83 (75 and 114)%, and, upon repeated application of adren aline without histidine, it was 104 (94 and 118)%. Herewith, the differences between the effects of the three tests with adrenaline were statistically nonsignif icant (р1,2,3 > 0.05 according to the Mann–Whitney U test). For the strips of the pregnant rats, these values were 99 (92 and 127), 125 (93 and 150), and 112 (105 and 126)% (р1,2,3 > 0.05 according to the Mann–Whit ney U test). These data prove the notion [5] that histi dine acting on the intact rat myocardium has no β1 or β2 adrenosensitizing activity. At the same time, our results demonstrate that this conclusion is correct for a wide range of histidine concentrations not only in regards to the myocardium of male rats [5], but also in regards to the myocardium of pregnant and nonpreg nant rats. We suggest that the β adrenosensitizing activity of histidine is prevented by the presence in car diac hystiocytes of rats of β3 ARs, their activation being known [10] to have a negative inotropic effect. The other series of experiments (series III–VIII) have revealed that adrenaline used at a concentration of 10–5 g/mL in the experiments with 40 myocardium strips of nonpregnant rats and 20 myocardium strips of pregnant rats has a positive inotropic effect. Its inten sity did not depend on the phase of the cycle and the presence of pregnancy. In particular, adrenaline statis DOKLADY BIOLOGICAL SCIENCES
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An amplitude of induced contractions (in mN and % to an experimental stage indicated, median and 25th and 75th percentiles) of myocardium strips of the right ventricle of the heart of nonpregnant (with the phase of the cycle not taken into account) and pregnant (5th to 20th day) rats upon application of adrenaline (10–5 g/mL) including that against application of an adrenoceptor antagonist (propranolol, atenolol, nicergoline) or an adrenoceptor antagonist combined with histidine (10–4 g/mL) Propranolol, 10–8 g/mL
Atenolol, 10–6 (series V) and 10–8 g/mL (series VI)
Nicergoline, 10–8 g/mL
Nicergoline + propra nolol, 10–8 g/mL
series III
series IV
series V
series VI
series VII
series VIII
nonpregnant rats (n = 10)
pregnant rats (n = 10)
nonpregnant rats (n = 10)
pregnant rats (n = 10)
nonpregnant rats (n = 10)
nonpregnant rats (n = 8)
Stage 1. Background amplitude under the conditions of perfusion with Krebs solution, mN 3.64 2.45 2.89 2.21 2.80 3.35 (1.85; 5.42) (2.06; 3.80) (2.03; 4.29) (1.80; 3.57)# (2.10; 3.65) (1.90; 5.70) Stage 2. Contraction amplitude upon application of adrenaline (10–5 g/mL), in % to the amplitude at stage 1 119 121 117 116 126 128 (111; 142)* (113; 126)* (109; 124)* (107; 129) (116; 170)* (119; 138)* Stage 4. Contraction amplitude upon application of an inhibitor, in % to the amplitude at stage 3 92 105 86 110 97 102 (89; 101) (99; 117)# (76; 101) (94; 135)# (85; 108) (88; 128) Stage 5. Contraction amplitude upon application of adrenaline (10–5 g/mL) and inhibitor, in % to the amplitude at stage 4 70 82 85 76 88 90 (65; 85)* (76; 100)* (75; 99)* (66; 98)* (77; 104) (82; 101) Stage 6. Contraction amplitude upon application of adrenaline (10–5 g/mL), inhibitor, and histidine (10–4 g/mL), in % to the amplitude at stage 4 96 91 98 105 114 103 (87; 103) (77; 110) (87; 107) (90; 142) (110; 125)* (88; 127) Stage 8. Contraction amplitude upon application of adrenaline (10–5 g/mL), in % to the amplitude at stage 1 128 119 108 118 128 Not studied (119; 144)* (99; 143)# (106; 113) (111; 155)* (119; 138)* p < 0.05 according to the Wilcoxon T test; # p < 0.05 according to the Mann–Whitney U test. Stages 3 and 7 are perfusion of the myocardium strips with Krebs solution.
