Normalization of the calcineurin pathway underlies the regression of hypertensive hypertrophy induced by Na+/H+ exchanger-1 (NHE-1) inhibition.

Na+/H+ exchanger-1 (NHE-1) inhibition induces cardiac hypertrophy regression and (or) prevention in several experimental models, although the intracellular events involved remain unclarified. We aimed to determine whether the calcineurin/NFAT pathway mediates this effect of NHE-1 inhibitors. Spontaneously hypertensive rats (SHR) with cardiac hypertrophy were treated with the NHE-1 inhibitors cariporide and BIIB723 for 30 days. Wistar rats served as normotensive controls. Their hearts were studied by echocardiography, immunoblotting, and real-time RT-PCR. Cytoplasmic Ca2+ and calcineurin Abeta expression were measured in cultured neonatal rat ventricular myocytes (NRVM) stimulated with endothelin-1 for 24 h. NHE-1 blockade induced cardiac hypertrophy regression (heart mass/body mass=3.63+/-0.07 vs. 3.06+/-0.05 and 3.02+/-0.13 for untreated vs. cariporide- and BIIB723-treated SHR, respectively; p<0.05) and decreased myocardial brain natriuretic peptide, calcineurin Abeta, and nuclear NFAT expressions. Echocardiographic evaluation demonstrated a reduction in left ventricular wall thickness without changes in cavity dimensions or a significant decrease in blood pressure. NHE-1-inhibitor treatment did not affect myocardial stiffness or endocardial shortening, but increased mid-wall shortening, suggesting that a positive inotropic effect develops after hypertrophy regression. Cariporide normalized the increased diastolic Ca2+ and calcineurin Abeta expression observed in ET-1-stimulated NRVM. In conclusion, our data suggest that inactivation of calcineurin/NFAT pathway may underlie the regression of cardiac hyper-trophy induced by NHE-1 inhibition.

Endothelin-1 induced hypertrophic effect in neonatal rat cardiomyocytes: involvement of Na+/H+ and Na+/Ca2+ exchangers.

Endothelin-1 (ET-1) is a potent agonist of cell growth that also stimulates Na(+)/H(+) exchanger isoform 1 (NHE-1) activity. It was hypothesized that the increase in intracellular Na(+) ([Na(+)](i)) mediated by NHE-1 activity may induce the reverse mode of Na(+)/Ca(2+) exchanger (NCX(rev)) increasing intracellular Ca(2+) ([Ca(2+)](i)) which in turn will induce hypertrophy. The objective of this work was to test whether the inhibition of NHE-1 or NCX(rev) prevents ET-1 induced hypertrophy in neonatal rat cardiomyocytes (NRVMs). NRVMs were cultured (24 h) in the absence (control) and presence of 5 nmol/L ET-1 alone, or combined with 1 mumol/L HOE 642 or 5 mumol/L KB-R7943. Cell surface area, (3)H-phenylalanine incorporation and atrial natriuretic factor (ANF) mRNA expression were increased to 131 +/- 3, 220 +/- 12 and 190 +/- 25% of control, respectively (P < 0.05) by ET-1. [Na(+)](i) and total [Ca(2+)](i) were higher (8.1 +/- 1.2 mmol/L and 636 +/- 117 nmol/L, respectively) in ET-1-treated than in control NRVMs (4.2 +/- 1.3 and 346 +/- 85, respectively, P < 0.05), effects that were cancelled by NHE-1 inhibition with HOE 642. The rise in [Ca(2+)](i) induced by extracellular Na(+) removal (NCX(rev)) was higher in ET-1-treated than in control NRVMs and the effect was prevented by co-treatment with HOE 642 or KB-R7943 (NCX(rev) inhibitor). The ET-1-induced increase in cell area, ANF mRNA expression and (3)H-phenylalanine incorporation in ET-1-treated NRVM were decreased by NHE-1 or NCX(rev) inhibition. Our results provide the first evidence that NCX(rev) is, secondarily to NHE-1 activation, involved in ET-1-induced hypertrophy in NRVMs.

Involvement of AE3 isoform of Na(+)-independent Cl(-)/HCO(3)(-) exchanger in myocardial pH(i) recovery from intracellular alkalization.

