Rabbit Coronary Artery Research Articles

Testosterone and dihydrotestosterone inhibit gallbladder motility through multiple signalling pathways

Testosterone (T) has been shown to cause vasodilation in rabbit coronary arteries through a nongenomic pathway. Part of this T-induced relaxation was shown to be mediated by opening voltage dependent K + channels. T infusion also reduces peripheral resistance in human males with heart failure. The effects of T or its active metabolite 5-α dihydrotestosterone (DHT) are not well studied. This study investigates the effect of T and DHT on contraction in guinea pig gallbladder strips. T or DHT induced a concentration-dependent relaxation of cholecystokinin octapeptide (CCK)-induced tension. Pretreatment of the strips with PKA inhibitor 14–22 amide myristolated had no significant effect on the relaxation induced by either T or DHT. Pretreatment of strips with 2-APB, an inhibitor of IP 3 induced Ca 2+ release, produced a significant ( p < 0.001) reduction in the T- or DHT-induced relaxation. Bisindolymaleimide IV and chelerythrine Cl − when used in combination had no significant effect on the amount of CCK-induced tension, but significantly ( p < 0.01) decreased the amount of T- or DHT-induced relaxation. The flavone chrysin, an aromatase inhibitor, and genistein, an isoflavone, each produced a significant ( p < 0.01) reduction in CCK-induced tension. Chrysin significantly ( p < 0.05) increased T-induced relaxation; however, genistein had no effect on T-induced relaxation. It is concluded that T and DHT inhibits gallbladder motility rapidly by nongenomic actions of the hormones. Multiple pathways that include inhibition of intracellular Ca 2+ release, inhibition of extracellular Ca 2+ entry, and the actions of PKC may mediate this effect.

Diverse properties of store‐operated TRPC channels activated by protein kinase C in vascular myocytes

In vascular smooth muscle, store-operated channels (SOCs) contribute to many physiological functions including vasoconstriction and cell growth and proliferation. In the present work we compared the properties of SOCs in freshly dispersed myocytes from rabbit coronary and mesenteric arteries and portal vein. Cyclopiazonic acid (CPA)-induced whole-cell SOC currents were sixfold greater at negative membrane potentials and displayed markedly different rectification properties and reversal potentials in coronary compared to mesenteric artery myocytes. Single channel studies showed that endothelin-1, CPA and the cell-permeant Ca(2+) chelator BAPTA-AM activated the same 2.6 pS SOC in coronary artery. In 1.5 mM [Ca(2+)](o) the unitary conductance of SOCs was significantly greater in coronary than in mesenteric artery. Moreover in 0 mM [Ca(2+)](o) the conductance of SOCs in coronary artery was unaltered whereas the conductance of SOCs in mesenteric artery was increased fourfold. In coronary artery SOCs were inhibited by the protein kinase C (PKC) inhibitor chelerythrine and activated by the phorbol ester phorbol 12,13-dibutyrate (PDBu), the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) and a catalytic subunit of PKC. These data infer an important role for PKC in activation of SOCs in coronary artery similar to mesenteric artery and portal vein. Anti-TRPC1 and -TRPC5 antibodies inhibited SOCs in coronary and mesenteric arteries and portal vein but anti-TRPC6 blocked SOCs only in coronary artery and anti-TRPC7 blocked SOCs only in portal vein. Immunoprecipitation showed associations between TRPC1 and TRPC5 in all preparations but between TRPC5 and TRPC6 only in coronary artery and between TRPC5 and TRPC7 only in portal vein. Finally, flufenamic acid increased SOC activity in coronary artery but inhibited SOCs in mesenteric artery and portal vein myocytes. These data provide strong evidence that vascular myocytes express diverse SOC isoforms, which are likely to be composed of different TRPC proteins and have different physiological functions.

