Inhibition of protein phosphatases attenuates A1-adenosine receptor-stimulation induced negative inotropic effects of cAMP-increasing agents in the isolated human atrium

Abstract

N⁶-(R)-Phenylisopropyladenosine (R-PIA), an agonist at A₁-adenosine receptors, alone exerts negative inotropic effects (NIE) in the human atrium. This NIE is augmented in the presence of cAMP-increasing agonists like phosphodiesterase inhibitors (cilostamide, rolipram) or a direct activator of adenylyl cyclase (forskolin). Cantharidin inhibits protein phosphatases 1 and 2A (PP1, PP2A). We hypothesized that cantharidin would attenuate this NIE of R-PIA in the presence of cilostamide or forskolin. During open heart surgery (patients were suffering from severe coronary heart disease), isolated human atrial preparations (HAP) were obtained. These HAP were mounted in organ baths and electrically stimulated (1 Hz). For comparison, we studied isolated electrically stimulated (1 Hz) left atrial preparations (LA) from wild type mice. We noted that R-PIA exerted negative inotropic effects in LA and HAP in the presence of cilostamide or rolipram and forskolin that were attenuated by cantharidin. We hypothesize that R-PIA in the presence of phosphodiesterase inhibitors or forskolin stimulates PP in the human atrium. Hence, R-PIA acts, at least in part, by stimulating PP in HAP.

Keywords: Adenosine receptor; CAMP; Cantharidin; Human atrium; Phosphatases.

Fig. 1

Phosphodiesterase (PDE) isoenzyme inhibition (by rolipram or cilostamide) and forskolin increase cAMP levels in the human atrium are depicted. This cAMP activates cAMP-dependent protein kinases (PKA). PKA then phosphorylates regulatory proteins in the human atrium. Cardiac relaxation is brought about by phosphorylation of phospholamban (PLB). Cardiac contraction is in part mediated by ryanodine receptors (RyR). The activities of protein phosphatases (PP) PP1 and PP2A are inhibited by cantharidin. R-PIA via stimulation of A₁-adenosine receptors may inhibit the enzymatic activity of adenylyl cyclase (AC) via a pertussis-toxin-sensitive G-protein (G_i) and may open potassium channels (PC) in the sarcolemma or may close L-type calcium ion (Ca²⁺) channels (LTCC) and may directly or indirectly activate PP

Fig. 2

Effects of R-PIA in the presence of rolipram on contractile parameters in left and right mouse atrium in the presence or absence of cantharidin. A Original recording of the effect of R-PIA in the presence of rolipram on the force of contraction in milli Newton (mN, Ordinate) over time in minutes (min, Abscissa). B Original recording of the effect of R-PIA in the presence of rolipram on the force of contraction in the additional presence of 30 µM cantharidin (CANT) in milli Newton (mN, Ordinate) over time in minutes (min, Abscissa). C Diagram for the negative inotropic effect of R-PIA in the presence of rolipram alone or the additional presence of cantharidin (30 µM) in isolated electrically driven mouse left atrial preparations. Ordinates and abscissa give the force of contraction in % of pre-drug value or applied concentration of R-PIA, respectively. The asterisk (*) and plus sign ( +) indicate the first significant difference versus 30 µM cantharidin or time-matched control values (Ctr2) or R-PIA in the presence of cantharidin, respectively. Number (n) indicates the number of experiments. Ctr1 indicates pre-drug value. Ctr2 (100%) indicates plus/minus cantharidin. D Line diagram of the effects of R-PIA in the presence of rolipram or in the additional presence of cantharidin (30 µM) on rate of tension development in isolated electrically driven mouse left atrial preparations. Ordinate and abscissa give the rate of tension development (dF/dt_max) in % of pre-drug value or applied concentration of R-PIA, respectively. The asterisk (*) and plus sign ( +) indicate the first significant difference versus 30 µM cantharidin or time-matched control values (Ctr2) or R-PIA in the presence of cantharidin, respectively. Number (n) indicates the number of experiments. Ctr1 indicates pre-drug value. Ctr2 (100%) indicates plus/minus cantharidin. E Line diagram of the effects of R-PIA in the presence of rolipram or in the additional presence of cantharidin (30 µM) on rate of tension relaxation in isolated electrically driven mouse left atrial preparations. Ordinate and abscissa give the rate of tension relaxation (dF/dt_min) in % of pre-drug value or applied concentration of R-PIA, respectively. The asterisk (*) and plus sign ( +) indicate the first significant difference versus 30 µM cantharidin or time-matched control values (Ctr2) or R-PIA in the presence of cantharidin, respectively. Number (n) indicates the number of experiments. Ctr1 indicates pre-drug value. Ctr2 (100%) indicates plus/minus cantharidin

