Nitroarginine does not inhibit lysophosphatidylcholine (LPC)-induced vascular relaxation and accumulation of cyclic GMP.

Lysophosphatidylcholine (LPC) is a potent endothelium-dependent vascular smooth muscle relaxant. The possibility that its action is mediated through endothelium-derived nitric oxide (EDNO), although suggestive, has not been proven. Both lysophosphatidylcholine and endothelium-derived nitric oxide relax by activating guanylate cyclase to form cyclic GMP. Based on the finding that EDNO formation is inhibited by NNA (N-omega-nitro-L-arginine), we followed cyclic GMP changes in bovine intrapulmonary arteries with LPC after incubation with NNA. Inhibition of cyclic GMP by LPC following NNA exposure would be suggestive of the production of EDNO by LPC. However, while NNA significantly inhibited accumulation of cyclic GMP after exposure to the calcium ionophore A23187 which releases EDNO, NNA failed to inhibit LPC-induced accumulation of cyclic GMP. The results indicate that LPC relaxes vascular smooth muscle through a non NO-mediated pathway.     

Parathyroid hormone-induced changes in cyclic nucleotide levels during relaxation of the rabbit [correction of rat] aorta

Parathyroid hormone is a potent vasodilator in vivo and relaxes vascular tissue in vitro. Since parathyroid hormone action in kidney and bone is thought to be mediated by stimulation of cellular cyclic AMP production, the present study was designed to monitor changes in cyclic AMP and cyclic GMP in vascular tissue during relaxation by parathyroid hormone. Rabbit aortic strips were quick-frozen at various times after exposure to parathyroid hormone and the percent relaxation and cyclic nucleotide levels were determined. Cyclic AMP concentrations were elevated about 3-fold within 30 seconds after treatment with hormone. This corresponded to a 10% relaxation of the norepinephrine-contracted tissue. After five minutes, cyclic AMP was still elevated 2-fold above basal and the relaxation response was maximal (36%). The cyclic AMP and relaxation responses to parathyroid hormone were markedly potentiated by forskolin or methylisobutylxanthine. Parathyroid hormone produced a small but significant increase in cyclic GMP concentrations only at early time points whereas sodium nitroprusside substantially increased cyclic GMP and relaxed the strips at all times studied. The increase in cyclic AMP levels after exposure to parathyroid hormone occurred prior to or coincident with the onset of relaxation of the aortic strips. These findings are supportive of the hypothesis that the vascular actions of parathyroid hormone involve cyclic AMP.     

A model to study physiological activation of phospholipase A2 and vasorelaxation by lysophosphatidylcholine

Earlier we demonstrated that micellar solutions of LPC caused endothelium-dependent relaxation of rabbit thoracic aorta and bovine intrapulmonary artery and vein through a cyclic GMP-dependent mechanism. The availability of LPC for vasorelaxation depends on its production by deacylation of PC by PLA2. We assessed the possible activation of PLA2 by commonly used vasorelaxants such as acetylcholine, bradykinin, calcium ionophore A23187 and thrombin and vasoconstrictors like histamine and phenylephrine in the presence of indomethacin in a model system where 14C PC was incorporated into bovine intrapulmonary arterial segments. Taking the ratio of 14C PC:LPC formed by exogenous PLA2 as an index of deacylation, we found that while all the agents relaxed the strips in an endothelium-dependent manner, only thrombin caused relaxation followed by an increase in 14C LPC and a concomittant decrease in 14C PC indicating activation of PLA2. Our data show that PC/PLA2 system can be activated to generate LPC for vascular relaxation under specific physiological conditions. This model system can be used to monitor PLA2 activity and LPC production to compensate flow and pressure induced changes in arteries.     