Myocardial pH(i) recovery from intracellular alkalization results in part from the acid load (-J(H+)) carried by Cl(-)/HCO(3)(-) anion-exchangers (AE). Three AE isoforms, AE1, AE2 and AE3, have been identified in cardiac membranes, but the function of each isoform on pH(i) homeostasis is still under investigation. This work explored, by means of specific antibodies, the role of AE3 isoform in myocardial pH(i) regulation. We developed rabbit polyclonal antibodies against the extracellular "loops": one connecting the fifth to sixth and the other one the seventh to eighth transmembrane domains (loops 3 and 4, respectively) of AE3, and their effect on pH(i) regulation was studied in rat papillary muscles. The anti-AE3 loop 3 antibody decreased -J(H+) in response to myocardial alkalization (from a mean control value of 1.06+/-0.26 to 0.32+/-0.13 mmol/L/min, n=7, P<0.05) without affecting the baseline pH(i) (7.22+/-0.03 vs. 7.21+/-0.04). The anti-AE3 loop 4 antibody did not modify either pH(i) recovery or baseline pH(i). Under control conditions, endothelin-1 (ET-1) increased -J(H+) in response to myocardial alkalization from 1.30+/-0.18 to 2.01+/-0.33 mmol/L /min (n=5, P<0.05). This effect of ET-1 on -J(H+) was abolished by anti-AE3 loop 3 antibody. In addition, the MgATP-induced stimulation of AE activity was reduced by the anti-AE3 loop 3 antibody. These data support the key role of the AE3 isoform in myocardial pH(i) recovery from alkaline loads and also in the stimulatory effect of ET-1 on AE activity. To a lesser extent, it may also contribute to the effect of MgATP on pH(i).

Involvement of the Na+-independent Cl-/HCO3- exchange (AE) isoform in the compensation of myocardial Na+/H+ isoform 1 hyperactivity in spontaneously hypertensive rats.

Enhanced activity of Na+/H+ isoform 1 (NHE-1) and the Na+-independent Cl-/HCO3- exchange (AE) is a feature of the hypertrophied myocardium in spontaneously hypertensive rats (SHR). The present study explored the possibility that sustained intracellular acidosis due to increased myocardial acid loading through AE causes NHE-1 enhancement. To this aim, SHR were treated for 2 weeks with a rabbit polyclonal antibody against an AE3 isoform that was recently developed and proven to have inhibitory effects on myocardial AE activity. We then compared the AE activity in the left ventricle papillary muscles isolated from untreated SHR with antiAE3-treated SHR; AE activity was measured in terms of the rate of intracellular pH recovery after an intracellular alkali load was introduced. AE activity was diminished by approximately 70% in SHR treated with the antiAE3 antibody, suggesting that the AE3 isoform is a major carrier of acid-equivalent influx in the hypertrophied myocardium. However, the antibody treatment failed to normalize NHE-1 activity that remained elevated in the myocardium of normotensive rats. The data therefore rule out the possibility that NHE-1 hyperactivity in hypertensive myocardium was due to sustained intracellular acidosis induced by increased AE activity that characterizes SHR myocardial tissue.

Endothelin 1 versus endothelin 3 in the development of the slow force response to myocardial stretch.

BACKGROUND: Myocardial stretch promotes an increase in developed force (DF) in two phases: a rapid initial phase, and a slowly developing second phase called the slow force response (SFR) to myocardial stretch. The SFR results from an autocrine/paracrine mechanism of angiotensin II and endothelin (ET) release that is triggered by the stretch. OBJECTIVE: To explore whether exogenous ET-1 and/or ET-3 could mimic the SFR. METHODS: Experiments were performed in isometrically contracting (0.2 Hz) rat papillary muscles at 30 degrees C. DF was measured either after stretch or after the addition of ET-1 or ET-3 (in doses that increase contractility to a similar magnitude as does the SFR), with or without the selective ETA receptor antagonist BQ123 (300 nmol/L). RESULTS AND CONCLUSIONS: After 15 min, the SFR was 17.6+/-1.4% greater than the initial rapid phase (n=4; P<0.05) and was abolished by BQ123. ET-1 (5.0 nmol/L) increased DF by 25.9+/-1.7% (n=4; P<0.05) after 30 min, an effect that was not altered by BQ123 (22.6+/-3.9%; n=5). ET-3 (5.0 nmol/L) increased DF by 23.8+/-3.2% (n=5; P<0.05), an effect that was suppressed by BQ123 (-5.4+/-1.9%; n=5; P<0.05). Given that BQ123 eliminated the SFR and the inotropic response to ET-3 but not to ET-1, the results suggest that the SFR that follows myocardial stretch is due to the endogenous release of ET-3 acting in an autocrine/paracrine fashion.