Progestins oppose the effects of estradiol on the endothelin-1 receptor type B in coronary arteries from ovariectomized hyperlipidemic rabbits

Progestins may be associated with the adverse cardiovascular outcomes observed with estrogen plus progestogen therapy, but the mechanism is not resolved. In this study we examined the effect of 17beta-estradiol (E2) alone and in combination with two progestins on the endothelin-1 (ET-1) system in coronary arteries. Watanabe heritable hyperlipidemic rabbits were treated orally with either E2 (4 mg/d), medroxyprogesterone acetate (MPA) (10 mg/d), norethisterone acetate (NETA) (2 mg/d), E2 + MPA, E2 + NETA, or placebo for 16 weeks (n=10 in each group). Coronary arteries were used for mRNA and myograph analyses. E2 alone but not in combination with MPA or NETA increased ETB mRNA expression in coronary arteries. Accordingly, E2 alone but not in combination with MPA or NETA reduced the maximal contraction to ET-1, and the reduction was sustained after ETA but not after ETB blockade. E2 reduced preproendothelin-1 mRNA expression; however, this effect was not blunted by MPA or NETA. ETA and ET-converting enzyme-1 mRNA expression was unaffected by treatment. The data suggest that long-term E2 treatment selectively attenuates ET-1-induced vasoconstriction, possibly by increasing ETB gene expression in rabbit coronary arteries and that this effect is abolished by the two progestins investigated. This observation may help to explain how progestins oppose the supposed beneficial effects of estrogen on the arterial wall.

Comparison of contractile mechanisms of sphingosylphosphorylcholine and sphingosine‐1‐phosphate in rabbit coronary artery

We investigated the difference of patterns and specific mechanisms between SPC‐ and S1P‐induced contraction in rabbit coronary artery. Contractile responses in normal and permeabilized strips, changes in [Ca2+]i, and phosphorylation of MYPT1 were measured. SPC induced a gradual and sustained contraction. S1P induced initial transient and secondary sustained force. In isolated single cells, SPC induced a slight increase in [Ca2+]i but S1P significantly increased [Ca2+]i. In the absence of extracellular Ca2+, S1P‐induced [Ca2+]i increase was significantly inhibited, but initial transient increase in [Ca2+]i was still remained, which was completely abolished by CPA. In constant Ca2+ levels, SPC induced significant augmentation of the pCa6.3‐induced force but S1P induced little or no augmentation. Pretreatment of fasudil significantly inhibited SPC‐induced contraction. SPC induced several folds increase in Thr696 and Thr853 phosphorylation of MYPT1 but S1P did not significantly affect. Our results suggest that SPC‐induced contraction shows gradual and sustained pattern but S1P‐induced contraction shows initial transient and secondary sustained pattern. Furthermore, activation of RhoA/ROCK pathway and phosphorylation of MYPT1 may be key mechanisms for SPC‐induced contraction but elevation of [Ca2+]i may be a key mechanism for S1P‐induced contraction.

Acute hypoxia induces vasodilation and increases coronary blood flow by activating inward rectifier K+ channels

We examined the effects of acute hypoxia on vascular tone and coronary blood flow (CBF) in rabbit coronary arteries. In the pressurized arterial preparation of small arteries (<100 mum) and the Langendorff-perfused rabbit hearts, hypoxia induced coronary vasodilation and increased CBF in the presence of glibenclamide (K(ATP) channel blocker), Rp-8-Br-PET-cGMPs [cyclic guanosine monophosphate (cGMP)-dependent protein kinase inhibitor, Rp-cGMPs], and methionyl transfer RNA synthetase (MRS) 1334 (adenosine A(3) receptor inhibitor); these increases were inhibited by the inward rectifier K(+) (Kir) channel inhibitor, Ba(2+). These effects were blocked by the adenylyl cyclase inhibitor SQ 22536 and by the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) inhibitors Rp-8-CPT-cAMPs (Rp-cAMPs) and KT 5720. However, cGMP-dependent protein kinase was not involved in the hypoxia-induced increases of the vascular diameter and CBF. In summary, our results suggest that acute hypoxia can induce the opening of Kir channels in coronary artery that has small diameter (<100 mum) by activating the cAMP and PKA signalling pathway, which could contribute to vasodilation and, therefore, increased CBF.