Atriopeptin II elevates cyclic GMP, activates cyclic GMP-dependent protein kinase and causes relaxation in rat thoracic aorta

Synthetic atriopeptin II, an atrial natriuretic factor with potent vasodilatory effects, was studied in isolated strips of rat thoracic aorta to determine its actions on contractility, cyclic nucleotide concentrations and endogenous activity of cyclic nucleotide-dependent protein kinases. Atriopeptin II was found to relax aortic strips precontracted with 0.3 microM norepinephrine whether or not the endothelial layer was present. Relaxation to atriopeptin II was closely correlated in a time- and concentration-dependent manner with increases in cyclic GMP concentrations and activation of cyclic GMP-dependent protein kinase (cyclic GMP-kinase). The threshold concentration for all three effects was 1 nM. Atriopeptin II (10 nM for 10 min) produced an 80% relaxation, an 8-fold increase in cyclic GMP concentrations and a 2-fold increase in cyclic GMP-kinase activity ratios. Atriopeptin II did not significantly alter cyclic AMP concentrations or cyclic AMP-dependent protein kinase activity. These data suggest that cyclic GMP and cyclic GMP-kinase may mediate vascular relaxation to a new class of vasoactive agents, the atrial natriuretic factors. Similar effects have been observed with the nitrovasodilator, sodium nitroprusside, and the endothelium-dependent vasodilator, acetylcholine. Therefore, a common biochemical mechanism of action that includes cyclic GMP accumulation and activation of cyclic GMP-kinase may be involved in vascular relaxation to nitrovasodilators, endothelium-dependent vasodilators and atrial natriuretic factors.     

Effects of potassium chloride and smooth muscle relaxants on tension and cyclic nucleotide levels in rat myometrium.

Ten minutes after KCl-depolarization of rat myometrial strips, at which time the muscles were in a state of sustained contracture, tissue levels of adenosine 3',5'-cyclic monophosphate (cyclic AMP) were increased by approximately 40% over relaxed controls, and levels of guanosine 3',5'-cyclic monophosphate (cyclic GMP) were decreased by 40%. At this point both nitroglycerin (4 X 10(-4) M) and papaverine (2 X 10(-5) M) were capable of relaxing the depolarized muscles without significantly increasing cyclic AMP levels. Isoproterenol, in concentrations from 5 X 10(-9) M to 10(-6) M, relaxed the depolarized muscles and significantly increased tissue levels of cyclic AMP. However, the magnitudes of the cyclic AMP increases seen after the lower concentrations of isoproterenol were small relative to the increases observed during KCl-contracture alone. For example, the 40% elevation of cyclic AMP seen 10 min after KCl-depolarization did not cause the muscles to relax, whereas 5 X 10(-9) M isoproterenol caused relaxation with an increase in cyclic AMP levels of only 16% over depolarized controls. It was concluded that changes in total tissue levels of cyclic AMP were not responsible for the uterine relaxation caused by nitroglycerin, papaverine or isoproterenol in these experiments. Cyclic GMP levels in the depolarized muscles were not significantly changed by isoproterenol or papaverine but were increased approximately 80% by nitroglycerin. The above results are not consistent with the previously suggested roles for cyclic GMP and cyclic AMP as mediators of smooth muscle contraction and relaxation, respectively.     

Prostacyclin increases cAMP in coronary arteries.

This study was designed to determine whether prostacyclin (PGI2)-induced relaxation in circular strips of coronary arteries might be mediated by cAMP. Partially depolarized circular strips of bovine coronary arteries were used and PGI2-induced changes in length were compared with tissue concentrations of cAMP and cGMP, measured by RIA. It was found that PGI2 produced significant and concentration dependent increases in cAMP levels which were closely associated with the relaxant effects produced by the same concentrations (0.3 - 26.7 muM). Cyclic GMP was not changed by these concentrations. The relaxant effects of PGI2 were not antagonized by propranolol. There was a significant linear correlation between log increases in cAMP and percent relaxation produced by PGI2 which was almost identical with similar correlations obtained with either isoprenaline or adenosine, indicating that the relaxant effects of PGI2 are in analogy to those of isoprenaline and adenosine likely to be mediated by cAMP.     

