Modulation of Xenopus laevis Ca-activated Cl currents by protein kinase C and protein phosphatases: implications for studies of anesthetic mechanisms.

Ca-activated Cl currents (I(Cl(Ca))) are used frequently as reporters in functional studies of anesthetic effects on G protein-coupled receptors using Xenopus laevis oocytes. However, because anesthetics affect protein kinase C (PKC), they could indirectly affect I(Cl(Ca)) if this current is regulated by phosphorylation. We therefore studied the effect of modulation of either PKC or protein phosphatases PP1alpha and PP2A on I(Cl(Ca)) stimulated either by lysophosphatidate (LPA) signaling or by microinjection of Ca.

Functional properties of the rat phosphatase 1alpha promoter.

The aim of this study was to investigate the functional properties of the promoter of the protein phosphatase 1alpha catalytic subunit. Luciferase plasmids with different fragments of the rat catalytic subunit of the protein phosphatase 1alpha promoter ranging from -3.7 kbp to -59 bp were transiently transfected into cells by the calcium-phosphate precipitation method.

Activation and inactivation of cAMP-response element-mediated gene transcription in cardiac myocytes.

Objective: Chronic beta-adrenergic stimulation of the cAMP-dependent signalling pathway is implicated in functionally relevant expressional changes in congestive heart failure. We studied activation and inactivation of the cardiac gene transcription mediated by the cAMP-response element (CRE) and the CRE-binding protein (CREB) as an important mechanism of a cAMP-dependent gene regulation.

Methods: We investigated the transcriptional activation by forskolin, an activator of the adenylyl cyclase, in chick embryonic cardiomyocytes transfected with a CRE-controlled luciferase construct in comparison to the phosphorylation and expression of CREB determined on immunoblots.

Enhanced protein phosphorylation in hypertensive hypertrophy.

Objective: Chronic pressure overload in spontaneously hypertensive rats (SHR) is accompanied by heart hypertrophy and signs of heart failure. Since there is growing evidence for a possible pathophysiological role of altered protein phosphorylation in heart hypertrophy and failure, we studied here cardiac regulatory phosphoproteins and the kinases and phosphatases which regulate their phosphorylation state.

Methods: The experiments were performed in ventricles of SHR (12-13 weeks old) and age-matched normotensive Wistar-Kyoto rats (WKY).

The inhibitory effect of bupivacaine on prostaglandin E(2) (EP(1)) receptor functioning: mechanism of action.

Prostaglandin E(2) receptors, subtype EP(1) (PGE(2)EP(1)) have been linked to several physiologic responses, such as fever, inflammation, and mechanical hyperalgesia. Local anesthetics modulate these responses, which may be due to direct interaction of local anesthetics with PGE(2)EP(1) receptor signaling. We sought to characterize the local anesthetic effects on PGE(2)EP(1) signaling and elucidate mechanisms of anesthetic action.

Role of protein phosphatases in regulation of cardiac inotropy and relaxation.

We studied the effects of the protein phosphatase (PP) inhibitor cantharidin (Cant) on time parameters and force of contraction (FOC) in isometrically contracting electrically driven guinea pig papillary muscles. We correlated the mechanical parameters of contractility with phosphorylation of the inhibitory subunit of troponin (TnI-P) and with the site-specific phosphorylation of phospholamban (PLB) at serine-16 (PLB-Ser-16) and threonine-17 (PLB-Thr-17). Cant (after 30 min) started to increase FOC (112 +/- 4% of control, n = 10) and TnI-P and PLB-Thr-17 (120 +/- 5 and 128 +/- 7% of control) without any alteration of relaxation time.

Regional expression of protein phosphatase type 1 and 2A catalytic subunit isoforms in the human heart.

In mammalian species, including man, the duration of myocardial contraction is shorter in atria than ventricles. Total contraction time depends at least in part on phosphorylation and dephosphorylation of cardiac regulatory proteins. Dephosphorylation reactions are mediated by protein phosphatases.

Protein phosphatase activity is increased in a rat model of long-term beta-adrenergic stimulation.

We tested the hypothesis that altered phosphorylation of Ca2+ regulatory proteins contributes to contractile anomalies in cardiac hypertrophy. Cardiac hypertrophy was induced in rats by chronic s.c.

Functional properties of transgenic mouse hearts overexpressing both calsequestrin and the Na(+)-Ca(2+) exchanger.

Overexpression of calsequestrin (CSQ) induces severe cardiac hypertrophy, whereas overexpression of Na(+)-Ca(2+) exchanger (NCX) does not affect cardiac weight. To investigate a possible beneficial effect of NCX in hypertrophy, we produced transgenic mice overexpressing both NCX and CSQ (NCX/CSQ). Surprisingly, these mice developed severe heart failure.

Cantharidin enhances norepinephrine-induced vasoconstriction in an endothelium-dependent fashion.