Endothelin‐1 activates a Ca2+‐permeable cation channel with TRPC3 and TRPC7 properties in rabbit coronary artery myocytes

In the present work we used patch pipette techniques to study the properties of a novel Ca(2+)-permeable cation channel activated by the potent coronary vasoconstrictor endothelin-1 (ET-1) in freshly dispersed rabbit coronary artery myocytes. With cell-attached recording bath application of 10 nm ET-1 evoked cation channel currents (I(cat)) with subconductance states of about 18, 34 and 51 and 68 pS, and a reversal potential of 0 mV. ET-1 evoked channel activity when extracellular Ca(2+) was the charge carrier, illustrating significant Ca(2+) permeability. ET-1-induced responses were inhibited by the ET(A) receptor antagonist BQ123 and the phospholipase C (PLC) inhibitor U73122. The diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) also stimulated I(cat), but the protein kinase C (PKC) inhibitor chelerythrine did not inhibit either the OAG- or ET-1-induced I(cat). Inositol 1,4,5-trisphosphate (IP(3)) did not activate I(cat), but greatly potentiated the response to OAG and this effect was blocked by heparin. Bath application of anti-TRPC3 and anti-TRPC7 antibodies to inside-out patches markedly inhibited ET-1-evoked I(cat), but antibodies to TRPC1, C4, C5 and C6 had no effect. Immunocytochemical studies demonstrated preferential TRPC7 expression in the plasmalemma, whereas TRPC3 was distributed throughout the myocyte, and moreover co-localization of TRPC3 and TRPC7 signals was observed at, or close to, the plasma membrane. Flufenamic acid, Gd(3+), La(3+) and extracellular Ca(2+) inhibited I(cat) with IC(50) values of 2.45 microm, 3.8 microm, 7.36 microm and 22 microm, respectively. These results suggest that in rabbit coronary artery myocytes ET-1 evokes a Ca(2+)-permeable non-selective cation channel with properties similar to TRPC3 and TRPC7, and indicates that these proteins may be important components of this conductance.

Endothelin–1 activates a cation conductance, with TRPC3/7 channel properties in rabbit coronary arteries

Endothelin–1 activates a cation conductance, with TRPC3/7 channel properties in rabbit coronary arteries

Role of L-type calcium channels and PKC in active tone development in rabbit coronary artery

The present study investigated active tone development in isolated ring segments of rabbit epicardial coronary artery. Endothelium-denuded (E-) or endothelium-intact (E+) vessels treated with the NO synthase inhibitor N(omega)-nitro-L-arginine (100 microM) developed active tone, which was enhanced by stretch and reversed by the NO donor sodium nitroprusside (SNP; IC(50)=9 nM). Nifedipine abolished active tone and the contractile response to phorbol dibutyrate (PDBu; 10 nM) with the same potency (IC(50)=8 nM), whereas 300 nM PDBu responses were only partially blocked by nifedipine. The classical and novel PKC inhibitors GF-109203X (IC(50)=1-2 microM) and chelerythrine (IC(50)=4-5 microM) and the classical PKC inhibitor Gö-6976 (IC(50)=0.3-0.4 microM) blocked both active tone and 10 nM PDBu responses with similar potency. Active tone development was associated with depolarization of membrane potential (E(m)) and a shift to the left of the E(m)-vs.-contraction relationship determined by varying extracellular potassium. The depolarization and leftward shift were reversed by either chelerythrine (10 microM) or SNP (30 nM). PDBu (100-300 nM) increased peak L-type calcium channel (Ca(v)) currents in isolated coronary myocytes, and this effect was reversed by chelerythrine (1 microM) or Gö-6976 (200 nM). SNP (500 nM) reduced Ca(v) currents only in the presence of the PKA blocker 8-bromo-2'-O-monobutyryl-cAMPS, Rp isomer (10 microM). In conclusion, active tone development in coronary artery is suppressed by basal NO release and is dependent on both enhanced Ca(v) activity and classical PKC activity. Both E(m)-dependent and -independent processes contribute to contraction. Our results suggest that one E(m)-independent process is direct enhancement of Ca(v) current by PKC.