Effects of LY83583, nordihydroguaiaretic acid, and quinacrine on cyclic GMP elevation and inhibition of tension by muscarinic agonists in rabbit aorta and left atrium

Elevation of cyclic GMP by muscarinic agonists has been suggested to be responsible for the negative inotropic effects of these agents in cardiac muscle, and for the endothelium-dependent relaxation caused by these agents in vascular smooth muscle. These relationships were studied by monitoring the effects of muscarinic agonists on tension and cyclic GMP levels in rabbit left atrial strips and aortic rings, in the presence and absence of the cyclic GMP lowering agent, LY83583. LY83583 completely blocked both the cyclic GMP increase and the relaxation caused by acetylcholine in rabbit aortic rings with intact endothelial cells. Acetylcholine-induced cyclic GMP elevation and relaxation in these preparations were also blocked by quinacrine and nordihydroguaiaretic acid (NDGA), but neither response was blocked by the 5-lipoxygenase inhibitor U-60257. In the experiments with rabbit left atrium, LY83583 blocked the acetylcholine-induced cyclic GMP elevation but did not block the negative inotropic effects of the drug. Quinacrine, NDGA, and a guanylate cyclase inhibitor, methylene blue, failed to block either the cyclic GMP increase or the decrease in contractile force caused by carbachol in atrial strips. These results support the suggestion that an increase in cyclic GMP may be responsible for the endothelium-dependent relaxation of rabbit aorta by muscarinic agonists, but not for the direct negative inotropic effects of these drugs in rabbit atrium. Muscarinic agents appear to increase cyclic GMP levels in rabbit atrium and aorta by different mechanisms. Although both are blocked by LY83583, they differ not only in their requirements for endothelial cells, but also in their susceptibility to other blocking agents.     

Albumin inhibits cytotoxic activity of lysophosphatidylcholine by direct binding

Fetal bovine serum (FBS) was found to protect Jurkat T cells from LPC-induced cytotoxicity. Twenty micromolar LPC-induced cytotoxicity of 80-90% of the cells in media without FBS for 3 h, whereas 50-70% in media with 0.5% FBS. However, LPC-induced cytotoxicity was not observed in the presence of 5% FBS in media. The cytotoxicity was specific for LPC among lysophospholipids tested and significantly observed with palmitoyl (C16:0) LPC, stearoyl (C18:0) LPC, and oleoyl (C18:1) LPC among 11 synthetic LPCs. Furthermore, the cytoprotective effect of FBS was observed only when it was added before the treatment, but not after the treatment of LPC, and premixing of FBS and LPC before addition to the cells ameliorated LPC-induced cytotoxicity. Finally, albumin, a major constituent of FBS, prevented completely LPC-induced cytotoxicity even at as low as 3 microM concentration. We also found that five molecules of LPC could sequentially bind to one BSA using isothermal titration calorimetry. The above results suggest that the cytotoxic activity of LPC could be attenuated by albumin in blood. Finally, it should be cautioned that, when experiments are conducted with LPC dissolved in assay buffers containing albumin, the albumin in the buffer could influence the results.     

Differences in the association of calmodulin with cyclic nucleotide phosphodiesterase in relaxed and contracted arterial strips

Changes in the concentration of cytosolic Ca2+ are assumed to alter the activity of Ca2+-calmodulin-sensitive cyclic nucleotide phosphodiesterase in intact cells. However, this assumption is based on indirect evidence and by analogy from studies of enzyme activities in broken cell systems. We have developed a procedure for estimating the fraction of Ca2+-calmodulin-sensitive phosphodiesterase that is in an activated, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) sensitive state in intact porcine coronary artery strips. The experimental approach involves homogenization of the strips and assay of cyclic guanosine monophosphate (cyclic GMP) phosphodiesterase activity under conditions that retard changes in the amount of the complex Ca2+-calmodulin-phosphodiesterase. Our findings indicate that cyclic GMP phosphodiesterase in intact coronary artery strips does associate with Ca2+-calmodulin and that interventions that change the concentration of Ca2+ in the cytosol of the intact strip change the extent of this functional association. Exposure to histamine (10 or 100 microM) or 50 mM KCl caused contraction and an increase in EGTA-sensitive cyclic GMP phosphodiesterase activity. Isoproterenol-induced relaxation of tissues that had been caused to contract with 10 microM histamine was accompanied by a reduction in EGTA-sensitive cyclic GMP phosphodiesterase activity to the same level as that present before contraction was initiated.     