Endothelin isoforms and the response to myocardial stretch.

Myocardial stretch elicits a biphasic increase in developed force with a first rapid force response and a second slow force response (SFR). The rapid phase is due to an increase in myofilament Ca(2+) responsiveness; the SFR, analyzed here, is ascribed to a progressive increase in Ca(2+) transients. Experiments were performed in cat papillary muscles to further elucidate the signaling pathway underlying the SFR. Although the SFR was diminished by BQ-123, a similar endothelin (ET)-1-induced increase in force was not affected: 23 +/- 2 vs. 23 +/- 3% (not significant). Instead, BQ-123 suppressed the contractile effects of ET-2 or ET-3 (21 +/- 2 and 25 +/- 3% vs. -1 +/- 1 and -7 +/- 3% respectively, P < 0.05), suggesting that ET-2 or ET-3, but not ET-1, was involved in the SFR. Each isoform activated the Na(+)/H(+) exchanger (NHE-1), increasing intracellular Na(+) concentration by 2.0 +/- 0.1, 2.3 +/- 0.1, and 2.1 +/- 0.4 mmol/l for ET-1, ET-2, and ET-3, respectively (P < 0.05). The NHE-1 inhibitor HOE-642 prevented the increases in force and intracellular Na(+) concentration induced by all the ET isoforms, but only ET-2 and ET-3 effects were sensitive to BQ-123. Real-time RT-PCR measurements of prepro-ET-1, -ET-2, and -ET-3 were performed before and 5, 15, and 30 min after stretch. No changes in ET-1 or ET-2, but an increase of approximately 60% in ET-3, mRNA after 15 min of stretch were detected. Stretch-induced ET-3 mRNA upregulation and its mechanical counterpart were suppressed by AT(1) receptor blockade with losartan. These data suggest a role for AT(1)-mediated ET-3 released in the early activation of NHE-1 that follows myocardial stretch.

A low dose of angiotensin II increases inotropism through activation of reverse Na(+)/Ca(2+) exchange by endothelin release.

OBJECTIVE: This work was aimed to prove that release/formation of endogenous endothelin acting in an autocrine/paracrine fashion contributes to the increase in contractility promoted by a low dose of angiotensin II. METHODS: Isolated cat papillary muscles were used for force, pH(i), [Na(+)](i) and [Ca(2+)](i) measurements and isolated cat myocytes for patch-clamp experiments. RESULTS: In papillary muscles, 1.0 nmol/l angiotensin II increased force by 23+/-2% (n=4, P<0.05), [Na(+)](i) by 2.2+/-0.2 mmol/l (n=4, P<0.05), and peak (but not diastolic) Ca(2+) from 0.674+/-0.11 to 0.768+/-0.13 micromol/l (n=4, P<0.05), without affecting pH(i). Force and [Na(+)](i) increase were abolished by inhibition of the Na(+)/H(+) exchanger (NHE) with the inhibitor HOE642, blockade of endothelin receptors with the nonselective antagonist TAK044 and by inhibition of the endothelin-converting enzyme with phosphoramidon. Force but not [Na(+)](i) increase was abolished by inhibition of reverse Na(+)/Ca(2+) exchange (NCX) with the inhibitor KB-R7943. Similar increase in force (21+/-2%, n=4, P<0.05) and in [Na(+)](i) (2.4+/-0.4 mmol/l, n=4, P<0.05) that were also suppressed by TAK044 and HOE642 were induced by exogenous 5.0 nmol/l endothelin-1. KB-R7943 reverted the endothelin-1 effect on force but not on [Na(+)](i). In isolated myocytes, exogenous endothelin-1 dose-dependently increased the NCX current and shifted the NCX reversal potential (E(NCX)) to a more negative value (DeltaE(NCX): -10+/-3 and -17+/-5 mV, with 1 and 10 nmol/l endothelin-1, respectively, n=12). The latter effect was prevented by HOE642. CONCLUSION: Taken together, the results indicate that a low dose of angiotensin II induces release of endothelin, which, in autocrine/paracrine fashion activates the Na(+)/H(+) exchanger, increases [Na(+)](i) and changes E(NCX), promoting the influx of Ca(2+) that leads to a positive inotropic effect (PIE).

Influence of Na+-independent Cl--HCO3- exchange on the slow force response to myocardial stretch.