Medroxyprogesterone acetate attenuates long-term effects of 17β-estradiol in coronary arteries from hyperlipidemic rabbits

Objective The progestin component in hormone replacement treatment may oppose the effects of estrogen on vascular function. This study examined the effect of long-term treatment with 17β-estradiol (E 2) alone and in combination with two progestins on K + and Ca 2+-mediated mechanisms in coronary arteries. Methods Watanabe heritable hyperlipidemic rabbits were treated orally with either E 2 (4 mg/day), medroxyprogesterone acetate (MPA) (10 mg/day), norethindrone acetate (NETA) (2 mg/day), E 2 + MPA, E 2 + NETA, or placebo for 16 weeks ( n = 10 in each group). Coronary arteries were used for mRNA and myograph studies. Results E 2 increased vasodilatation induced by sodium nitroprusside and decreased vasocontraction induced by potassium. The first but not the latter response was opposed by MPA. The combination of MPA and E 2, but neither compound alone enhanced nimodipine-induced vasodilatation and increased the expression of L-type voltage-gated Ca 2+ channel mRNA. NETA had no opposing effects. Hormone treatment did not affect large-conductance Ca 2+ activated or ATP-sensitive K + channels or cGMP-dependent protein kinase mRNA expression. Hyperlipidemia had no effect on vascular reactivity. Conclusion When E 2 is administered with MPA, effects of E 2 on nitric oxide and Ca 2+-mediated vascular reactivity in rabbit coronary arteries are modulated. The results suggest that the progestin component in hormone replacement treatment may interfere with the supposed beneficial vascular effects of estrogen.

Endothelin-1 Inhibits Inward Rectifier K+ Channels in Rabbit Coronary Arterial Smooth Muscle Cells Through Protein Kinase C

We studied inward rectifier K+ (Kir) channels in smooth muscle cells isolated from rabbit coronary arteries. In cells from small- (<100 microm, SCASMC) and medium-diameter (100 approximately 200 microm, MCASMC) coronary arteries, Kir currents were clearly identified (11.2 +/- 0.6 and 4.2 +/- 0.6 pA pF at -140 mV in SCASMC and MCASMC, respectively) that were inhibited by Ba(2+) (50 microm). By contrast, a very low Kir current density (1.6 +/- 0.4 pA pF) was detected in cells from large-diameter coronary arteries (>200 microm, LCASMC). The presence of Kir2.1 protein was confirmed in SCASMC in a Western blot assay. Endothelin-1 (ET-1) inhibited Kir currents in a dose-dependent manner. The inhibition of Kir currents by ET-1 was abolished by pretreatment with the protein kinase C (PKC) inhibitor staurosporine (100 nM) or GF 109203X (1 microm). The PKC activators phorbol 12,13-dibutyrate (PDBu) and 1-oleoyl-2-acetyl-sn-glycerol (OAG) reduced Kir currents. The ETA-receptor inhibitor BQ-123 prevented the ET-1-induced inhibition of Kir currents. The amplitudes of the ATP-dependent K+ (KATP), Ca(2+)-activated K+ (BKCa), and voltage-dependent K+ (KV) currents, and effects of ET-1 on these channels did not differ between SCASMC and LCASMC. From these results, we conclude that Kir channels are expressed at a higher density in SCASMC than in larger arteries and that the Kir channel activity is negatively regulated by the stimulation of ETA-receptors via the PKC pathway.

Selective down-regulation of sub-endocardial ryanodine receptor expression in a rabbit model of left ventricular dysfunction

Defective sarcoplasmic reticulum (SR) Ca 2+ handling is evident in cardiomyopathy and may be mediated by selective dysregulation of SR Ca 2+ handling proteins. To assess whether regulation of SR Ca 2+ release may vary regionally within the normal and diseased heart, left ventricular transmural expression and activity of the ryanodine receptor (RyR2) was studied in a rabbit coronary artery ligation model of left ventricular dysfunction (LVD). Tissue/cells were isolated from both the sub-endocardial and subepicardial layers of the left ventricular free wall from sham-operated and coronary artery ligated rabbit hearts. Three independent methods were used to study alterations in RyR2 mRNA (real-time quantitative PCR (RT-PCR)) and protein expression (quantitative immunoblotting and [ 3H] ryanodine binding). These biochemical data were compared with functional measurements of fractional SR Ca 2+ release from both of these regions. Data from RT-PCR revealed lower RyR2 mRNA levels in the sub-endocardium compared with subepicardium in both experimental groups with the reduction being significantly lower in the sub-endocardium from the LVD group. Quantitative analysis of RyR2 protein levels revealed the same expression patterns. Calsequestrin mRNA and protein levels showed no significant changes. This study demonstrates a lower expression level of RyR2 in the sub-endocardium of the left ventricle of rabbit hearts and is the first to show a further specific reduction in LVD. There is a corresponding decrease in fractional SR Ca 2+ release in cells isolated from the sub-endocardium of hearts from the LVD group, relating these selective biochemical alterations to changes in function.