tically significantly (p < 0.05 according to the Wil coxon T test) increased the amplitude of contractions of the myocardium strips of the nonpregnant rats to 117 (108 and 127)% of the initial level and that of the pregnant rats to 121 (113 and 126)% (pPR–NPR > 0.05 according to the Mann–Whitney U test). Propranolol and atenolol alone did not affect (p > 0.05 according to the Wilcoxon T test) the amplitude of contractions of the strips of the pregnant and nonpregnant rats; the same was observed for a mixture of nicergoline and propranolol and nonpregnant rats (table). At further stages of series III–VIII, it was found that, under the action of each of the adrenoceptor antagonists, adrenaline (10–5 g/mL) had a negative inotropic effect instead of a positive one, and addi tional introduction of histidine (10–4 g/mL) to the medium inhibited the negative inotropic effect of adrenaline, though did not restore its ability to have a positive inotropic effect. Thus, in the experiments with propranolol (10–8 g/mL, series III, table) adrenaline first (stage 2) DOKLADY BIOLOGICAL SCIENCES
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statistically significantly (p < 0.05 according to the Wilcoxon T test) revealed a positive inotropic effect increasing the amplitude of contractions to 119 (111 and 142)% of the initial level. Propranolol (stage 4) alone did not affect the contraction amplitude: it was 92 (89 and 101)% of the level under the PKS condi tions (stage 3). However, adrenaline applied against the background of propranolol (stage 5) had a negative inotropic effect instead of positive one: it statistically significantly (p < 0.05 according to the Wilcoxon T test) decreased the contraction amplitude to 70 (65 and 85)% of the level at stage 4. The addition of histidine (10–4 g/mL) to the mixture of adrenaline and propranolol (stage 6) prevented the negative inotropic effect of adrenaline but did not restore its ability to exert a positive inotropic effect. Under these condi tions, the contraction amplitude was 96 (87 and 103)% of the amplitude observed against the background of propranolol at stage 4. The washing of the strip with Krebs solution from propranolol and histidine was accompanied by restoration of the positive inotropic effect of adrenaline (stage 8): it statistically signifi
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cantly (p < 0.05 according to the Wilcoxon T test) increased the contraction amplitude to 128 (119 and 144)% of the level observed during PKS at stage 7. Similar data were obtained in the experiments with the myocardium strips of pregnant rats (series IV). Ini tially, adrenaline had a positive inotropic effect; pro pranolol alone did not change the contraction ampli tude; adrenaline in the presence of propranolol induced a negative inotropic effect, and histidine inhibited it. However, restoration of the positive ino tropic effect occurred after propranolol and histidine were removed at the final stage 8 (table). Similar results were observed in the experiments with atenolol (table) acting on the myocardium strips of nonpreg nant rats (series V) and pregnant rats (series VI). In the experiments with nicergoline (10–8 g/mL, series VII, table) performed on the myocardium strips of nonpregnant rats, we demonstrated that, in the presence of nicergoline, which itself had no effects on the contraction amplitude, adrenaline lost its ability to exert a positive inotropic effect. Herewith, in contrast to the series with propranolol and atenolol, nicer goline did not induce a negative inotropic effect. Addi tion of histidine (10–4 g/mL) restored the ability of adrenaline to exert a positive inotropic effect even in the presence of nicergoline. In the experiments with the myocardium of nonpregnant rats where a mixture of nicergoline and propranolol was used (10–8 g/mL, series VIII, table), it was also demonstrated that the antagonists themselves did not affect the contraction amplitude but prevented a positive inotropic effect of adrenaline, which, however, was not restored com