Inotropic effects of diadenosine monophosphate (AP1A) in isolated human cardiac preparations.

Dependent on the number of phosphate residues, diadenosine polyphosphates (APnP) exert divergent inotropic effects in the human heart. We studied the inotropic effects of the smallest member of this family, diadenosine monophosphate (AP1A). Force of contraction was measured in an isometric setup in isolated electrically driven (0.5 Hz) preparations from human atria. AP1A exerted a concentration-dependent negative inotropic effect. The IC50 value was 20.2 microM and the IC50 value was 3.1 microM (n = 5-8). At 100 microM AP1A, force of contraction declined to 50% of the predrug value after 2.5 +/- 0.5 min of incubation (n = 8). AP1A antagonized the positive inotropic effect of the beta-adrenoceptor agonist isoprenaline (10 nM). For 100 microM AP1A, the time to 50% of the predrug force in the presence of isoprenaline amounted to 2.3 +/- 0.2 min (n = 5). The positive inotropic and lusitropic effects of isoprenaline were antagonized by AP1A. The direct (AP1A alone) and indirect (AP1A in the presence of isoprenaline) negative inotropic effects of AP1A were blocked by the A1-adenosine receptor antagonist 1,3-dipropyl-cyclopentyl-xanthine (DPCPX, 0.3 microM). The inotropic effect of AP1A was not blocked by adenosine deaminase. In conclusion, AP1A exerts indirect and direct negative inotropic effects in the human heart through A1-adenosine receptors. These effects might protect the heart against excessive beta-adrenergic stimulation.

On the contractile function of protein phosphatases in isolated human coronary arteries.

It is unknown whether protein phosphatases types 1 and 2A are present in and can regulate the tone of human vascular tissue. The expression and possible function of serine/threonine protein phosphatases (PP) type 1 (PP1) and type 2A (PP2A) were studied in isolated human coronary arteries. Catalytic subunits of PPI and PP2A were identified by means of phosphatase activity measurement in tissue homogenates, by separation of enriched extracts through affinity column chromatography, by immunoblotting with specific antibodies, by hybridization of mRNA with specific DNA probes and PCR of reverse transcribed mRNA. Based on these methods, the catalytic subunits of PP1(alpha,beta,gamma) and PP2A(alpha,beta) were identified. Appropriately, cantharidin, an inhibitor of PP1 and PP2A, increased basal tone of human isolated coronary artery rings with an EC50 of about 16 micromol/l by increasing the phosphorylation state of the regulatory light chains of myosin. In summary, PP1 and PP2A are expressed in human coronary arteries and they can alter vascular tone.

The protein phosphatase inhibitor cantharidin alters vascular endothelial cell permeability.

In this study, we characterized the effects of the protein phosphatases type 1 (PP 1) and type 2A (PP 2A) inhibitor cantharidin in endothelial cells. We identified catalytic subunits of PP 1alpha, PP 2Aalpha, and PP 2Abeta immunologically in bovine aortic endothelial cells. Moreover, we detected mRNAs coding for catalytic subunits of PP 1alpha, PP 1beta, and PP 2Aalpha by hybridization with specific DNA probes in total RNA from these cells. Okadaic acid and cantharidin inhibited the activities of catalytic subunits of PP 1 (okadaic acid, 0.01-1 microM; cantharidin, 1-100 microM) and PP 2A (okadaic acid, 0.1 nM to 1 microM; cantharidin, 0.1-100 microM) separated by column chromatography in a concentration-dependent manner. Moreover, cantharidin (1 microM to 1 mM) increased the phosphorylation state of endothelial proteins including the regulatory light chains of myosin without affecting cytosolic calcium concentrations. Cantharidin (5-100 microM) increased the permeability of cultured endothelial cells in a time- and concentration-dependent manner. We suggest that inhibition of PP 1 and PP 2A activities by cantharidin increases endothelial permeability by enhancing the phosphorylation state of endothelial regulatory proteins. Thus, cantharidin might be a useful tool to study the function of protein phosphatases in endothelial barrier function.

Contractility and inhibition of protein phosphatases by cantharidin.

1. Cantharidin is a natural defensive toxicant produced by blister beetles. 2. Cantharidin shares structural similarity with highly toxic commercial herbicides (e.g., endothall, endothall anhydride and endothall thioanhydride). 3. Cantharidin inhibits the activity of purified catalytic subunits of serine/threonine protein phosphatases (PP) type 1 and type 2A. 4. Cantharidin increases force of contraction in isolated myocardial and vascular preparations. 5. Cantharidin enhances the phosphorylation state of myocardial and vascular regulatory proteins. 6. Cantharidin is a valuable tool for studying the function of PP in regulatory phosphorylation-dephosphorylation events.