Previous work demonstrated that the slow force response (SFR) to stretch is due to the increase in calcium transients (Ca2+T) produced by an autocrine-paracrine mechanism of locally produced angiotensin II/endothelin activating Na+-H+ exchange. Although a rise in pHi is presumed to follow stretch, it was observed only in the absence of extracellular bicarbonate, suggesting pHi compensation through the Na+-independent Cl--HCO3- exchange (AE) mechanism. Because available AE inhibitors do not distinguish between different bicarbonate-dependent mechanisms or even between AE isoforms, we developed a functional inhibitory antibody against both the AE3c and AE3fl isoforms (anti-AE3Loop III) that was used to explore if pHi would rise in stretched cat papillary muscles superfused with bicarbonate after AE3 inhibition. In addition, the influence of this potential increase in pHi on the SFR was analyzed. In this study, we present evidence that cancellation of AE3 isoforms activity (either by superfusion with bicarbonate-free buffer or with anti-AE3Loop III) results in pHi increase after stretch and the magnitude of the SFR was larger than when AE was operative, despite of similar increases in [Na+]i and Ca2+T under both conditions. Inhibition of reverse mode Na+-Ca2+ exchange reduced the SFR to the half when the AE was inactive and totally suppressed it when AE3 was active. The difference in the SFR magnitude and response to inhibition of reverse mode Na+-Ca2+ exchange can be ascribed to a pHi-induced increase in myofilament Ca2+ responsiveness.

Regression of isoproterenol-induced cardiac hypertrophy by Na+/H+ exchanger inhibition.

Cardiac hypertrophy is often associated with an increased sympathetic drive, and both in vitro and in vivo studies have demonstrated the development of cardiomyocyte hypertrophy in response to either alpha- or beta-adrenergic stimulation. Because an association between the Na+/H+ exchanger and cellular growth has been proposed, this study aimed to analyze the possible role of the antiporter in isoproterenol-induced cardiac hypertrophy. Isoproterenol alone (5 mg/kg IP once daily) or combined with a selective inhibitor of the Na+/H+ exchanger activity (3 mg x kg(-1) x d(-1) BIIB723) was given to male Wistar rats for 30 days. Sex- and age-matched rats that received 0.9% saline IP daily served as controls. Echocardiographic follow-up showed a 33% increase in left ventricular mass in the isoproterenol-treated group, whereas it did not increase in the isoproterenol+BIIB723-treated group. Heart weight-to-body weight ratio at necropsy was 2.44+/-0.11 in controls and increased to 3.35+/-0.10 (P<0.05) with isoproterenol, an effect that was markedly attenuated by BIIB723 (2.82+/-0.07). Intense cardiomyocyte enlargement and severe subendocardial fibrosis were found in isoproterenol-treated rats, and both effects were attenuated by BIIB723. Myocardial Na+/H+ exchanger activity and protein expression significantly increased in isoproterenol-treated rats compared with the control group (1.45+/-0.11 vs 0.91+/-0.05 arbitrary units, P<0.05). This effect was significantly reduced by BIIB723 (1.17+/-0.02, P<0.05). In conclusion, our results show that Na+/H+ exchanger inhibition prevented the development of isoproterenol-induced hypertrophy and fibrosis, providing strong evidence in favor of a key role played by the antiporter in this model of cardiac hypertrophy.

Stretch-elicited Na+/H+ exchanger activation: the autocrine/paracrine loop and its mechanical counterpart.

The stretch of the cardiac muscle is immediately followed by an increase in the contraction strength after which occurs a slow force increase (SFR) that takes several minutes to fully develop. The SFR was detected in a wide variety of experimental preparations including isolated myocytes, papillary muscles and/or trabeculae, left ventricle strips of failing human myocardium, in vitro isovolumic and in vivo volume-loaded hearts. It was established that the initial increase in force is due to an increase in myofilament Ca2+ responsiveness, whereas the SFR results from an increase in the Ca2+ transient. However, the mechanism(s) for this increase in the Ca2+ transient has remained undefined until the proposal of Na+/H+ exchanger (NHE) activation by stretch. Studies in multicellular cardiac muscle preparations from cat, rabbit, rat and failing human heart have shown evidence that the stretch induces a rise in intracellular Na+ ([Na+]i) through NHE activation, which subsequently leads to an increase in Ca2+ transient via reverse-mode Na+/Ca2+ (NCX) exchange. These experimental data agree with a theoretical ionic model of cardiomyocytes that predicted an increased Na+ influx and a concurrent increase in Ca2+ entry through NCX as the cause of the SFR to muscle stretch. However, there are aspects that await definitive demonstration, and perhaps subjected to species-related differences like the possibility of an autocrine/paracrine loop involving angiotensin II and endothelin as the underlying mechanism for stretch-induced NHE activation leading to the rise in [Na+]i and reverse-mode NCX.

