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H M Piper

Publications and source records attributed to H M Piper.

At least 19 recordsLinked to original sources
PubMed2001

Hypertrophy-associated gene induction after beta-adrenergic stimulation in adult cardiomyocytes.

In isolated cardiomyocytes, hypertrophic responsiveness to beta-adrenergic stimulation can be induced by pre-exposure of the cells to TGF-beta. To characterize genes involved in beta-adrenergically mediated hypertrophy, mRNA expression patterns in isoprenaline-stimulated cardiomyocytes which were pre-exposed to TGF-beta were analysed by differential display RT-PCR analysis. Eighteen fragments, upregulated by isoprenaline, were identified. Six of them, which code for proteins with known function, were further analysed by RT-PCR (1) to verify their induction after beta-adrenergic stimulation, (2) to restrict their number to genes only upregulated after hypertrophy inducing beta-adrenergic stimulation, and (3) to study their expression in stroke-prone spontaneous hypertrophic rats (SHR-sp), an in vivo model of myocardial hypertrophy, in which elevated levels of TGF-beta are found. Induction by isoprenaline could be proved for all but one of the six genes. Further analysis of these genes in freshly isolated myocytes, which respond with hypertrophic growth only to alpha--but not beta--adrenergic stimulation, revealed that three of them, coding for the translation initiation factor sui 1, the cis-golgi transport protein p28 and the mitochondrial NADH-dehydrogenase II subunit, are specifically induced in TGF-beta-pre-exposed cardiomyocytes after beta-adrenergic stimulation. Their induction is therefore closely associated with a beta-adrenergic growth response in isolated cardiomyocytes. p28-mRNA is also markedly increased in SHR-sp rats. Antisense experiments revealed a functional importance of p28 for the beta-adrenergic growth response in isolated cardiomyocytes. Therefore, p28 seems causally involved in this beta-adrenergic growth response.

Adrenergic beta-Agonists↗
PubMed2001

Beta-adrenoceptor stimulation attenuates the hypertrophic effect of alpha-adrenoceptor stimulation in adult rat ventricular cardiomyocytes.

OBJECTIVES: The study investigated whether beta-adrenoceptor antagonists augment the hypertrophic response of cardiomyocytes evoked by norepinephrine. BACKGROUND: In adult ventricular cardiomyocytes, stimulation of alpha- but not beta-adrenoceptors induces myocardial hypertrophy. Natural catecholamines, like norepinephrine, stimulate simultaneously alpha- and beta-adrenoceptors. We investigated whether beta-adrenoceptor stimulation interferes with the hypertrophic response caused by alpha-adrenoceptor stimulation. METHODS: Adult ventricular cardiomyocytes isolated from rats were used as an experimental model. Hypertrophic parameters under investigation were stimulation of phenylalanine incorporation and protein mass, stimulation of 14C-uridine incorporation and RNA mass, and increases in cell shape. RESULTS: Norepinephrine (0.01 to 10 micromol/liter) increased concentration-dependent phenylalanine incorporation; pEC50 value was 5.9 +/- 0.1 (n = 8). The alpha1-adrenoceptor antagonist prazosin (0.1 micromol/liter) suppressed norepinephrine-induced increase in rate of protein synthesis. Conversely, propranolol (1 micromol/liter) and the beta1-adrenoceptor selective antagonists CPG 20712A (300 nmol/liter) or atenolol (1 micromol/liter) augmented increases in phenylalanine incorporation caused by norepinephrine. Addition of the beta2-adrenoceptor antagonist ICI 118,551 (55 nmol/liter) did not influence the hypertrophic effect of norepinephrine. Atenolol augmented the norepinephrine-induced increases of all hypertrophic parameters investigated (i.e., protein mass, uridine incorporation, RNA mass, cell volume, and cross-sectional area). In the presence of norepinephrine, inhibition of beta1-adrenoceptors increased the amount of protein kinase C-alpha and -delta isoforms translocated into the particulate fraction. The effect of pharmacological inhibition of beta1-adrenoceptors could be mimicked by Rp-cAMPS (adenosine-3', 5'-cyclic phosphorothiolate-Rp). The inhibitory effect of beta1-adrenoceptor stimulation on the alpha-adrenoceptor-mediated effect persisted in cardiomyocytes isolated from hypertrophic hearts of rats submitted to aortic banding. CONCLUSIONS: In isolated ventricular cardiomyocytes from rats, beta1-adrenoceptor stimulation attenuates the hypertrophic response evoked by alpha1-adrenoceptor stimulation.

