Chun-Yang Xiao

Characterization of prostanoid receptors mediating contraction of the gastric fundus and ileum: studies using mice deficient in prostanoid receptors

Receptors mediating prostanoid‐induced contractions of longitudinal sections of gastric fundus and ileum were characterized by using tissues obtained from mice deficient in each type and subtype of prostanoid receptors. The fundus and ileum from mice deficient in either EP3 (EP3−/− mice), EP1 (EP1−/− mice) and FP (FP−/− mice) all showed decreased contraction to PGE2 compared to the tissues from wild‐type mice, whereas contraction of the fundus slightly increased in EP4−/− mice. 17‐phenyl‐PGE2 also showed decreased contraction of the fundus from EP3−/−, EP1−/− and FP−/− mice. Sulprostone showed decreased contraction of the fundus from EP3−/− and FP−/− mice, and decreased contraction of the ileum to this compound was seen in tissues from EP3−/−, EP1−/− and FP−/− mice. In DP−/− mice, sulprostone showed increased contraction. DI‐004 and AE‐248 caused the small but concentration‐dependent contraction of both tissues, and these contractions were abolished in tissues obtained from EP1−/− and EP3−/− mice, respectively, but not affected in other mice. Contractions of both fundus and ileum to PGF2α was absent at lower concentrations (10−9 to 10−7 M), and suppressed at higher concentrations (10−6 to 10−5 M) of the agonist in the FP−/− mice. Suppression of the contractions at the higher PGF2α concentrations was also seen in the fundus from EP3−/−, EP1−/− and TP−/− mice and in the ileum from EP3−/− and TP−/− mice. Contraction of the fundus to PGD2 was significantly enhanced in DP−/− mice, and contractions of the fundus and ileum to this PG decreased in FP−/− and EP3−/− mice. Contractions of both tissues to I‐BOP was absent at 10−9 to 10−7 M and much suppressed at higher concentrations in TP−/− mice. Slight suppression to this agonist was also observed in the tissues from EP3−/− mice. PGI2 induced small relaxation of both tissues from wild‐type mice. These relaxation reactions were much potentiated in EP3−/− mice. On the other hand, significant contraction to PGI2 was observed in both tissues obtained from IP−/− mice. These results show that contractions of the fundus and ileum induced by each prostanoid agonist are mediated by actions of this agonist on multiple types of prostanoid receptors and in some cases modified by its action on relaxant receptors.

Effects of the prostanoids on the proliferation or hypertrophy of cultured murine aortic smooth muscle cells

Effects of the prostanoids on the growth of cultured aortic vascular smooth muscle cells (VSMCs) were examined using mice lacking prostanoid receptors. Proliferation of VSMCs was assessed by measuring [3H]‐thymidine incorporation and the cell number, and their hypertrophy by [14C]‐leucine incorporation and protein content. In VSMCs from wild‐type mice, expressions of mRNAs for the EP4 and TP were most abundant, followed by those for the IP, EP3 and FP, when examined by competitive reverse transcriptase‐PCR. Those for the EP1, EP2 and DP, however, could not be detected. AE1‐329, an EP4 agonist, and cicaprost, an IP agonist, inhibited platelet derived growth factor (PDGF)‐induced proliferation of VSMCs from wild‐type mice; these inhibitory effects disappeared completely in VSMCs from EP4−/− and IP−/− mice, respectively. In accordance with these effects, AE1‐329 and cicaprost stimulated cAMP production in VSMCs from wild‐type mice, which were absent in VSMCs from EP4−/− and IP−/− mice, respectively. Effects of PGE2 on cell proliferation and adenylate cyclase were almost similar with those of AE1‐329 in VSMCs from wild‐type mice, which disappeared in VSMCs from EP4−/− mice. PGD2 inhibited PDGF‐induced proliferation of VSMCs from both wild‐type and DP−/− mice to a similar extent. This action of PGD2 was also observed in VSMCs from EP4−/− and IP−/− mice. In VSMCs from wild‐type mice, I‐BOP, a TP agonist, showed potentiation of PDGF‐induced hypertrophy. I‐BOP failed to show this action in VSMCs from TP−/− mice. The specific agonists for the EP1, EP2 or EP3, and PGF2α showed little effect on the growth of VSMCs. These results show that PGE2, PGI2 and TXA2 modulate PDGF‐induced proliferation or hypertrophy of VSMCs via the EP4, IP and TP, respectively, and that the inhibitory effect of PGD2 on PDGF‐induced proliferation is not mediated by the DP, EP4 or IP.

Differential effects of Ca2+ channel blockers on Ca2+ overload induced by lysophosphatidylcholine in cardiomyocytes.