pletely upon addition of histidine. The results of series III–VIII confirm our earlier data [4] that, upon inhibition of β1 ARs by atenolol and that of β1 and β2 ARs by propranolol, adrenaline in the myocardium strips of nonpregnant rats had a negative inotropic effect instead of positive one. In this study, we demonstrated that this reversion of the adrenaline effect was also observed for the myocar dium strips of pregnant rats. Taking into account liter ature data on a decrease in the contraction amplitude of the myocardium during activation of β3 ARs [10] and α1 ARs [9], we suggest that the negative inotropic effect upon application of propranolol, atenolol, and nicergoline (as a nonselective antagonist of β1 and β2 ARs) is caused by the activation of β3 ARs and α1 ARs. Removal of the negative inotropic effect of adrenaline and even restoration of the ability of adrenaline to exert a positive inotropic effect upon application of histidine including those observed in the myocardium of pregnant rats were caused by an increase in the effectiveness of activation of β1 ARs and β2 ARs upon application of histidine due to β1 and β2 adrenosensi tizing activity of histidine. It cannot be excluded that the presence of β3 ARs in this case prevents the com
plete restoration of the ability of adrenaline to exert a positive inotropic effect. The results of the experiments with histidine and data obtained by us earlier [4] make it possible to for mulate three assumptions. (1) Patients taking antago nists of β ARs upon stress could have, instead of an increase in the heart functioning, its decrease and, as a result, develop acute heart failure, since catechola mines against the background of inhibition of β1 ARs and β2 ARs could activate β3 ARs and α1 ARs. Thus, in these cases, it could be reasonable to use analogs of ESBAR (e.g., mildronat or histidine). This is also proved by clinical experience of using mildronat (as a metabolitic drug) together with antagonists of β1 ARs in myocardial infarction [2]. (2) It is reasonable to use histidine, mildronat, and other analogs of ESBAR in chronic heart failure, which is known [11] to be char acterized by a decreased effectiveness of activation of β ARs and in the cases of threatened preterm birth to increase the effectiveness of the socalled β AR inhibi tion mechanism [6]. (3) It is necessary to create exog enous sensitizers of β1 ARs and β2 ARs, including those based on histidine, tryptophan, or tyrosine. REFERENCES 1. Glantz, S., Primer of Biostatistics, New York: McGraw Hills, 2012, 7th edition. 2. Gordeev, I.G., Lyusov, V.A., Il’ina, E.E., et al., Kardi ologiya, 2007, vol. 47, no. 2, pp. 22–24. 3. Korotaeva, K.N., Nozdrachev, A.D., Vyaznikov, V.A., et al., Vestn. S.Peterb. Gos. Univ. Ser. 3 Biol., 2011, no. 2, pp. 45–57. 4. Korotaeva, Yu.V. and Tsirkin, V.I., Vyat. Med. Vestn., 2013, no. 3, pp. 24–30. 5. Penkina, Yu.A., Nozdrachev, A.D., and Tsirkin, V.I., Vestn. S.Peterb. Gos. Univ. Ser. 3 Biol., 2008, no. 1, p. 551. 6. Toropov, A.L., Nozdrachev, A.D., and Tsirkin, V.I., Vestn. S.Peterb. Gos. Univ. Ser. 3 Biol., 2011, no. 1, pp. 27–42. 7. Tsirkin, V.I., Sizova, E.N., Kaisina, I.G., et al., Ros. Vestn. Akush.Ginek., 2004, no. 2, pp. 4–9. 8. Shekhtman, M.M., Rukovodstvo po ekstragenital’noi patologii u beremennykh (Handbook on Extragenital Pathology in Pregnancy), Moscow: Triada, 2011. 9. Chu, C., Thai, K., Park, K., et al., Am. J. Physiol. Heart Circ. Physiol., 2013, vol. 304, no. 7, pp. 946–953. 10. Gill, R., Cheung, P., Yu, X., et al., Clin. Med. Insights. Cardiol., 2012, vol. 6, pp. 45–51. 11. Guggilam, A., Hutchinson, K., West, T., et al., J. Mol. Cell. Cardiol., 2013, vol. 57, pp. 47–58. 12. SirykBathgate, A., Dabul, S., and Lymperopoulos, A., Drug Des. Devel. Ther., 2013, vol. 7, pp. 1209–1222. 13. VirgenOrtiz, A., Marin, J., Elizalde, A., et al., J. Phys iol. Sci., 2009, vol. 59, no. 5, pp. 391–396. 14. Wehling, M., Herz, 2002, vol. 27, suppl. 1, pp. 16–25.
Translated by E. Berezhnaya DOKLADY BIOLOGICAL SCIENCES
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