Animals↗
PubMed2001

A N-terminal PTHrP peptide fragment void of a PTH/PTHrP-receptor binding domain activates cardiac ET(A) receptors.

1. Adult ventricular cardiomyocytes show an unusual structure-function relationship for cyclic AMP-dependent effects of PTHrP. We investigated whether PTHrP(1 - 16), void of biological activity on classical PTHrP target cells, is able to mimic the positive contractile effect of PTHrP(1 - 34), a fully biological agonist on cardiomyocytes. 2. Adult ventricular cardiomyocytes were paced at a constant frequency of 0.5 Hz and cell contraction was monitored using a cell-edge-detection system. Twitch amplitudes, expressed as per cent cell shortening of the diastolic cell length, and rate constants for maximal contraction and relaxation velocity were analysed. 3. PTHrP(1 - 16) (1 micromol l(-1)) mimicked the contractile effects of PTHrP(1 - 34) (1 micromol l(-1)). It increased the twitch amplitude from 5.33+/-0.72 to 8.95+/-1.10 (% dl l(-1)) without changing the kinetic of contraction. 4. PTH(1 - 34) (10 micromol l(-1)) affected the positive contractile effect of PTHrP(1 - 34), but not that of PTHrP(1 - 16). 5. RpcAMPS (10 micromol l(-1)) inhibited the positive contractile effect of PTHrP(1 - 34), but not that of PTHrP(1 - 16). 6. The positive contractile effect of PTHrP(1 - 16) was antagonized by the ET(A) receptor antagonist BQ123. 7. Sarafotoxin 6b and PTHrP(1 - 16), but not PTHrP(1 - 34), replaced (3)H-BQ123 from cardiac binding sites. 8. We conclude that N-terminal PTHrP peptides void of a PTH/PTHrP-receptor binding domain are able to bind to, and activate cardiac ET(A) receptors.

Adrenergic alpha-Agonists↗
PubMed2001

Modulation of early [Ca2+]i rise in metabolically inhibited endothelial cells by xestospongin C.

When energy metabolism is disrupted, endothelial cells lose Ca(2+) from endoplasmic reticulum (ER) and the cytosolic Ca(2+) concentration ([Ca(2+)](i)) increases. The importance of glycolytic energy production and the mechanism of Ca(2+) loss from the ER were analyzed. Endothelial cells from porcine aorta in culture and in situ were used as models. 2-Deoxy-D-glucose (2-DG, 10 mM), an inhibitor of glycolysis, caused an increase in [Ca(2+)](i) (measured with fura 2) within 1 min when total cellular ATP contents were not yet affected. Stimulation of oxidative energy production with pyruvate (5 mM) did not attenuate this 2-DG-induced rise of [Ca(2+)](i), while this maneuver preserved cellular ATP contents. The inhibitor of ER-Ca(2+)-ATPase, thapsigargin (10 nM), augmented the 2-DG-induced rise of [Ca(2+)](i). Xestospongin C (3 microM), an inhibitor of D-myo-inositol 3-phosphate [Ins(3)P]-sensitive ER-Ca(2+) release, abolished the rise. The results demonstrate that the ER of endothelial cells is very sensitive to glycolytic metabolic inhibition. When this occurs, the ER Ca(2+) store is discharged by opening of the Ins(3)P-sensitive release channel. Xestospongin C can effectively suppress the early [Ca(2+)](i) rise in metabolically inhibited endothelial cells.

Adenosine Triphosphate↗
PubMed2000

Expression, release, and biological activity of parathyroid hormone-related peptide from coronary endothelial cells.