Endothelin-1 Acts via Protein Kinase C to Block KATP Channels in Rabbit Coronary and Pulmonary Arterial Smooth Muscle Cells

We investigated the effects of the vasoconstrictor endothelin-1 (ET-1) on the whole-cell ATP-sensitive K+ (KATP) currents of smooth muscle cells that were isolated enzymatically from rabbit coronary artery (CASMCs) and pulmonary artery (PASMCs). The size of the KATP current did not differ significantly between CASMCs and PASMCs. ET-1 reduced the KATP current in a concentration-dependent manner, and this inhibition was greater in PASMCs than in CASMCs (half-inhibition values of 12.20 nM and 1.98 nM in CASMCs and PASMCs, respectively). However, the level of inhibition induced by other vasoconstrictors (angiotensin II, norepinephrine, and serotonin) were not significantly different between CASMCs and PASMCs. Pretreatment with the protein kinase C (PKC) inhibitors staurosporine (100 nM) and GF 109203X (1 microM) prevented ET-1-induced inhibition of the KATP current in both arterial smooth muscle cell preparations. The PKC activators phorbol-12,13-dibutyrate (PDBu) and 1-olelyl-2-acetyl-sn-glycerol (OAG) reduced the KATP current in dose-dependent manner. Although the numbers of ET receptors were not significantly different between the 2 arterial smooth muscle cell preparations, the effects of PDBu and OAG were greater on PASMCs. ET-1-induced inhibition of the KATP current was unaffected by the PKA inhibitor Rp-cAMPs (100 microM) and PKA inhibitory peptide (5 microM).

KATP‐channel‐induced vasodilation is modulated by the Na,K‐pump activity in rabbit coronary small arteries

The purpose of the study was to evaluate the importance of the Na,K-pump in relaxations induced by K(ATP)-channel openers in rabbit coronary small arteries. Arterial segments were mounted in myographs for recording of isometric tension. Whole-cell patch clamp was used to assess K(ATP)-channel currents in isolated smooth muscle cells from the arteries. In arteries preconstricted with the thromboxane A(2) analogue U46619 pinacidil and cromakalim induced concentration-dependent relaxations. In arteries preconstricted with potassium (124 mM) only high concentrations of pinacidil had a small relaxant effect. In arteries preconstricted with U46619 pinacidil-induced relaxations were unaffected by pretreatment with N(omega)-nitro-L-arginine (L-NNA) and only slightly reduced after mechanical removal of the endothelium. Pinacidil induced relaxations were not significantly affected by 1 microM glibenclamide. However, the relaxations were partly inhibited in potassium-free media and by 1 microM ouabain. In contrast, the concentration-dependent relaxation to cromakalim was partly blocked by 1 microM glibenclamide and partly by 1 microM ouabain and when both drugs were present the inhibition increased. Ouabain (1 microM) and glibenclamide (1 microM) each partly inhibited an ATP-sensitive current induced by pinacidil and cromakalim. In the presence of both inhibitors a greater inhibition was seen. When the solution in the patch pipette was sodium-free the current was reduced and ouabain had no effect. The study suggests that the relaxation to cromakalim and most likely pinacidil is mediated through opening of K(ATP) channels. Inhibition of the Na,K-pump, however, may change the local environment for the K(ATP) channels (i.e. increases the ATP/ADPratio and/or decreases the transmembrane potassium gradient), which partly prevents the activation of the K(ATP)-channel current.

Extracellular Mg(2+) blocks endothelin-1-induced contraction through the inhibition of non-selective cation channels in coronary smooth muscle.