Myosin light chain phosphorylation in contraction and relaxation of intact rat thoracic aorta

Myosin light chain phosphorylation in intact rat thoracic aorta was elevated during contraction induced by 0.3 microM norepinephrine, but was not maintained. Addition of 0.5 microM sodium nitroprusside to norepinephrine treated rat aorta strips led to elevation of cyclic GMP levels, relaxation of tension, and dephosphorylation of myosin light chain. Depletion of extracellular calcium or addition of calmodulin antagonists trifluoperazine and W7 diminished the contraction and phosphorylation of myosin light chain by norepinephrine, but did not prevent dephosphorylation by sodium nitroprusside or the elevated levels of cyclic GMP. Isoproterenol, 8-bromo cyclic GMP, and dibutyryl cyclic AMP all caused dephosphorylation of myosin light chain and induced relaxation during the period of development of tone. Eight other proteins had increased phosphorylation following norepinephrine treatment and one protein had less phosphorylation. The different proteins phosphorylated by norepinephrine showed varying degrees of sensitivity to Ca2+-free solution and to the calmodulin antagonists. The pattern of protein phosphorylation caused by sodium nitroprusside was best mimicked by 8-bromo cyclic GMP, rather than isoproterenol and dibutyryl cyclic AMP. These proteins were, generally, unaffected by Ca2+-free solution and the calmodulin antagonists. The present observations support the hypothesis that vasodilators inhibit tone development through myosin light chain dephosphorylation. Furthermore, the nitrovasodilators act through elevation of cyclic GMP and phosphorylation of proteins by cyclic GMP-dependent protein kinase.     

Non-selective cation channel activity is required for lysophosphatidylcholine-induced monocyte migration

Lysophosphatidylcholine (LPC) is a major atherogenic lipid which stimulates the recruitment of monocytes to atherosclerotic lesions. The physiological mechanisms underlying LPC-induced monocyte migration are poorly understood. Here we demonstrate that LPC activates non-selective cation channels, which are significantly involved in LPC-induced chemotaxis of monocytes. External LPC elicited the activation of non-selective cation currents in THP-1 monocytes, which occurred in a G protein and phospholipase C-independent manner. LPC-activated currents were almost completely inhibited by Gd³⁺, La³⁺, and TRAM-34. Furthermore, currents were partially reduced by either 2-aminoethoxydiphenyl borate (2-APB) or ruthenium red, while combined application of 2-APB and ruthenium red abolished LPC-activated currents. The 2-APB-sensitive current component was potentiated by flufenamic acid and Ca²⁺-free extracellular solution, while the ruthenium red-sensitive current component was abolished by capsazepine. This pharmacological profile suggests that LPC simultaneously activates TRPC6 and TRPV1 channels in monocytes. Furthermore, in the presence of Gd³⁺, La³⁺, TRAM-34, 2-APB, ruthenium red or capsazepine, LPC-induced chemotaxis of monocytes was substantially inhibited, indicating that activation of both channel types is required for optimal migration of LPC-stimulated monocytes. Thus, ion channel inhibition may represent a powerful strategy to attenuate the progression of atherosclerosis by reducing monocyte infiltration. J. Cell. Physiol. 221: 325–334, 2009. © 2009 Wiley-Liss, Inc.     

NG-nitro-L-arginine inhibits non-adrenergic, non-cholinergic relaxation in rabbit urethral smooth muscle.

Electrical field stimulation induced a relaxation response in female rabbit urethral smooth muscle strips precontracted with phenylephrine. The relaxation response was inhibited by tetrodotoxin, but not by atropine, propranolol, or hexamethonium. The relaxation response thus results from stimulation of inhibitory non-adrenergic, non-cholinergic nerves. The electrically induced relaxation response was inhibited by an inhibitor of nitric oxide biosynthesis, NG-nitro-L-arginine. This inhibition was overcome by addition of a precursor of nitric oxide, L-arginine. An inhibitor of soluble guanylate cyclase, methylene blue, reduced the relaxation response, and a selective cyclic GMP phosphodiesterase inhibitor, M & B 22948, potentiated the relaxation response. These data indicate that agents which affect the biosynthesis of nitric oxide are associated with the urethral relaxation response evoked by electrical field stimulation, and that cyclic GMP may mediate the relaxation response.     