Ventricular cardiomyocytes have previously been identified as potential target cells for parathyroid hormone-related peptide (PTHrP). Synthetic PTHrP peptides exert a positive contractile effect. Because systemic PTHrP levels are normally negligible, this suggests that PTHrP is expressed in the ventricle and acts as a paracrine mediator. We investigated the ventricular expression of PTHrP and its expression in cultured cells isolated from the ventricle, studied the release of PTHrP from hearts and cultures, and investigated whether this authentic PTHrP mimics the biological effects previously described for synthetic PTHrP on ventricular cardiomyocytes. We found PTHrP expressed in ventricles of neonatal and adult rat hearts. In cells isolated from adult hearts, we found PTHrP expression exclusively in coronary endothelial cells but not in cardiomyocytes. The latter, however, are target cells for PTHrP. PTHrP was released from isolated perfused hearts during hypoxic perfusion and from cultured coronary endothelial cells under energy-depleting conditions. This PTHrP was biologically active; ie, it exerted a positive contractile and lusitropic effect on cardiomyocytes. Authentic PTHrP was glycosylated and showed a slightly higher potency than synthetic PTHrP. These results suggest that PTHrP is an endothelium-derived modulator of ventricular function.

Animals↗
PubMed2000

Central role for ornithine decarboxylase in beta-adrenoceptor mediated hypertrophy.

OBJECTIVE: TGF-beta stimulation of cardiac myocytes induces a hypertrophic responsiveness to beta-adrenoceptor stimulation. This study investigates whether this beta-adrenoceptor mediated effect depends on induction of ornithine decarboxylase (ODC). METHODS: Isolated adult ventricular cardiomyocytes from rats were used as an experimental model. Cells were either cultured in 20% (v/v) FCS to activate autocrine released TGF-beta or used without pre-treatment. The hypertrophic response was characterized by an increased 14C-phenylalanine incorporation, RNA and protein mass or by an increased expression of atrionatriurectic factor and ODC. The results on cell cultures were compared to those achieved by isoprenaline perfused mice hearts from transgenic mice overexpressing TGF-beta 1. RESULTS: ODC activity and expression increased within 2 h in TGF-beta 1 pre-treated cells under isoprenaline. In the presence of ODC inhibitors (alpha-methylornithine or difluoromethylornithine) this increase remained absent and the increases in 14C-phenylalanine incorporation, protein and RNA mass under isoprenaline were abolished. In cells not exposed to TGF-beta no induction of ODC was observed. Isoprenaline also induced ODC in isolated perfused ventricles from transgenic mice overexpressing TGF-beta 1, but not in ventricles from their nontransgenic counterparts. CONCLUSIONS: This study shows first, a pivotal role for ODC induction in the hypertrophic response of cardiomyocytes to beta-adrenoceptor stimulation and second, that ODC induction in vivo and in vitro requires pre-treatment of cardiomyocytes with TGF-beta. It is concluded that TGF-beta induces a hypertrophic responsiveness to beta-adrenoceptor stimulation that is characterized by ODC induction.

Adrenergic beta-Agonists↗
PubMed2000

Mechanism of Ca(2+) overload in endothelial cells exposed to simulated ischemia.

OBJECTIVE: Several studies have shown that myocardial ischemia leads to functional failure of endothelial cells (EC) whereby disturbance of Ca(2+) homeostasis may play an important role. The mechanisms leading to Ca(2+) disbalance in ischemic EC are not fully understood. The aim of this study was to test effects of different components of simulated ischemia (glucose deprivation, anoxia, low extracellular pH (pH(o)) and lactate) on Ca(2+) homeostasis in EC. METHODS: Cytosolic Ca(2+) (Ca(i)), cytosolic pH (pH(i)) and ATP content were measured in cultured rat coronary EC. RESULTS: In normoxic cells 60 min glucose deprivation at pH(o) 7.4 had no effect on pH(i). It only slightly increased Ca(i) and decreased ATP content. Reduction of pH(o) to 6.5 under these conditions led to marked cytosolic acidosis and Ca(i) overload, but had no effect on ATP content. Anoxia at pH(o) 6.5 had no additional effect on Ca(i) overload, but significantly reduced cellular ATP. Addition of 20 mmol/l lactate to anoxia at pH(o) 6.5 accelerated Ca(i) overload due to faster cytosolic acidification. Acidosis-induced Ca(i) overload was prevented by inhibition of Ca(2+) release channels of endoplasmic reticulum (ER) with 3 micromol/l ryanodine or by pre-emptying the ER with thapsigargin. Re-normalisation of pH(o) for 30 min led to recovery of pH(i), but not of Ca(i). CONCLUSION: The ischemic factors leading to cytosolic acidosis (low pH(o) and lactate) cause Ca(i) overload in endothelial cells, while anoxia and glucose deprivation play only a minor role. The ER is the main source for this Ca(i) rise. Ca(i) overload is not readily reversible.