This study investigated the effects of changing the extracellular [Mg(2+)] ([Mg(2+)](o)) on endothelin-1 (ET-1)-induced contraction of rabbit coronary artery smooth muscle and the involvement of non-selective cation (NSC) channels in this response. Increased [Mg(2+)](o) shifted the concentration/contraction relationship curve of ET-1 to the right. In whole-cell patch clamp recordings, ET-1 (10(-7) M) induced a long-lasting inwards current (94.7+/-7.2 pA) that was inhibited by 8 mM [Mg(2+)](o) (45.3+/-4.4%) and NSC channel blockers (10(-3) M streptomycin and 10(-3) M La(3+)), but not by the voltage-dependent Ca(2+) channel blocker nicardipine. The current/voltage (I/V) curve was linear. Furthermore, in pressurized arteries, the ET-1-induced contraction was also inhibited by La(3+) and streptomycin, but not by nicardipine. U-73122, a selective phospholipase C (PLC) inhibitor and staurosporine and GF 109203X, which block protein kinase C (PKC), reduced ET-1-activated NSC currents by 54.2+/-5.1%, 60.3+/-5.5% and 48.5+/-2.9%, respectively. The inwards current was increased by 1-oleoyl-2-acetyl-sn-glycerol (OAG) and phorbol 12,13-dibutyrate (PDBu), which activate PKC selectively. Like transient receptor potential channel (TRPC3) currents, ET-1-activated NSC currents had a linear I/V relationship, were blocked by flufenamate and activated by a diacylglycerol analogue. These results suggest that [Mg(2+)](o) blocks ET-1-induced contraction of coronary arteries by inhibiting NSC channels. Activation of PLC and PKC might be involved in activation of NSC channels.

Tibolone and its metabolites acutely relax rabbit coronary arteries in vitro

Objectives: To compare the acute effects of estradiol, tibolone and its metabolites on coronary arteries in vitro and to investigate possible vascular mechanisms. Methods: Coronary artery ring segments from female rabbits were mounted in myographs for isometric tension recordings. Concentration–response curves to tibolone, 3α-OH-tibolone, 3β-OH-tibolone, Δ4-isomer and 17β-estradiol were obtained after precontraction with potassium 30 mmol/l and after addition of N ω -nitro- l-arginine methyl ester 10 −4 mol/l ( l-NAME, an inhibitor of endothelial nitric oxide (NO) synthase) or tetraethylammonium chloride 10 −2 mol/l (TEA, an unspecific inhibitor of potassium channels). The effects of the different substances to calcium concentration–response curves were evaluated. Responses are expressed as maximal contraction ( E max), concentration giving half maximal contraction (log EC 50) or area under curve (AUC). Results: Tibolone and its metabolites induced a concentration-dependent vasodilatation comparable to that of 17β-estradiol with the rank of potency: 3β-OH-tibolone ≅ tibolone > 3α-OH-tibolone > Δ4-isomer (ANOVA). l-NAME partly inhibited the relaxation to all substances. TEA induced a slight rightward shift of the relaxation to 3α-OH-tibolone (log EC 50: −5.05 versus −5.20; P<0.05; Student’s t-test), but not to the other substances. Calcium concentration-dependent contraction curves were inhibited by all substances compared to controls (AUC, P<0.05, ANOVA). Conclusions: Our data indicate that the acute relaxation induced by tibolone and its metabolites in coronary arteries in vitro are probably mediated by endothelium independent inhibition of calcium channels but may also involve an endothelium-dependent mechanism via nitric oxide. The effect of tibolone is comparable to that of 17β-estradiol in this set-up.

Effect of Ischemic Preconditioning Based on Different Epicardial Branching Patterns of the Left Coronary Artery in the Rabbit Heart