A study on sensing and adaptation in Dictyostelium discoideum: guanosine 3'',5''-phosphate accumulation and light-scattering responses

Cells of Dictyostelium discoideum respond to extracellular cyclic AMP with marked changes in intracellular cyclic GMP levels and light scattering. In this work, defined temporal increases in cyclic AMP were produced by the continuous addition of cyclic AMP to agitated suspensions of cells; concomitant hydrolysis of cyclic AMP by the cells subsequently established a constant, steady state concentration. The cells responded to the initial increase in extracellular cyclic AMP with a rapid increase in the intracellular cyclic GMP concentration and a rapid decrease in light scattering. At cyclic AMP input rates of 0.5-5 nM X s-1, the fast reactions of cyclic GMP and light scattering had already relaxed while the cyclic AMP concentration in the cell suspension was still increasing. The cells responded to constant concentrations of cyclic AMP with constant elevated cyclic GMP concentrations and constant decreased levels of light scattering. Our results are consistent with the existence of two types of perception systems, one of which adapts to constant stimuli and one of which does not adapt.     

Characteristics of lysophosphatidylcholine-induced Ca2+ response in human neuroblastoma SH-SY5Y cells

Lysophosphatidylcholine (LPC) is an important bioactive lipid. In the nervous system, elevated levels of LPC have been shown to produce demyelination. In the present study, we examined the effect of exogenous LPC on intracellular Ca2+ mobilization in human neuroblastoma SH-SY5Y cells. In Ca2+-containing medium, introduction of LPC induced a steady rise in cytosolic Ca2+ levels ([Ca2+]i) in a dose-dependent manner, and this rise was provoked by LPC itself, not by its hydrolysis product produced by lysophospholipase. The increase in [Ca2+]i was reduced by 36% by removal of extracellular Ca2+, while preincubation of the cells with verapamil, an L-type Ca2+ channel blocker, inhibited the response by 23%, part of the Ca2+ influx. Conversely, Ni2+, which inhibits the Na+-Ca2+ exchanger, or Na+-deprivation did not affect LPC-induced Ca2+ influx. In Ca2+-free medium, depletion of Ca2+ stores in the endoplasmic reticulum (ER) by thapsigargin, an ER Ca2+-ATPase inhibitor, abolished the Ca2+ increase. Moreover, LPC-induced [Ca2+]i increase was fully blocked by ruthenium red and procaine, inhibitors of ryanodine receptor (RyR), but was not affected by 2-aminoethoxydiphenyl borate, an inhibitor of inositol triphosphate receptor, or by pertussis toxin, a G(i/o) protein inhibitor. Combined treatment with verapamil plus thapsigargin markedly inhibited but did not abolish the LPC-induced Ca2+ response. These findings indicate that LPC-induced [Ca2+]i increase depends on both external Ca2+ influx and Ca2+ release from ER Ca2+ stores, in which L-type Ca2+ channels and RyRs may be involved. However, in digitonin-permeabilized SH-SY5Y cells, LPC could not induce any [Ca2+]i increase in Ca2+-free medium, suggesting that LPC may act indirectly on RyRs of ER.     

Lysophosphatidylcholine increases endothelial permeability: role of PKCalpha and RhoA cross talk

Lysophosphatidylcholine (LPC) is a bioactive proinflammatory lipid that can be generated by pathological activities. We investigated the hypothesis that LPC signals increase in endothelial permeability. Stimulation of human dermal microvascular endothelial cells and bovine pulmonary microvascular endothelial cells with LPC (10-50 microM) induced decreases (within minutes) in transendothelial electrical resistance and increase of endothelial permeability. LPC activated (within 5 min) membrane-associated PKC phosphotransferase activity in the absence of translocation. Affinity-binding analysis indicated that LPC induced increases (also by 5 min) of GTP-bound RhoA, but not Rac1 or Cdc42. By 60 min, both signaling pathways decreased toward baseline. Inhibition of RhoA with C3 transferase inhibited approximately 50% of LPC-induced resistance decrease. Pretreatment with PKC inhibitor G?-6983 (concentrations selective for classic PKC), PMA-induced depletion of PKCalpha, and transfection of antisense PKCalpha oligonucleotide each prevented 40-50% of the LPC-induced resistance decrease. Furthermore, these three PKC inhibition strategies inhibited 60-80% of the LPC-induced GTP-bound RhoA. These results show that LPC directly impairs the endothelial barrier function that was dependent, at least in part, on cross talk of PKCalpha and RhoA signals. The evidence indicates that elevated LPC levels can contribute to the activation of a proinflammatory endothelial phenotype.     