Adenosine Triphosphate↗
PubMed2000

Apoptosis induction by nitric oxide in adult cardiomyocytes via cGMP-signaling and its impairment after simulated ischemia.

OBJECTIVE: Nitric oxide (NO) has been shown to induce apoptosis in cardiomyocytes under normoxic conditions. The ability of NO to induce apoptosis after ischemia-reperfusion, a situation of increased NO release in vivo, has not been investigated. The present study was undertaken to characterize the pathway of induction of apoptosis by NO and the influence of ischemia on this pathway in cardiomyocytes. METHODS: The study was performed on isolated adult cardiomyocytes of the rat. Ischemia was simulated by anoxia in a glucose free medium, pH 6.4. Induction of apoptosis was detected (1) by annexinV-fluorescein isothiocyanate (annexinV-FITC) binding to cells under exclusion of propidium iodide and (2) by laddering of genomic DNA. RESULTS: Incubation of cardiomyocytes with the NO-donor (+/-)-S-nitroso-N-acetylpenicillamine (SNAP, 100 microM) induced apoptosis in 14.1 +/- 1.9% of the cells and necrosis in 24.4 +/- 4.6%. The induction of apoptosis but not necrosis could be blocked by inhibition of soluble guanylyl cyclase or of protein kinase G. Apoptosis induction was mimicked by incubation of cardiomyocytes with 8-pCPT-cGMP (100 microM, 9.6 +/- 0.6% apoptotic cells) or YC-1 (75 microM, 14.6 +/- 2.8% apoptotic cells), a direct activator of soluble guanylyl cyclase. After 3 h of anoxia, cardiomyocytes were transiently protected against apoptosis induced by NO, but not by 8-pCPT-cGMP or YC-1 (8.9 +/- 0.7% or 13.4 +/- 2.4% apoptotic cells). A correlation of the apoptotic response to SNAP or YC-1 with an increased activity of soluble guanylyl cyclase, determined by measurements of intracellular cGMP contents, was found. CONCLUSIONS: NO induces apoptosis in a cGMP dependent manner in isolated adult cardiomyocytes whereas induction of necrosis seems cGMP-independent. After simulated in vitro ischemia the activation of soluble guanylyl cyclase by NO is transiently inhibited resulting in a transient anti-apoptotic protection.

Analysis of Variance↗
PubMed2000

Hypertrophic effect of selective beta(1)-adrenoceptor stimulation on ventricular cardiomyocytes from adult rat.

We investigated whether selective beta(1)-adrenoceptor stimulation causes hypertrophic growth on isolated ventricular cardiomyocytes from adult rat. As parameters for the induction of hypertrophic growth, the increases of [(14)C]phenylalanine incorporation, protein and RNA mass, and cell size were determined. Isoproterenol (Iso, 10 microM) alone had no growth effect. In the presence of the beta(2)-adrenoceptor antagonist ICI-118551 (ICI, 10 microM), Iso caused an increase in [(14)C]phenylalanine incorporation, protein and RNA mass, cell volume, and cross-sectional area. We showed for phenylalanine incorporation that the growth effect of Iso+ICI could be antagonized by beta(1)-adrenoceptor blockade with atenolol (10 microM) or metoprolol (10 microM), indicating that it was caused by selective beta(1)-adrenoceptor stimulation. The growth response to Iso+ICI was accompanied by an increase in ornithine decarboxylase (ODC) activity and expression. Inhibition of ODC by the ODC antagonist difluoromethylornithine (1 mM) attenuated this hypertrophic response, indicating that ODC induction is causally involved. The growth response to Iso+ICI was found to be cAMP independent but was sensitive to genistein (100 microM) or rapamycin (0.1 microM). The reaction was enhanced in the presence of pertussis toxin (10 microM). We conclude that selective beta(1)-adrenoceptor stimulation causes hypertrophic growth of ventricular cardiomyocytes by a mechanism that is independent of cAMP but dependent on a tyrosine kinase and ODC.

Adrenergic beta-Antagonists↗
PubMed2000

ATP induces dephosphorylation of myosin light chain in endothelial cells.