Regression of hypertensive myocardial fibrosis by Na(+)/H(+) exchange inhibition.

We have recently reported that the inhibition of the Na(+)/H(+) exchanger (NHE) during 1 month in spontaneously hypertensive rats (SHR) is followed by regression of cardiomyocyte hypertrophy but not of myocardial fibrosis. The aim of this study was to evaluate whether a treatment of longer duration could reduce myocardial fibrosis and stiffness. SHR received 3.0 mg/kg per day of the specific NHE-1 inhibitor cariporide; the effect on cardiomyocyte cross-sectional area, myocardial collagen volume fraction, collagen synthesis, and myocardial stiffness (length-tension relation in left papillary muscles) was evaluated at several time points (after 1, 2, or 3 months). A slight decrease of approximately 5 mm Hg in systolic blood pressure was observed after 1 month of treatment with no further changes. After 2 and 3 months of treatment, the size of cardiomyocytes remained within normal values and myocardial fibrosis progressively decreased to normal level. Accordingly, myocardial stiffness and the serum levels of the carboxyterminal propeptide of procollagen type I, a marker of collagen type I synthesis, were normalized after 3 months. Left ventricular weight decreased from 910+/-43 (in untreated SHR) to 781+/-21 mg (treated SHR) after 3 months of treatment. No difference in body weight between treated and untreated SHR was observed after this period of treatment. The present data allow us to conclude that in the SHR the administration of an NHE-1 inhibitor for 2 or 3 months leads to the normalization of collagen type I synthesis, myocardial collagen volume fraction, and stiffness.

Upregulation of myocardial Na+/H+ exchanger induced by chronic treatment with a selective inhibitor.

Rats exposed to prolonged administration of the NHE-1 inhibitor cariporide showed enhanced activity of the exchanger in cardiac tissue, as assessed by the rise in the steady-state pHi value in the absence of bicarbonate (7.15+/-0.01 in control vs 7.49+/-0.06 and 7.41+/-0.05 in cariporide-treated for 1 or 2 months, respectively, P<0.05). In the presence of bicarbonate, the change in pHi was blunted due to a compensatory activation of acid loading pHi regulatory mechanisms. The enhancement of NHE activity disappeared after 1 week of the inhibitor withdrawal. The kinetic analysis of H+ fluxes after an acid load revealed an increased net H+ efflux (JH+) at any given pHi value and an alkaline shift of the apparent "set-point" of the exchanger (from 7.11+/-0.02 to 7.38+/-0.04,P <0.05) in treated rats. In the presence of the PKC inhibitor chelerythrine, the "set-point" of the exchanger was normalized in the cariporide-treated rats while JH+ at acidic pHi values persisted elevated. Cardiac NHE-1 mRNA levels and protein expression were increased in cariporide-treated rats. In addition to the increased protein expression after the treatment, the normalization of the augmented "set-point" by chelerythrine suggests an increased turnover rate of the units through a PKC dependent pathway. These data demonstrate that long-term treatment with the NHE-1 inhibitor cariporide enhances the antiporter activity in cardiac tissue through an increase of the number and turnover of functional units. This finding deserves further experimental and clinical evaluations to consider whether it would be advisable a gradual withdrawal of prolonged NHE inhibition to avoid an enhanced response when the exchanger is stimulated.

Regression of cardiomyocyte hypertrophy in SHR following chronic inhibition of the Na(+)/H(+) exchanger.