Effects of isoproterenol,theophylline and carbachol on cyclic nucleotide levels and relaxation of bovine tracheal smooth muscle

The effect of theophylline and isoproterenol on bovine tracheal smooth muscle tension and cyclic AMP levels was investigated. Concentrations of isoproterenol (4 × 10^?6 M) and theophylline (10 mM) that relaxed carbachol-contracted tracheal muscle by 85–95% did not significantly elevate control levels of cyclic AMP. In the absence of carbachol, several-fold increases in cyclic AMP were caused by isoproterenol although no elevations by theophylline were measurable. However, when isoproterenol and theophylline were administered together, theophylline potentiated the rise in cyclic AMP caused by isoproterenol. Phosphodiesterase studies in tracheal muscle showed the presence of a high and a low K_m enzyme which were inhibited by theophylline. Cyclic GMP levels were elevated in muscles contracted by carbachol as well as in carbachol-contracted muscles that had been relaxed by theophylline. In non-tension studies, in which the tracheal muscle was not under isometric tension, carbachol or theophylline alone increased cyclic GMP and together they synergistically elevated cyclic GMP. Atropine blocked the elevation caused by carbachol but not that caused by theophylline. In contrast to theophylline, isoproterenol did not elevate cyclic GMP, and in carbachol-contracted muscles that had been relaxed by isoproterenol, cyclic GMP levels were no different from control. Also, in non-tension studies, isoproterenol decreased basal cyclic GMP and antagonized the increase in cyclic GMP due to carbachol.The results indicate that whole-tissue levels of cyclic AMP and cyclic GMP do not correlate with the state of tracheal smooth muscle tension. Cyclic GMP levels do not clearly correlate with either contraction or relaxation. The inhibition by carbachol of increases in cyclic AMP due to isoproterenol and the inhibition by isoproterenol of increases in cyclic GMP due to carbachol provide evidence for a reciprocal cholinergic-adrenergic antagonism of cyclic AMP and cyclic GMP levels. The antagonism did not appear to be due to either cyclic nucleotide affecting the elevation of the other since the levels of both cyclic nucleotides were depressed.     

Pilus backbone protein PitB of Streptococcus pneumoniae contains stabilizing intramolecular isopeptide bonds

Naturally occurring CD4⁺CD25⁺ regulatory T cells (nTregs) play a pivotal role in the maintenance of self-tolerance and immune homeostasis. To gain insight into the mechanism of action of nTregs in pathological and physiological immune responses, it is important to analyze bioactive molecules that modulate the maintenance and function of nTregs. From a library of bioactive lipids, we obtained lysophosphatidylcholine (LPC) as a molecule that enhanced the Foxp3 expression and suppressive function of human nTregs significantly in comparison with those of DMSO-treated nTregs (control). The expression levels of TGF-β1 mRNA and protein in LPC-treated nTregs were significantly higher than those in control nTregs. After treatment with anti-TGF-β1 antibody, the increases in Foxp3 expression and the suppressive properties of LPC-treated nTregs returned to the levels observed in control nTregs. These findings indicate that LPC enhances Foxp3 expression and the suppressive function of nTregs through TGF-β1 produced by nTregs themselves. Experimental knockdown of G2A and GPR4 showed that this LPC-induced TGF-β1 expression in nTregs was due to G2A signaling, and did not involve GPR4. Moreover, JNK was a major contributor to LPC-induced TGF-β1 expression in nTregs, although LPC activated MAPKs including ERK1/2, p38 MAPK, and JNK via G2A. LPC is a bioactive lysolipid highly abundant in the circulation. Therefore, LPC may contribute to the maintenance and function of human nTregs in vivo.     

Procyanidins in crataegus extract evoke endothelium-dependent vasorelaxation in rat aorta

The extract of Crataegus, a mixture of flavonoids and procyanidins extracted from hawthorn, Crataegus oxyacantha, L. and C. monogyna Jacq., relaxed vascular tone or increased production of cyclic GMP in the rat aorta, but flavonoid components of Crataegus extract, hyperoside, rutin and vitexin, did not affect the vascular tone. The aim of the present study was to characterize the endothelium-dependent relaxation elicited by procyanidins fractionated from Crataegus extract in isolated rat aorta. Procyanidins caused endothelium-dependent relaxation which was associated with the production of cyclic GMP. Both responses to these procyanidins were inhibited by methylene blue or N(G)-nitro-L-arginine, but not by indomethacin. Relaxation in response to procyanidins was not affected by atropine, diphenhydramine, [D-Pro2,D-Trp7,9]substance P, propranolol, nifedipine, verapamil and glibenclamide, but were markedly reduced by tetraethylammonium. These findings showed that procyanidins in Crataegus extract may be responsible for the endothelium-dependent nitric oxide-mediated relaxation in isolated rat aorta, possibly via activation of tetraethylammonium-sensitive K+ channels.     