Fig. 3

Effects of R-PIA in the presence of forskolin on contractile parameters in left and right mouse atrium in the presence or absence of cantharidin. A Original recording of the effect of R-PIA (1 µM) in the presence of forskolin (1 µM) on the force of contraction in milli Newton (mN, Ordinate) over time in minutes (min, Abscissa). B Original recording of the effect of R-PIA (1 µM) in the presence of forskolin (1 µM) on the force of contraction in the additional presence of cantharidin (30 µM) in milli Newton (mN, Ordinate) over time in minutes (min, Abscissa). C Line diagram of the negative inotropic effect of R-PIA (1 µM) in the presence of forskolin (1 µM) or with first applied cantharidin (30 µM) in isolated electrically driven left atrial preparations. Ordinate and abscissa give the force of contraction in % of pre-drug value or applied concentration of R-PIA (1 µM), respectively. The asterisk (*) and number sign (#) indicate the first significant difference versus 30 µM cantharidin or time-matched control values (Ctr2) or R-PIA (1 µM) in the presence of cantharidin, respectively. Number (n) indicates the number of experiments. Ctr1 indicates pre-drug value. Ctr2 (100%) indicates plus/minus cantharidin. D Line diagram of the effects of R-PIA (1 µM) alone or in additional presence of cantharidin (30 µM) on the rate of tension development in isolated electrically driven mouse left atrial preparations. Ordinate and abscissa give the rate of tension development (dF/dt_max) in % of pre-drug value or applied concentration of R-PIA, respectively. The asterisk (*) and number sign (#) indicate the first significant difference versus 30 µM cantharidin or time-matched control values (Ctr2) or R-PIA in the presence of cantharidin, respectively. Number (n) indicates the number of experiments. Ctr1 indicates pre-drug value. Ctr2 (100%) indicates plus/minus cantharidin. E Line diagram of the effects of R-PIA (1 µM) in the presence of forskolin (1 µM) or in additional presence of cantharidin (30 µM) on rate of tension relaxation in isolated electrically driven human right atrial preparations. Ordinate and abscissa give the rate of tension relaxation (dF/dt_min) in % of pre-drug value or applied concentration of R-PIA, respectively. The asterisk (*) and number sign (#) indicate the first significant difference versus 30 µM cantharidin or time-matched control values (Ctr2) or R-PIA in the presence of cantharidin, respectively. Number (n) indicates the number of experiments. Ctr1 indicates pre-drug value. Ctr2 (100%) indicates plus/minus cantharidin

Fig. 4

Cantharidin attenuates the negative inotropic effect of R-PIA of cilostamide on contractile parameters in the human right atrium. A Original recording of the effect of R-PIA (1 µM) and 1 µM cilostamide on the force of contraction in milli Newton (mN, Ordinate) over time in minutes (min, Abscissa). B Original recording of the effect of R-PIA and 1 µM cilostamide on the force of contraction in the presence of 100 µM cantharidin (CANT) in milli Newton (mN, Ordinate) over time in minutes (min, Abscissa). C Line diagram of the negative inotropic effect of R-PIA and 1 µM cilostamide or after first applied cantharidin (100 µM) in isolated electrically driven mouse left atrial preparations. Ordinates and abscissa give the force of contraction in % of pre-drug value or applied concentration of R-PIA, respectively. The asterisk (*) and plus sign ( +) indicate the first significant difference versus 100 µM cantharidin or time-matched control values (Ctr2) or R-PIA in the presence of cantharidin, respectively. Number (n) indicates the number of experiments. Ctr1 indicates pre-drug value. Ctr2 (100%) indicates plus/minus cantharidin. D Diagram of the effects of R-PIA in the presence and 1 µM cilostamide or in additional presence of cantharidin (100 µM) on rate of tension development in isolated electrically driven mouse left atrial preparations. Ordinate and abscissa give the rate of tension development (dF/dt_max) in % of pre-drug value or applied concentration of R-PIA, respectively. The asterisk (*) and plus sign ( +) indicate the first significant difference versus 100 µM cantharidin or time-matched control values (Ctr2) or R-PIA in the presence of cantharidin, respectively. Number (n) indicates the number of experiments. Ctr1 indicates pre-drug value. Ctr2 (100%) indicates plus/minus cantharidin

Fig. 5

Cantharidin does not attenuate the negative inotropic effect of R-PIA in the presence of forskolin on contractile parameters in the human right atrium. A Original recording of the effect of R-PIA (1 µM) in the presence of forskolin (1 µM) on the force of contraction in milli Newton (mN, Ordinate) over time in minutes (min, Abscissa). B Original recording of the effect of R-PIA (1 µM) in the presence of forskolin (1 µM) on the force of contraction in the additional presence of cantharidin (100 µM) in milli Newton (mN, Ordinate) over time in minutes (min, Abscissa). C Line diagram of the negative inotropic effect of R-PIA (1 µM) in the presence of forskolin (1 µM) or with first applied cantharidin (100 µM) in isolated electrically driven human right atrial preparations. Ordinate and abscissa give the force of contraction in % of pre-drug value or applied concentration of R-PIA, respectively. The asterisk (*) and number sign (#) indicate the first significant difference versus 100 µM cantharidin or time-matched control values (Ctr2) or R-PIA in the presence of cantharidin, respectively. Number (n) indicates the number of experiments. Ctr1 indicates pre-drug value. Ctr2 (100%) indicates plus/minus cantharidin. D Line diagram of the effects of R-PIA (1 µM) in the presence of forskolin (1 µM) or in additional presence of cantharidin (100 µM) on rate of tension development in isolated electrically driven human right atrial preparations. Ordinate and abscissa give the rate of tension development (dF/dt_max) in % of pre-drug value or applied concentration of R-PIA, respectively. The asterisk (*) and number sign (#) indicate the first significant difference versus 100 µM cantharidin or time-matched control values (Ctr2) or R-PIA in the presence of cantharidin, respectively. Number (n) indicates the number of experiments. Ctr1 indicates pre-drug value. Ctr2 (100%) indicates plus/minus cantharidin. E Line diagram of the effects of R-PIA (1 µM) in the presence of forskolin (1 µM) or in additional presence of cantharidin (100 µM) on rate of tension relaxation in isolated electrically driven human right atrial preparations. Ordinate and abscissa give the rate of tension relaxation (dF/dt_min) in % of pre-drug value or applied concentration of R-PIA, respectively. The asterisk (*) and number sign (#) indicate the first significant difference versus 100 µM cantharidin or time-matched control values (Ctr2) or R-PIA in the presence of cantharidin, respectively. Number (n) indicates the number of experiments. Ctr1 indicates pre-drug value. Ctr2 (100%) indicates plus/minus cantharidin