OBJECTIVE: Experiments were performed to examine the effect of chronic inhibition of the Na(+)/H(+) exchanger isoform-1 (NHE-1) on cardiac hypertrophy of spontaneously hypertensive rats (SHR). METHODS: SHR were orally treated during 1 month with two different doses (0.3 and 3.0 mg/kg/day) of the NHE-1 inhibitor, cariporide, or nifedipine (10.0 mg/kg/day). RESULTS: The two doses of cariporide did not differ in their effects after 1 month of treatment, since both induced a slight decrease in systolic blood pressure (SBP) of approximately 6 mmHg and regression of the heart weight to body weight ratio (mg/g) from 3.28+/-0.05 to 3.04+/-0.05 (0.3 mg) and 2.99+/-0.10 (3.0 mg, P<0.05). Nifedipine, given for the same period, produced similar reduction in the hypertrophy index (3.03+/-0.05), but with a much greater decrease in arterial pressure (35.6+/-7.4 mmHg). Chronic treatment with cariporide induced a complete regression of the augmented cross sectional area of left ventricular myocytes without significant changes in collagen content, serum procollagen 1 propeptide levels or myocardial distensibility. CONCLUSIONS: NHE inhibition represents a novel approach to induce regression of pathological hypertrophy of the heart. The finding can be rationalized mechanistically by previous in vitro studies suggesting a role of the NHE in the development of myocardial hypertrophy.

Efecto paradojico del calcio en presencia de verapanil / Paradoxical effect of calcium in the presence of verapamil

Se estudió la capacidad de la administración de calcio para revertir los efectos cardiovasculares del verapamilo en ratas anestesiadas y en aurículas aisladas latiendo espontaneamente. El verapamilo produjo descenso de la presión arterial media (Pa) de 31,0 ñ 5,6 mmHg, prolongación de la duración del ciclo cardíaco (RR) de 84,5 + 15,7 mseg y del intervalo PR (PR) de 22,2 + 5,5 mseg. La administración de calcio provocó el retorno de la Pa a su valor basal pero paradójicamente intensificó el efecto bradicardizante del verapamil y fue incapaz para revertir el alargamiento del PR. En animales previamente bloqueados con atropina en calcio revirtió tanto el efecto hipotensor como el enlentecimiento de la frecuencia cardíaca y de la conducción AV producidos por el verapamil. En aurículas aisladas latiendo espontaneamente el aumento de Ca2+ extracelular de 1,0 a 6,0 mmoles/1 aumenta la frecuencia espontanea en 41,5 ñ 13 lat/min. El mismo aumento de Ca2+ en presencia de verapamil disminuye la frecuencia en 50 ñ 15 lat/min en aurículas sin bloqueo, mientras que en las preparaciones con bloqueo atropínico revierte el efecto cronotrópico negativo del verapamil. Los resultados obtenidos permiten concluír que la administración de calcio es eficaz para revertir el efecto hipotensor del verapamil pero paradójicamente aumenta su efecto bradicardizante. El calcio tampoco revierte el efecto enlentecedor de la conducción AV provocado por verapamil. Estos efectos paradójicos del calcio pueden ser prevenidos mediante el bloqueo con atropina

Efecto paradojico del calcio en presencia de verapanil / Paradoxical effect of calcium in the presence of verapamil

Se estudió la capacidad de la administración de calcio para revertir los efectos cardiovasculares del verapamilo en ratas anestesiadas y en aurículas aisladas latiendo espontaneamente. El verapamilo produjo descenso de la presión arterial media (Pa) de 31,0 ñ 5,6 mmHg, prolongación de la duración del ciclo cardíaco (RR) de 84,5 + 15,7 mseg y del intervalo PR (PR) de 22,2 + 5,5 mseg. La administración de calcio provocó el retorno de la Pa a su valor basal pero paradójicamente intensificó el efecto bradicardizante del verapamil y fue incapaz para revertir el alargamiento del PR. En animales previamente bloqueados con atropina en calcio revirtió tanto el efecto hipotensor como el enlentecimiento de la frecuencia cardíaca y de la conducción AV producidos por el verapamil. En aurículas aisladas latiendo espontaneamente el aumento de Ca2+ extracelular de 1,0 a 6,0 mmoles/1 aumenta la frecuencia espontanea en 41,5 ñ 13 lat/min. El mismo aumento de Ca2+ en presencia de verapamil disminuye la frecuencia en 50 ñ 15 lat/min en aurículas sin bloqueo, mientras que en las preparaciones con bloqueo atropínico revierte el efecto cronotrópico negativo del verapamil. Los resultados obtenidos permiten concluír que la administración de calcio es eficaz para revertir el efecto hipotensor del verapamil pero paradójicamente aumenta su efecto bradicardizante. El calcio tampoco revierte el efecto enlentecedor de la conducción AV provocado por verapamil. Estos efectos paradójicos del calcio pueden ser prevenidos mediante el bloqueo con atropina (AU)