The aim of this study was to characterize the effect of increasing numbers of preconditioning cycles on the different branching patterns (bifurcation/trifurcation) of the coronary artery in the rabbit heart. Fifty-six NZW rabbits were assigned to a bifurcation group and subjected to 0, 1, 3, 5, or 7 (B0, B1, B3, B5, or B7 subgroup, respectively) cycles of preconditioning (PC) (5 min of regional ischemia plus 10 min of reperfusion/cycle) and this was followed by 45 min of sustained ischemia and 72 h of reperfusion; 16 rabbits were assigned to a trifurcation group and subjected to 0, 1, 3, or 5 (T0, T1, T3, or T5 subgroup, respectively) cycles of PC. The ratio of necrotic zone (NZ) to ischemic zone (IZ) was calculated. The bifurcation group showed higher mortality (28.6%) than the trifurcation group (0%). The volume of the ischemic zone (expressed as a percentage of volume of the left ventricle) and the volume of the necrotic zone were larger in the bifurcation group than in the trifurcation group. The ratio of the necrotic zone to the ischemic zone was significantly lower in the B5 and B7 subgroups than in the B0, B1, and B3 subgroups. In the trifurcation group, the ratio of the necrotic zone to the ischemic zone showed a diminishing tendency in the subgroups as the PC cycle number increased, but without statistical significance. Thus, in the trifurcation pattern of the rabbit coronary artery there could be little effect on preconditioning, but in the bifurcation pattern the number of preconditioning is recommended to be 5 to 7 cycles. In this regard, different branching patterns of the epicardial branching of the rabbit coronary artery should be considered when interpreting experimental results on ischemic preconditioning.

Altered oscillatory work by ventricular myofilaments from a rabbit coronary artery ligation model of heart failure.

Understanding the changed ability of cardiac myofilaments to produce pump work requires knowledge of kinetics of crossbridge function as well as more widely studied parameters such as Ca-sensitivity and isometric force development. We tested the hypothesis that altered crossbridge kinetics contribute to reduced myofilament work in early-stage heart failure (left ventricular dysfunction, LVD). The sinusoidal oscillation technique can yield insights into crossbridge function. Dynamic stiffness, oscillatory work and power were assessed in chemically skinned, Ca-activated trabeculae from rabbit ventricles in early-stage failure, 8 weeks after infarction induced by coronary artery ligation (LIG). Results were compared with sham-operated controls (SH). LVD was assessed by echocardiography. Ca-activated force and myofilament Ca-sensitivity were not significantly altered at this early stage of LVD. In maximally Ca-activated preparations, the frequency of minimal dynamic stiffness (f(min)) was 23% lower in LIG. f(min) increases by >80% between pCa 5.8 and 4 in SH but not in LIG. Maximal phase lead and lag angles (between length and tension) were lower in LIG at frequencies near f(min), lowering oscillatory work and power. The Lissajous figures (oscillatory work loops) of imposed length vs. tension are often asymmetric near f(min). The degree of asymmetry was greater in LIG. Reduced capacity for mechanical power, consistent with depressed haemodynamic performance in LVD hearts, is only partially attributable to crossbridge slowing; changes in the phase relationship will also contribute. These changes are not readily attributable to known alterations in contractile protein isoforms. Some deductions are drawn about which steps in the crossbridge cycle are modified in this model of LVD. Altered cardiac myocyte Ca-transients, reported to be associated with LVD, will be translated into pump work by a contractile machinery that is functionally altered, even though isometric force and myofilament Ca-sensitivity might remain near-normal at this stage.

Inhibitory effects of ursodeoxycholic acid on the induction of nitric oxide synthase in vascular smooth muscle cells

The expression of inducible nitric oxide synthase (iNOS) and the resultant increased nitric oxide production are associated with endotoxemia and atherosclerotic lesions observed in transplant hearts or balloon-injured artery. Ursodeoxycholic acid has been shown to have cardiovascular protective effects, such as inhibition of the development of transplant arteriosclerosis, but its mechanism remains unclear. Here, we investigated the effects of ursodeoxycholic acid on nitric oxide production and the expression of iNOS in vascular smooth muscle cells isolated from adult rat aorta and rabbit coronary artery. Nitrite released from cells in the culture medium was measured with the Griess reaction. iNOS mRNA and protein were measured by Northern and Western blot analyses. Treatment with ursodeoxycholic acid (30–1000 μM) significantly inhibited lipopolysaccharide plus interferon-γ-induced nitric oxide production in a concentration-dependent manner, but ursodeoxycholic acid showed only small inhibitory effects on nitric oxide production that had already been induced by lipopolysaccharide plus interferon-γ. Ursodeoxycholic acid by itself did not affect basal nitric oxide production. Ursodeoxycholic acid also suppressed lipopolysaccharide plus interferon-γ-induced expression of iNOS mRNA and protein. Ursodeoxycholic acid had the most potent inhibitory effect among various kinds of bile acids examined, i.e. chenodeoxycholic acid, deoxycholic acid, cholic acid and conjugated bile acids such as tauroursodeoxycholic acid. These results suggest that ursodeoxycholic acid inhibits the induction of iNOS and then nitric oxide production in aortic and coronary artery smooth muscle cells, suggesting a possible mechanism for the cardiovascular protective effect of ursodeoxycholic acid under various pathophysiological conditions such as endotoxemia and atherosclerosis.