The mechanism of action of cantharidin in smooth muscle.

1. The aim of this study was to investigate the mechanism(s) of the vasoconstrictor effect of cantharidin in bovine preparations. 2. Catalytic subunits of protein phosphatase type 1 (PP 1) and type 2A (PP 2A) were immunologically identified in coronary arteries, isolated smooth muscle cells and ventricular myocardium. 3. The mRNAs coding for catalytic subunits of PP 1alpha, PP 1beta and PP 2Aalpha were identified by hybridization with specific cDNA-probes in total RNA from coronary arteries, isolated smooth muscle cells and ventricles. 4. The activities of catalytic subunits of PP 1 and PP 2A separated by column chromatography from coronary arteries, isolated smooth muscle cells and ventricles were inhibited by cantharidin in a concentration-dependent manner. 5. Cantharidin increased the phosphorylation state of smooth muscle proteins including the regulatory light chains of myosin in 32P-labelled intact smooth muscle cells in a concentration-dependent manner. 6. Cantharidin did not affect cytosolic calcium concentrations in aortic smooth muscle cells. 7. It is suggested that cantharidin contracts smooth muscle preparations by increasing the phosphorylation state of regulatory proteins due to inhibition of phosphatase activities. Thus, cantharidin might be a useful tool to study the function of phosphatases in smooth muscle.

The effect of the protein phosphatases inhibitor cantharidin on beta-adrenoceptor-mediated vasorelaxation.

1. Cantharidin, an inhibitor of protein phosphatase types 1 (PP1) and 2A (PP2A), increased basal tone of bovine isolated coronary artery rings (CARs) with and without endothelium in a time- and concentration-dependent manner with pEC50 values of about 5.1 and 5.2, respectively, for both preparations. 2. Beta-Adrenoceptor stimulation with isoprenaline (Iso; 0.03-100 microM) or inhibition of phosphodiesterase activity by 3-isobutyl-1-methylxanthine (IBMX; 10-1000 microM), respectively, relaxed CARs precontracted with KCl (75 mM). CARs with and without endothelium showed no difference in the relaxing response to Iso and IBMX, respectively. 3. Cantharidin (3 microM) attenuated vasorelaxation induced by Iso (0.03-100 microM) in CARs with and without endothelium in a time-dependent manner, whereas vasorelaxation induced by IBMX (10-1000 microM) was not attenuated by 3 microM cantharidin. 4. Cantharidin (3 microM) did not affect cyclic AMP content in bovine cultured vascular cells, i.e. coronary artery smooth muscle cells (BCs), aortic endothelial cells (BAECs) and aortic smooth muscle cells (BASMCs), either under basal conditions, after beta-adrenoceptor stimulation (Iso) or inhibition of phosphodiesterase activity (IBMX), respectively. 5. Cantharidin inhibited protein phosphatase activity in homogenates from bovine coronary artery rings with a pIC50 of about 6.0. In homogenates of bovine cultured vascular cells pIC50 values of cantharidin amounted to about 6.5 for BCs, 6.7 for BAECs and 6.7 for BASMCs, respectively. 6. It was concluded that cantharidin differently affects vasorelaxation due to stimulation of beta-adrenoceptors (Iso) or inhibition of phosphodiesterase activity (IBMX), respectively. The attenuation of beta-adrenoceptor-mediated vasorelaxation by phosphatase inhibition is not due to diminished adenosine 3':5'-cyclic monophosphate (cyclic AMP) generation but could be evidence for different subcellular compartments of cyclic AMP.

The effect of bradykinin and the bradykinin antagonist Hoe 140 on kinematic parameters of human spermatozoa.

Bradykinin (BK) has been suggested to be an active substance in the disputed therapeutic use of kallikrein to improve semen quality. The effects of exogenous BK and its antagonist Hoe 140, which acts on one of the bradykinin receptors (BK2), were examined in two groups of patients attending the fertility clinic: those with asthenozoospermia (group I) and normozoospermia (group II). Bradykinin (10nM-1 microM) added to washed human spermatozoa had no effect on most kinematic parameters and caused only a marginal increase (7%) in curvilinear velocity at 50 nM in group I patients; however, this increase was not suppressed by concomitant addition of the BK antagonist. The bradykinin antagonist itself had no effect on the percentage motility or kinematic motility parameters of washed human spermatozoa in either group of patients. The motility of spermatozoa in semen was also unaffected by the presence of the bradykinin antagonist. It is concluded that bradykinin does not act exogenously on washed spermatozoa nor endogenously on spermatozoa in semen to stimulate motility via BK2 receptors, regardless of the initial quality of the sperm motility.