The effects of Ca2+ channel blockers (verapamil, diltiazem, nicardipine, bepridil and flunarizine) on Ca2+ overload induced by lysophosphatidylcholine were examined in rat isolated cardiomyocytes. Addition of lysophosphatidylcholine (15 microM) produced Ca2+ overload as evidenced by a marked increase in the concentration of intracellular Ca2+ and hypercontracture of cells. Verapamil, flunarizine and bepridil concentration dependently inhibited the lysophosphatidylcholine-induced Ca2+ overload, whereas diltiazem and nicardipine did not. Lysophosphatidylcholine increased the release of creatine kinase, which was significantly attenuated by verapamil, flunarizine or bepridil (5 microM for each), but not by diltiazem or nicardipine (20 microM for each). Verapamil, flunarizine, bepridil (which attenuated the lysophosphatidylcholine-induced Ca2+ overload) and nicardipine (which did not) inhibited the veratridine-induced increase in the concentration of intracellular Na+ (indicated by the increase in fluorescence ratio of Na(+)-binding benzofuran isophthalate) and cell contracture, whereas diltiazem did not. These results suggest that verapamil, bepridil and flunarizine attenuate the Ca2+ overload induced by lysophosphatidylcholine, and that the Ca2+ channel blocking action of these drugs does not contribute substantially to this effect. The Na+ channel inhibition together with high lipophilicity of these drugs may be important for the attenuation of the lysophosphatidylcholine-induced Ca2+ overload.

PALMITOYL-L-CARNITINE MODIFIES THE MYOCARDIAL LEVELS OF HIGH-ENERGY PHOSPHATES AND FREE FATTY ACIDS

Long-chain acylcarnitines, such as palmitoyl-L-carnitine (PALCAR), are known to accumulate in the myocardium during ischemia. We examined whether exogenous PALCAR modifies the myocardial levels of high-energy phosphates (HEP) and free fatty acids (FFA) in the heart, and whetherd-cis-diltiazem andl-cis-diltiazem, an optical isomer having less potent Ca2+ channel blocking action thand-cis-diltiazem, attenuate the PALCAR-induced myocardial changes. Rat hearts were perfused aerobically at a constant flow according to the Langendorffs technique, while being paced electrically. PALCAR (5 μM) decreased the tissue levels of adenosine triphosphate and creatine phosphate and increased the tissue level of adenosine monophosphate, and produced mechanical dysfunction. In addition, PALCAR (5 μM) increased markedly the tissue levels of FFA, especially those of arachidonic and palmitoleic acids, and the release of creatine kinase (CK) from the myocardium. These alterations in the myocardial levels of HEP and FFA induced by PALCAR were significantly attenuated byd-cis-diltiazem (15 μM) orl-cis-diltiazem (15 μM). Both drugs also attenuated the PALCAR-induced CK release. The present study demonstrates that PALCAR modifies the tissue levels of HEP and FFA in the heart and that bothd-cis andl-cis-diltiazem protect the myocardium against the PALCAR-induced changes through mechanisms other that Ca2+ channel blocking action.

A new approach to the development of anti-ischemic drugs: Protective drugs against cell injury induced by lysophosphatidylcholine

Recent studies have revealed that lysophosphatidylcholine (LPC) produces mechanical and metabolic derangements in perfused working rat hearts and Ca2+-overload in isolated cardiac myocytes. Thus, LPC possesses an ischemia-like effect on the heart. Therefore, a drug that possesses an anti-LPC action would protect or improve ischemia/reperfusion damage. We examined the effects of various anti-ischemic drugs on the Ca2+ overload induced by LPC. Our data suggest that a drug with high lipophilicity possesses a protective effect on cell injury induced by LPC, probably because of preservation of membrane integrity.

Phospholipase A2 is not responsible for lysophosphatidylcholine-induced damage in cardiomyocytes

Lysophosphatidylcholine (LPC) is known to increase the intracellular concentration of Ca2+([Ca2+]i), leading to cell damage. In the present study we examined whether LPC activates phospholipase A2(PLA2) and whether the activation of PLA2 is responsible for the LPC-induced cell damage in isolated rat cardiomyocytes. LPC (15 μM) produced an increase in [Ca2+]i, a change in cell shape from rod to round, and the release of creatine kinase (CK) accompanied by a significant elevation of the cellular level of nonesterified fatty acids (NEFA), especially arachidonic acid. Three PLA2 inhibitors, 7,7-dimethyl-(5 Z,8 Z)-eicosadienoic acid (DEDA), 3-(4-octadecylbenzoyl)acrylic acid (OBAA), and manoalide, attenuated the LPC-induced accumulation of unsaturated NEFA to a similar degree. Nevertheless, whereas both DEDA and OBAA attenuated the LPC-induced increase in [Ca2+]i, change in cell shape, and release of CK, manoalide attenuated none of them. In the Ca2+-free solution, LPC did not increase [Ca2+]iwith significantly less accumulation of NEFA, but it changed the cell shape from rod to round and increased the release of CK. These results suggest that exogenous LPC increases the PLA2 activity, which, however, may not be responsible for the LPC-induced damage in cardiomyocytes.