Lysophosphatidylcholine-induced myocardial damage is inhibited by pretreatment with poloxamer 188 in isolated rat heart

Lysophosphatidylcholine (LPC) accumulates in myocardial tissues and coronary sinus during ischemia, and plays important role in the development of ischemia-reperfusion injury and ischemic ventricular arrhythmia. The aim of this study was to examine whether pretreatment of poloxamer 188 (P-188), a nonionic and non-toxic surfactant, can prevent the cardiac dysfunction induced by exogenous LPC perfusion in Langendorff perfused rat heart model. LPC (6 M) significantly (p < 0.05) decreased heart rate (HR) and left ventricular developed pressure (LVDP) from 274.3 ± 23.2 to 175.0 ± 42.9/min and from 115.9 ± 11.3 to 26.7 ± 7.1 mmHg, respectively. The LPChyphen;induced reduction of HR and LVDP did not recover by washout of LPC. Pretreatment with P-188 (1 mM for 30 min) inhibited completely the LPC-induced decreases of HR and LVDP. The pretreatment with P-188 also prevented the LPC-induced increases of left ventricular end-diastolic pressure (LVEDP) and GOT release, significantly (p < 0.05). The coronary perfusion pressure (CPP) rose (p < 0.01) by the LPC perfusion from 71.9 ± 5.3 to 121.9 ± 13.0 mmHg, significantly, but pretreatment of P-188 did not affect the LPC-induced vasoconstriction. Our results suggest that exogenous LPC causes irreversible cardiac injury by the sarcolemmal membrane disruption followed by Ca overload, and this LPC-induced cardiac injury, probably, can be prevented by the pretreatment with poloxamer 188.     

Release of prostacyclin,endothelium-derived relaxing factor and endothelin by freshly harvested cells attached to microcarrier beads

Summary Cultured endothelial cells have been used in the past as a source of endothelium-derived relaxing factor (EDRF) and of prostacyclin (PGI₂). Although cell cultures are essential for observation of prolonged exposure to media or when there is delayed response, they are time consuming and sterile conditions are essential. In the present study, we report that endothelial cells, freshly harvested from bovine aortas, readily attached themselves to cytodex-3 microcarrier beads and released an endothelium-derived relaxing factor (EDRF), prostacyclin (PGI₂) and increased the amount of cyclic GMP in vascular smooth muscle. Attachment to microcarrier beads was essential since it increased the surface area and the number of attached cells and permited collection of cell free filtrates because of the formation of dense networks of cells and beads. As a result superfusion of cells and beads on the filter did not dislodge bound cells which remain on the filter. Conditioned filtrates from freshly harvested endothelial cells attached to microcarrier beads caused marked relaxation of endothelium-deprived bovine pulmonary artery strips. The degree of relaxation depended on the number of cells; maximal relaxation occurred with 50 million cells at ED₅₀ of 14 million. High values of cyclic GMP were found in vascular smooth muscle exposed to conditioned filtrate. The calcium ionophore A23187 further increased the amount of cyclic GMP. Large amounts of PGI₂ were released by freshly harvested endothelial cells particularly after stimulation with the calcium ionophore. In contrast, endothelin production by freshly harvested cells attached to microcarrier beads was barely detectable after 30 min incubation and was beyond the limit of detection by bioassay procedures. Freshly harvested endothelial cells attached to microcarrier beads appear to be a useful adjunct to tissue cultures under specific experimental conditions.Abbreviations EDRF Endothelium-Derived Relaxing Factor - PGI₂ Prostacyclin - K-H Krebs-Henseleit solution - cyclic GMP cyclic Guanosine Monophosphate - fmoles femtomoles - IB Ibuprofen