Cantharidin and sodium fluoride attenuate the negative inotropic effects of carbachol in the isolated human atrium

Figures

• 

  Fig. 1. Scheme: The cardiac effects of carbachol: The Ca2+ for force generation is derived in part from “trigger” Ca2+ entering the cardiomyocyte through the L-type Ca2+ channel (LTCC) and in larger part by subsequent release of Ca2+ from the sarcoplasmic reticulum (SR) via ryanodine receptors (RYR). Cardiac relaxation is brought about by phosphorylation of phospholamban (PLB) and of inhibitory subunit of troponin (TnI). Phosphorylated PLB de-inhibits the activity of the SR—Ca2+−ATPase (SERCA) and Ca2+is more rapidly removed from the cytosol into the SR, supporting relaxation. The activities of protein phosphatases called PP1 and PP2A are inhibited by cantharidin (see structural formula as inset, a naturally occurring molecule) and by sodium fluoride. Carbachol is an agonist at cardiac M2-muscarinic receptors (M2-R). Carbachol may inhibit the enzymatic activity of adenylyl cyclases (AC) via a pertussis toxin sensitive inhibitory guanosine-triphosphate binding-protein (Gi). Carbachol opens potassium channels (K+), may close the L-type Ca2+ channel (LTCC) and may activate PP1 and/or PP2A

• 

  Fig. 2. Cantharidin attenuates the negative inotropic effect of carbachol on force of contraction in mouse left atrium. Original recording of the buffer, Tyrode’s solution, alone (A), in the presence of 100 µM cantharidin alone (B), after application of carbachol (10, 30, 100, 300 nM, 1 µM) alone (C) or with cantharidin (100 µM) first applied (D). Concentrations of carbachol applied before negative inotropic effect was reversed by subsequent positive inotropic effect to carbachol and after application of carbachol (1, 3, 10 µM) alone (E), or if applying first cantharidin (100 µM) and, when plateau was reached, thereafter carbachol for longer periods of time such that the positive inotropic effects of carbachol that followed the negative inotropic effect of carbachol reached plateau (1, 3, 10 µM, F). The ordinates give force of contraction in milli Newton (mN) over time in minutes (min, abscissae) in electrically stimulated wild type mouse left atrium. The ratio of force at label b divided by force at label a marks the negative inotropic effect of carbachol. G Summarized data indicating the positive inotropic effect of 100 µM cantharidin over time. Ordinate and abscissa give force of…

• 

  Fig. 3. Cantharidin attenuates the negative inotropic effect of carbachol on contractile parameters in mouse left atrium. A Negative inotropic effect of carbachol (10, 30, 100, 300 nM, 1 µM) alone or after previous application of cantharidin (100 µM) in isolated electrically driven mouse left atrial preparations. In the pipetting of the concentration response curves, the subsequent concentration of carbachol was applied just before the negative inotropic effect was reversed to a positive inotropic effect. Ordinate and abscissa give force of contraction in % of pre-drug value or applied concentration of carbachol, respectively. * and + first significant difference versus zero time point (Ctr) or carbachol in presence of cantharidin, respectively. Numbers in brackets indicate the number of experiments. B Bar diagram of the negative inotropic effect of carbachol (1 µM) alone or in the additional presence of cantharidin (100 µM) in isolated electrically driven mouse left atrial preparations. The effect of 100 µM cantharidin or time matched control values on force of contraction were set as 100 percent (left two columns). Negative inotropic effects of carbachol in the absence (-) or in the…

• 

  Fig. 4. Cantharidin attenuates the negative inotropic effect of carbachol on force of contraction in human right atrium. Original recordings of the buffer, Tyrode’s solution, alone (A), in the presence of 100 µM cantharidin alone (B), after application of carbachol (10, 30, 100, 300 nM, 1 µM) alone (C) or with cantharidin (100 µM) first applied (D) followed by a carbachol-induced concentration response curve. Concentrations of carbachol were applied before the negative inotropic effect was reversed and after application of carbachol (1, 3, 10 µM) alone (E), or if applying first cantharidin (100 µM) and, when plateau was reached, thereafter carbachol (1, 3, 10 µM, F). The ordinates give force of contraction in milli Newton (mN, ordinates) over time in minutes (min, abscissae) in electrically stimulated mouse left atrium. The ratio of force at label b divided by force at label a marks the negative inotropic effect of carbachol. G Summarized data indicating the positive inotropic effect of 100 µM cantharidin over time. Data are given in mean values in percentage ± SEM. * and + first significant difference versus zero time point or Tyrode´s solution, respectively. Number of experiments n =…