In cultured porcine aortic endothelial monolayers, the effect of ATP on myosin light chain (MLC) phosphorylation, which controls the endothelial contractile machinery, was studied. ATP (10 microM) reduced MLC phosphorylation but increased cytosolic Ca(2+) concentration ([Ca(2+)](i)). Inhibition of the ATP-evoked [Ca(2+)](i) rise by xestospongin C (10 microM), an inhibitor of the inositol trisphosphate-dependent Ca(2+) release from endoplasmic reticulum, did not affect the ATP-induced dephosphorylation of MLC. MLC dephosphorylation was prevented in the presence of calyculin A (10 nM), an inhibitor of protein phosphatases PP-1 and PP-2A. Thus ATP activates MLC dephosphorylation in a Ca(2+)-independent manner. In the presence of calyculin A, MLC phosphorylation was incremented after addition of ATP, an effect that could be abolished when cells were loaded with the Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N, N,N',N'-tetraacetic acid acetoxymethyl ester (10 microM). Thus ATP also activates a Ca(2+)-dependent kinase acting on MLC. In summary, ATP simultaneously stimulates a functional antagonism toward both phosphorylation and dephosphorylation of MLC in which the dephosphorylation prevails. In endothelial cells, ATP is the first physiological mediator identified to activate MLC dephosphorylation by a Ca(2+)-independent mechanism.

Adenosine Triphosphate↗
PubMed2000

Influence of simulated ischemia on apoptosis induction by oxidative stress in adult cardiomyocytes of rats.

Oxidative stress may cause apoptosis of cardiomyocytes in ischemic-reperfused myocardium. We investigated whether ischemia-reperfusion modifies the susceptibility of cardiomyocyte induction of apoptosis by oxidative stress. Ischemia was simulated by incubating isolated cardiomyocytes from adult rats in an anoxic, glucose-free medium, pH 6.4, for 3 h. Annexin V-fluorescein isothiocyanate/propidium iodide staining and the detection of DNA laddering were used as apoptotic markers. H(2)O(2) (7.5 micromol/l) induced apoptosis in 20.1 +/- 1.8% of cells under normoxic conditions but only 14.4 +/- 1.6% (n = 6, P < 0.05) after ischemia-reoxygenation. This partial protection of ischemic-reoxygenated cells was observed despite a reduction in their cellular glutathione content, from 11.4 +/- 1.9 in normoxic controls to 2.9 +/- 0.8 nmol/mg protein (n = 3, P < 0.05). Elevation of end-ischemic glutathione contents by pretreatment with 1 mmol/l N-acetylcysteine entirely protected ischemic-reoxygenated cells against induction of apoptosis by H(2)O(2). In conclusion, ischemia-reperfusion can protect cardiomyocytes against induction of apoptosis by exogenous oxidative stress. This endogenous protective effect is most clearly demonstrated when control and postischemic cardiomyocytes are compared at similar glutathione levels.

Acetylcysteine↗
PubMed2000

Importance of bicarbonate transport for protection of cardiomyocytes against reoxygenation injury.

Isolated cardiomyocytes from adult rats were incubated in anoxic bicarbonate-buffered media at extracellular pH (pH(o)) 6.4 until a cytosolic Ca(2+) overload and intracellular pH (pH(i)) of 6.4 were reached. On reoxygenation, the pH of the medium was changed to 7.4 to activate the Na(+)/H(+)exchanger (NHE) and the Na(+)-HCO(-)(3) symporter (NBS). The reoxygenation was performed in the absence or presence of the NHE inhibitor HOE-642 (3 micromol/l) and/or the NBS inhibitor DIDS (0.5 mmol/l), as in bicarbonate-free media. In reoxygenated control cells pH(i) rapidly recovered to the preanoxic level, and a burst of spontaneous oscillations of cytosolic Ca(2+) occurred, accompanied by the development of hypercontracture. When NBS and NHE were simultaneously inhibited during reoxygenation, pH(i) recovery was prevented, Ca(2+) oscillations were attenuated, and hypercontracture was abolished. Sole inhibition of NBS or NHE showed no protection against hypercontracture. In the absence of cytosolic acidosis, HOE-642 or DIDS did not prevent hypercontracture induced by Ca(2+) overload. The results demonstrate that simultaneous inhibition of NHE and NBS is needed to protect myocardial cells against reoxygenation-induced hypercontracture.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2000

Inhibition of NHE protects reoxygenated cardiomyocytes independently of anoxic Ca(2+) overload and acidosis.