Testosterone causes simultaneous decrease of [Ca2+]I and tension in rabbit coronary arteries: by opening voltage dependent potassium channels.

The relationship between the level of testosterone and the incidence of coronary heart disease is still controversial in the view of the results of clinical and epidemiologic studies. This uncertainty might be partly due to relatively small number of experimental studies undertaken to investigate the cellular mechanism underlying the vascular responses to testosterone. To further investigate the cellular mechanisms of testosterone with respect to vascular response, we investigated the effect of testosterone on contractility and intracellular Ca2+ regulation in a rabbit coronary artery and evaluated the underlying mechanism of testosterone-induced changes of coronary vascular tone by using various pharmacological blockers. Testosterone was found to relax rabbit coronary arteries in a dose-dependent manner, and no significant difference was found in the relaxation response to testosterone with or without endothelium. Similar results were obtained in male and non-pregnant female rabbit coronary arteries. The relaxation response of rabbit coronary arteries to testosterone was greater for PGF2alpha-contracted rings than for KCl contracted rings, which suggest the involvement of K+ channels. Furthermore, the relaxation response to testosterone was significantly reduced by 4-aminopyridine, a sensitive blocker of voltage dependent K+ channels, but not by low doses of tetraethylammonium or iberiotoxin, a Ca2+ activated K+ channel blocker. Testosterone simultaneously reduced the intracellular Ca2+ concentration ([Ca2+]i) and tension, and 4-AP effectively antagonized the testosterone-induced change of [Ca2+]i and tension. Therefore, it may be concluded that the stimulation of voltage dependent K channels is responsible, at least in part, for the testosterone-induced relaxation of rabbit coronary arteries.

Intracellular Mg(2+) hyperpolarizes rabbit coronary artery smooth muscle cells by differential modulation of Ca(2+)(i)-dependent ion channels.

We investigated the role of intracellular Mg(2+) ([Mg(2+)](i)) in the regulation of membrane potential ( V(m)) in rabbit coronary artery smooth muscle cells. V(m), membrane currents and intracellular Ca(2+) ([Ca(2+)](i)) were measured using standard patch-clamp and microfluorometry techniques. When [Ca(2+)](i) was increased by caffeine, V(m) depolarized at low [Mg(2+)](i) (0.1 mM), but hyperpolarized at high [Mg(2+)](i) (> or =1.2 mM). Effects of [Mg(2+)](i) on caffeine-induced currents were investigated. [Mg(2+)](i) selectively facilitated the activation of Ca(2+)-activated K(+) currents ( I(K,Ca)), while Ca(2+)-activated Cl(-) currents ( I(Cl,Ca)) were unaffected. Simultaneous recording of [Ca(2+)](i) and I(K,Ca) at different [Mg(2+)](i) showed that [Mg(2+)](i) increased the Ca(2+) sensitivity of I(K,Ca). [Ca(2+)](i) also inhibited voltage-dependent K(+) (K(V)) currents, although this effect was significant only at low [Mg(2+)](i). These results imply that the relative contributions of I(K,Ca), I(Cl,Ca) and K(V) currents to V(m) during an increase in [Ca(2+)](i) are affected by [Mg(2+)](i): at low [Mg(2+)](i), activation of I(Cl,Ca) and inhibition of K(V) currents depolarized V(m); at high [Mg(2+)](i) the activation of I(K,Ca) predominated, resulting in hyperpolarization of V(m). In conclusion, [Mg(2+)](i) hyperpolarizes V(m) by selective facilitation of I(K,Ca) and may thus possibly contributes to the relaxation of the coronary artery.