In this study we characterized the effects of the protein phosphatase (PP) type 1 and type 2A inhibitor cantharidin (Cant) and its structural analogs cantharidic acid and endothall on PP activity, force of contraction, and myosin light chain phosphorylation in rat aorta. All compounds inhibited PP activity in homogenates of rat aorta with a rank order of potency of Cant = cantharidic acid > endothall. However, only Cant increased force of contraction and myosin light chain phosphorylation in intact isolated rat aortic rings.

Biochemical mechanism(s) of stunning in conscious dogs.

The mechanism(s) underlying contractile dysfunction in cardiac stunning is not completely understood. The expression and/or the phosphorylation state of cardiac Ca(2+) homoeostasis-regulating proteins might be altered in stunning. We tested this hypothesis in a well-characterized model of stunning.

On the cardiac contractile, electrophysiological and biochemical effects of endothall, a protein phosphatase inhibitor.

Protein phosphatase inhibitors, e.g. cantharidin, exert positive inotropic effects in mammalian heart preparations.

The early response genes c-jun and HSP-70 are induced in regional cardiac stunning in conscious mammals.

Objectives: A reversible contractile dysfunction without necrosis after transient myocardial ischemia has been termed stunning. The molecular mechanisms underlying this phenomenon are only now beginning to be unraveled. It is conceivable that the expression of early-response genes may play a crucial role in stunning.

Functional studies in atrium overexpressing A1-adenosine receptors.

1. Adenosine and the A1-adenosine receptor agonist R-PIA, exerted a negative inotropic effect in isolated, electrically driven left atria of wild-type mice. 2.

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.

Inositol-1,4,5-trisphosphate increase by diadenosine tetraphosphate in preparations from failing human myocardium.

In human ventricular trabeculae carneae 100 microM AP4A (diadenosine tetraphosphate) increased force of contraction to 162.8+/-15.7% of predrug value (n=9).

Regional expression of phospholamban in the human heart.

Background: Several independent lines of evidence indicate that phospholamban (PLB) expression correlates positively with depression of force of contraction and duration of contraction in isolated cardiac preparations of several animal species. Here, we studied whether PLB levels correlate with attenuation of contractility and enhancement of contractile time parameters in different parts of the human heart.

Methods: Force of contraction was measured in isolated electrically driven atrial and ventricular preparations from human hearts.

Expression of cardiac calcium regulatory proteins in atrium v ventricle in different species.

The duration of contraction in isolated electrically driven preparations from atrium and ventricle of mouse, rat, rabbit, guinea-pig and dog was consistently shorter in atrial compared to ventricular preparations. Overexpression of phospholamban (PLB) in transgenic mice prolonged duration of contraction, underscoring the importance of PLB for kinetics of cardiac contractility. The expression of regulatory proteins was studied by Western and Northern blot analysis.

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.

Quantitation of clobazam in human plasma using high-performance liquid chromatography.

A rapid and simple reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of clobazam concentrations in human blood samples is developed and validated. Solid-phase column extraction is performed to clean up blood samples before running the analytical HPLC system. The chromatography is isocratic with a mobile phase consisting of acetonitrile (20%, v/v), methanol (23%, v/v), and 0.

Inotropic effects of diadenosine tetraphosphate (AP4A) in human and animal cardiac preparations.

Diadenosine tetraphosphate (AP4A) is an endogenous compound and exerts diverse physiological effects in animal systems. However, the effects of AP4A on inotropy in ventricular cardiac preparations have not yet been studied. The effects of AP4A on force of contraction (FOC) were studied in isolated electrically driven guinea pig and human cardiac preparations.

Contractility and inhibition of protein phosphatases by cantharidin.

1. Cantharidin is a natural defensive toxicant produced by blister beetles. 2.

Long-term beta adrenoceptor-mediated alteration in contractility and expression of phospholamban and sarcoplasmic reticulum Ca(++)-ATPase in mammalian ventricle.

We studied the influence of prolonged administration of the beta adrenoceptor agonist isoproterenol on contractile parameters and expression of sarcoplasmic reticulum (SR) Ca(++)-ATPase and phospholamban, genes important for Ca++ uptake into the SR. Isoproterenol (Iso), 0.9% NaCl (Ctr), propranolol (Prop) or Iso plus Prop were administered to rats by subcutaneous infusion with osmotic minipumps for 1, 2, 3, 4, 8, 13 and 26 days, respectively.

Functional comparison of the three isoforms of the Na+/Ca2+ exchanger (NCX1, NCX2, NCX3).

Three distinct mammalian Na+/Ca2+ exchangers have been cloned: NCX1, NCX2, and NCX3. We have undertaken a detailed functional comparison of these three exchangers. Each exchanger was stably expressed at high levels in the plasma membranes of BHK cells.