We investigated the question of whether inhibition of the Na(+)/H(+) exchanger (NHE) during ischemia is protective due to reduction of cytosolic Ca(2+) accumulation or enhanced acidosis in cardiomyocytes. Additionally, the role of the Na(+)-HCO(3)(-) symporter (NBS) was investigated. Adult rat cardiomyocytes were exposed to simulated ischemia and reoxygenation. Cytosolic pH [2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)], Ca(2+) (fura 2), Na(+) [sodium-binding benzolfuran isophthatlate (SBFI)], and cell length were measured. NHE was inhibited with 3 micromol/l HOE 642 or 1 micromol/l 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), and NBS was inhibited with HEPES buffer. During anoxia in bicarbonate buffer, cells developed acidosis and intracellular Na and Ca (Na(i) and Ca(i), respectively) overload. During reoxygenation cells underwent hypercontracture (44.0 +/- 4.1% of the preanoxic length). During anoxia in bicarbonate buffer, inhibition of NHE had no effect on changes in intracellular pH (pH(i)), Na(i), and Ca(i), but it significantly reduced the reoxygenation-induced hypercontracture (HOE: 61.0 +/- 1.4%, EIPA: 68.2 +/- 1.8%). The sole inhibition of NBS during anoxia was not protective. We conclude that inhibition of NHE during anoxia protects cardiomyocytes against reoxygenation injury independently of cytosolic acidification and Ca(i) overload.

Amiloride↗
PubMed1999

Propagation of cardiomyocyte hypercontracture by passage of Na(+) through gap junctions.

Prolonged ischemia increases cytosolic Ca(2+) concentration in cardiomyocytes. Cells with severely elevated cytosolic Ca(2+) may respond to reperfusion, developing hypercontracture, sarcolemmal disruption, and death. Cardiomyocytes are efficiently connected through gap junctions (GJs) to form a functional syncytium, and it has been shown that hypercontracture can be propagated to adjacent myocytes through a GJ-mediated mechanism. This study investigated the mechanism of propagation of cell injury associated with sarcolemmal rupture in end-to-end connected pairs of isolated rat cardiomyocytes. Microinjection of extracellular medium into one of the cells to simulate sarcolemmal disruption induced a marked increase in cytosolic Ca(2+) (fura-2) and Na(+) (SBFI) in the adjacent cell and its hypercontracture in <30 seconds (22 of 22 cell pairs). This process was not modified when Ca(2+) release from the sarcoplasmic reticulum was blocked with 10 micromol/L ryanodine (5 of 5 cell pairs), but it was fully dependent on the presence of Ca(2+) in the extracellular buffer. Blockade of L-type Ca(2+) channels with 10 micromol/L nifedipine did not alter propagation of hypercontracture. However, the presence of 15 to 20 micromol/L KB-R7943, a highly selective blocker of reverse Na(+)/Ca(2+) exchange, prevented propagation of hypercontracture in 16 of 20 cell pairs (P<0.01) without interfering with GJ permeability, as assessed by the Lucifer Yellow transfer method. Addition of the Ca(2+) chelator EGTA (2 mmol/L) to the injection solution prevented hypercontracture in the injected cell but not in the adjacent one (n=5). These results indicate that passage of Na(+) through GJ from hypercontracting myocytes with ruptured sarcolemma to adjacent cells, and secondary entry of [Ca(2+)](o) via reverse Na(+)/Ca(2+) exchange, can contribute to cell-to-cell propagation of hypercontracture. This previously unrecognized mechanism could increase myocardial necrosis during ischemia-reperfusion in vivo and be the target of new treatments aimed to limit it.

Animals↗
PubMed1999

Pretreatment with PKC activator protects cardiomyocytes against reoxygenation-induced hypercontracture independently of Ca2+ overload.