Lysophosphatidylcholine induces Ca2+-independent cellular injury attenuated by d-propranolol in rat cardiomyocytes.

In isolated rat cardiomyocytes, exogenous lysophosphatidylcholine (LPC) (15 microM) increased the intracellular Ca2+ concentration (Ca2+]i) from 72 +/- 5 to 3042 +/- 431 nM accompanied by cell injury as indicated by the hypercontracture of the cells and the increase in creatine phosphokinase (CPK) release. In order to understand whether the cell injury induced by LPC was a consequence of the elevation of [Ca2+]i, the effect of LPC was examined in the Ca2+-free solution containing EGTA. Under the Ca2+ -free conditions, LPC did not increase [Ca2+]i, whereas it still inflicted injury on the cells in terms of cell-shape change and CPK release to the same degree as that under the Ca2+-present condition. Addition of ryanodine (10 microM) failed to prevent the changes in cell-shape and CPK release induced by LPC under both Ca2+-free and Ca2+-present conditions. Preincubation of the myocytes with d-propranolol (50 microM) inhibited the LPC-induced changes in cell-shape and CPK release under both Ca2+ -free and Ca2+ -present conditions (p < 0.05). Our study provides clear evidence that the cellular injury induced by LPC could be independent of the increase in [Ca2+]i, and the Ca2+-independent cellular injury induced by LPC could be attenuated by d-propranolol, although the mechanism remains unknown.

Inhibition of Na+ Channel or Na+/H+ Exchanger Attenuate the Hydrogen Peroxide-induced Derangements in Isolated Perfused Rat Heart

The effect of tetrodotoxin, a specific inhibitor of the Na+ channel, and 5‐(N,N‐dimethyl)‐amiloride, a specific inhibitor of the Na+/H+ exchanger, on the mechanical and metabolic derangements induced by hydrogen peroxide (H2O2) was studied in the isolated perfused rat heart. The isolated rat heart was perfused aerobically at a constant flow rate and driven electrically.

Lysophosphatidylcholine: A Possible Modulator of Ischemic Injury in the Heart

Lysophosphatidylcholine (LPC) is an amphiphilic metabolite produced from phosphatidylcholine, a membrane phospholipid, by activation of phospholipase A2, which accumulates in the heart during ischemia and reperfusion. Because of its amphiphilic property, LPC is readily incorporated into the lipid bilayers of the cell membrane, changing the physicochemical property of the cell membrane and thereby affecting the enzymes and ion channels embedded in the membrane. Recent studies have revealed that exogenous LPC produces mechanical and metabolic derangements in the perfused working heart and also induces Ca2+ overload in the isolated cardiac myocyte, suggesting that LPC possesses an ischemia-like deleterious effect on the heart. In this chapter, we review the deleterious effects of LPC on the myocardium and isolated cardiomyocytes. Further, we discuss the possible mechanisms of action of drugs that can protect the heart against LPC-induced myocardial damage.

Both D-cis- and L-cis-diltiazem attenuate hydrogen peroxide-induced derangements in rat hearts

The effects of D-cis- and L-cis-diltiazem on the hydrogen peroxide (H2O2)-induced derangements of mechanical function and energy metabolism, and accumulation of intracellular Na+ were studied in isolated rat hearts. The intracellular concentration of Na+ ([Na+]i) in the myocardium was measured using a nuclear magnetic resonance technique. H2O2 (600 microM) increased the left ventricular end-diastolic pressure, decreased the tissue level of ATP, and increased the release of lactate dehydrogenase from the myocardium. These alterations induced by H2O2 were significantly attenuated by D-cis-diltiazem (15 microM) or L-cis-diltiazem (15 microM). H2O2 (1 mM) produced a marked increase in the myocardial [Na+]i, which was effectively inhibited by tetrodotoxin (3 microM), D-cis-diltiazem (15 microM) or L-cis-diltiazem (15 microM). These results suggest that both D-cis- and L-cis-diltiazem protect the myocardium against the H2O2-induced derangements in the isolated, perfused rat heart. The protective action of D-cis- and L-cis-diltiazem may be due to their ability to inhibit the H2O2-induced increase in [Na+]i, at least in part.