• 

  Fig. 5. Cantharidin attenuates the contractile effects of carbachol on force of contraction in human right atrium. A Line diagram of the negative inotropic effect of carbachol (10, 30, 100, 300 nM, 1 µM) alone or with first applied cantharidin (100 µM) in isolated electrically driven human right atrial preparations. Subsequent concentrations of carbachol were applied to the organ bath before the negative inotropic effect was reversed. Ordinate and abscissa give force of contraction in % of pre-drug value or applied concentration of carbachol, respectively. * and + first significant difference versus zero time point (Ctr) or carbachol in presence of cantharidin, respectively. Numbers (n) indicate number of experiments. B Bar diagram of the negative inotropic effect of carbachol (1 µM) alone or in the additional presence of cantharidin (100 µM) in isolated electrically driven human right atrial preparations. The effect of 100 µM cantharidin or time matched control values on force of contraction were set as 100 percent (left two columns). Negative inotropic effects of carbachol in the absence (-) or in the presence (+) of 100 µM cantharidin are plotted. * p < 0.05 vs. zero time point (Ctr).…

• 

  Fig. 6. Sodium fluoride attenuates the negative inotropic effect of carbachol on force of contraction in isolated electrically driven mouse left atrial preparations. Original recording of the buffer, Tyrode’s solution, alone (A), in the presence of 3 mM sodium fluoride alone (B), after application of carbachol (10, 30, 100, 300 nM, 1 µM) alone (C) or with sodium fluoride (3 mM) first applied (D). Concentrations of carbachol were applied before negative inotropic effect was reversed at longer incubation times or after application of carbachol (1, 3, 10 µM) alone for a prolonged time such that a positive inotropic effect could appear, or with 3 mM sodium fluoride first applied. The ordinates give force of contraction in milli Newton (mN, ordinates) over time in minutes (min, abscissae) in electrically stimulated mouse left atrium. The ratio of force at label b divided by force at label a marks the negative inotropic effect of carbachol. G Summarized data indicating the positive inotropic effect of 3 mM sodium fluoride over time. Data are given in mean values in percentage ± SEM. * and # first significant difference versus zero time point or Tyrode´s solution, respectively. Number of…

• 

  Fig. 7. Sodium fluoride attenuates the effect of carbachol on contractile parameters in isolated electrically driven mouse left atrial preparations. A Line diagram of the negative inotropic effect of carbachol (10, 30, 100, 300 nM, 1 µM) alone or with first applied sodium fluoride (3 mM) in isolated electrically driven mouse left atrial preparations. Concentrations of carbachol applied before negative inotropic effect was reversed. Ordinate and abscissa give force of contraction in % of pre-drug value or applied concentration of carbachol, respectively. * and # first significant difference versus zero time point (Ctr) or carbachol in presence of sodium fluoride, respectively. Numbers (n) indicate number of experiments. B Bar diagram of the negative inotropic effect of carbachol (1 µM) alone or in the additional presence of sodium fluoride (3 mM) in isolated electrically driven mouse left atrial preparations. The effect of 3 mM sodium fluoride or time matched control values on force of contraction were set as 100 percent (left two columns). Negative inotropic effects of carbachol in the absence (-) or in the presence (+) of 3 mM sodium fluoride are plotted. * p < 0.05 vs. zero time point…

• 

  Fig. 8. Sodium fluoride attenuates the negative inotropic effect of carbachol on force of contraction in human atrium. Original recording of the buffer, Tyrode´s solution, alone (A), in the presence of 3 mM sodium fluoride alone (B), after application of carbachol (10, 30, 100, 300 nM, 1 µM) alone (C) or with sodium fluoride (3 mM) first applied (D). Concentrations of carbachol applied before negative inotropic effect was reversed and after application of carbachol (1, 3, 10 µM) alone (E), or with first sodium fluoride and, when plateau was reached, thereafter carbachol (1, 3, 10 µM, F) applied. The ordinates give force of contraction in milli Newton (mN, ordinates) over time in minutes (min, abscissae) in electrically stimulated mouse left atrium. The ratio of force at label b divided by force at label a marks the negative inotropic effect of carbachol. G Summarized data indicating the positive inotropic effect of sodium fluoride over time. Data are given in mean values in percentage ± SEM. * and # first significant difference versus zero time point or Tyrode´s solution, respectively. Number of experiments n = 3. H Summarized data indicating the negative and positive inotropic effect of…

• 

  Fig. 9. Sodium fluoride attenuates the effect of carbachol on contractile parameters in human atrium. A Line diagram of the negative inotropic effect of carbachol (10, 30, 100, 300 nM, 1 µM) alone or with first applied sodium fluoride (3 mM) in isolated electrically driven human right atrial preparations. Concentrations of carbachol applied before negative inotropic effect was reversed. Ordinate and abscissa give force of contraction in % of pre-drug value or applied concentration of carbachol, respectively. * and # first significant difference versus zero time point (Ctr) or carbachol in presence of sodium fluoride, respectively. Numbers (n) indicate number of experiments. B Bar diagram of the negative inotropic effect of carbachol (1 µM) alone or in the additional presence of sodium fluoride (3 mM) in isolated electrically driven human right atrial preparations. The effect of 3 mM sodium fluoride or time matched control values on force of contraction were set as 100 percent (left two columns). Negative inotropic effects of carbachol in the absence (-) or in the presence (+) of 3 mM sodium fluoride are plotted. * p < 0.05 vs. zero time point (Ctr). # p < 0.05 vs. without sodium…