OBJECTIVE: Although several studies have shown that activation of protein kinase C (PKC) plays an important role in protection through ischemic preconditioning, little is known about the effects of direct PKC activation on the course of ischemia-reperfusion injury. The aim of this study was to analyse the effects of a pretreatment with the PKC activator 1,2-dioctanoyl-sn-glycerol (1,2DOG). METHODS: Isolated adult Wistar rat cardiomyocytes were exposed to 80 min of simulated ischemia (anoxia, pHo 6.4) and 20 min of reoxygenation (pHo 7.4). Cytosolic Ca2+ (fura-2), cytosolic pH (BCECF), Mg2+ (Mg-fura-2), lactate and cell length were measured and compared between control cells and cells treated with 20 mumol/l 1,2DOG before anoxia (10 min treatment and 10 min wash out). RESULTS: 1,2DOG-pretreatment delayed the time to extreme ATP depletion, but had no effect on lactate production and cytosolic pH. The accumulation of cytosolic Ca2+ was markedly accelerated in pretreated cells that developed rigor shortening, but reoxygenation-induced hypercontracture was significantly reduced. 1,2DOG, therefore, completely abolished Ca(2+)-dependence of hypercontracture. The effects of pretreatment were fully abolished with 1 mumol/l bisindolylmalcimide (PKC inhibitor). We conclude that PKC preactivation leads to (1) reduction of energy demand, (2) acceleration of Ca2+ overload during anoxia and (3) prevention of reoxygenation-induced hypercontracture independent of anoxic changes in cytosolic Ca2+ and pH.

Analysis of Variance↗
PubMed1999

Effect of factor XIII on endothelial barrier function.

The effect of factor XIII on endothelial barrier function was studied in a model of cultured monolayers of porcine aortic endothelial cells and saline-perfused rat hearts. The thrombin-activated plasma factor XIII (1 U/ml) reduced albumin permeability of endothelial monolayers within 20 min by 30 +/- 7% (basal value of 5.9 +/- 0.4 x 10(-6) cm/s), whereas the nonactivated plasma factor XIII had no effect. Reduction of permeability to the same extent, i.e., by 34 +/- 9% could be obtained with the thrombin-activated A subunit of factor XIII (1 U/ml), whereas the iodoacetamide-inactivated A subunit as well as the B subunit had no effect on permeability. Endothelial monolayers exposed to the activated factor XIII A exhibited immunoreactive deposition of itself at interfaces of adjacent cells; however, these were not found on exposure to nonactivated factor XIII A or factor XIII B. Hyperpermeability induced by metabolic inhibition (1 mM potassium cyanide plus 1 mM 2-deoxy-D-glucose) was prevented in the presence of the activated factor XIII A. Likewise, the increase in myocardial water content in ischemic-reperfused rat hearts was prevented in its presence. This study shows that activated factor XIII reduces endothelial permeability. It can prevent the loss of endothelial barrier function under conditions of energy depletion. Its effect seems related to a modification of the paracellular passageways in endothelial monolayers.

Animals↗
PubMed1999

Autocrine regulation of TGF beta expression in adult cardiomyocytes.

As shown before, TGF beta acts in an autocrine manner on the induction of hypertrophic responsiveness to beta-adrenoceptor stimulation in cultured ventricular cardiomyocytes of adult rat. We now investigated how TGF beta expression and activation is regulated in these cultures and how beta-adrenoceptor stimulation influences TGF beta -mRNA expression. It was found that freshly isolated cardiomyocytes secrete latent TGF beta in the culture medium. Supplementation of the cultures with 20% FCS resulted in activation of the secreted TGF beta to 4.1+/-0.2 ng/ml active TGF beta after 6 days. Presence of the protease inhibitor aprotinin (50 microg/ml) reduced TGF beta activity by 44+/-5% (n=5, P<0.05). In cultures supplemented with 5% FCS, TGF beta was not activated. Active TGF beta downregulated its mRNA-expression: after 6 days TGF beta(1)-mRNA was reduced to 55.1+/-11.0%, TGF beta(2)-mRNA to 30.1+/-16.5%, and TGF beta(3)-mRNA to 0.3+/-0.4% in 20% FCS-cultures as compared to their expression in freshly isolated cells (n=4, P<0.05). TGF beta-mRNA expression did not change in cultures without active TGF beta. Isoprenaline (1 microm) increased TGF beta(1)-mRNA only in cultures which had been pre-exposed to active TGF beta. This effect was also seen when hearts from normal mice were compared with hearts from transgenic mice overexpressing TGF beta(1): only in hearts from transgenic animals perfusion with isoprenaline increased TGF beta(1)-mRNA. In conclusion, isolated cardiomyocytes release latent TGF beta, which is activated by external proteases. Active TGF beta downregulates its own mRNA expression. Preexposure to TGF beta is necessary for a beta-adrenoceptor-mediated increase in TGF beta(1)-mRNA in cardiomyocytes.

Animals↗