Porcine Coronary Artery Strips Research Articles

Transduction of the N-terminal fragments of MYPT1 enhances myofilament Ca2+ sensitivity in an intact coronary artery.

The region of the 110 kDa regulatory subunit (MYPT1) of smooth muscle myosin phosphatase involved in the regulation of contraction was determined under physiological conditions. Using HIV Tat protein-mediated protein transduction, the N-terminal fragments of MYPT1 were introduced to the intact porcine coronary arterial strips. Pre-incubation with 3 micromol/L TAT-MYPT1(1-374), a construct containing the Tat peptide and the residues 1 to 374 of MYPT1, for 15 minutes augmented (2.4-fold) the subsequent contraction induced by adding 1.25 mmol/L of extracellular Ca2+ under 118 mmol/L K+ depolarization, with no augmentation of the [Ca2+]i elevation. The deletion of the Tat peptide, MYPT1(1-374), abolished the augmenting effect. TAT-MYPT1(1-296) demonstrated a weaker but significant augmentation (1.7-fold). However, TAT-MYPT1(1-171), TAT-MYPT1(39-374), TAT-MYPT1(39-296), and TAT-MYPT1(297-374) had no augmenting activity. The myosin light chain phosphorylation level as a function of extracellular Ca2+ concentrations was shifted to the left in the strips pretreated with TAT-MYPT1(1-374) compared with the control. Region 1 to 296 was the minimal region involved in the enhancement of contraction, and region 297 to 374 played a supplemental role. These results suggested that the interaction mainly between catalytic subunit and MYPT1 play a critical role in the regulation of the endogenous myosin phosphatase in intact smooth muscle.

Sildenafil induced vasorelaxation is associated with increases in the phosphorylation of heat shock protein 20 (hsp20)

Background: Sildenafil is an oral phosphodiesterase type 5 inhibitor used in the treatment of erectile dysfunction. Phosphorylated heat shock protein 20 (pHSP20) mediates cyclic nucleotide induced vasorelaxation. The purpose of this study was to determine if sildenafil induced vasorelaxation is mediated through pHSP20. Methods: Peptides containing an 11 amino acid enhanced protein transduction domain (PTD) and the functional 13 amino acid sequence of HSP20 with a phosphoserine (PTD-pHSP20) were synthesized using F-MOC technology. Rings of porcine coronary artery were suspended in a muscle bath and maximally contracted with serotonin. Increasing concentrations of PTD-HSP20 (0.1 – 1.0mM), sodium nitroprusside (SNP; 0.1 - 10μM), or sildenafil (0.1 – 100 μM) were added to the baths and the percent relaxation was recorded. To determine if sildenafil induced vasorelaxation is associated with an increase in HSP20 phosphorylation, strips of porcine coronary artery were untreated (control), or treated with SNP (100 μM) or sildenafil (100 μM) for 5 minutes and then snap frozen. Extracted proteins were then separated using 2-dimensional SDS-PAGE and probed for HSP20. Results: Transduction of peptide analogues of pHSP20 led to a dose dependent relaxation of pre-contracted porcine coronary artery (open triangles). Sildenafil induced vasorelaxation of pre-contracted coronary artery in a dose dependent manner (closed triangles). Sildenafil induced vasorelaxation was associated with an increase in the phosphorylation of HSP20 (See graph). Conclusions: Transduction of PTD-pHSP20 results in a dose dependent relaxation of porcine coronary artery similar to the relaxation induced by Sildenafil. Sildenafil treatment led to increases in the phosphorylation of HSP20. Taken together, these findings suggest that protein transduction of PTD-pHSP20 may be an effective method of treating erectile dysfunction.

Inhibitory effects of brefeldin A, a membrane transport blocker, on the bradykinin-induced hyperpolarization-mediated relaxation in the porcine coronary artery.

1. To elucidate the mechanism of the relaxation mediated by endothelium-derived hyperpolarizing factors (EDHFs), the effect of brefeldin A, a membrane transport blocker, on cytosolic Ca(2+) concentration ([Ca(2+)]i) and tension was determined in the porcine coronary arterial strips. We also examined the effect of brefeldin A on [Ca(2+)]i in the endothelial cells of the porcine aortic valve. 2. In the presence of 10 microM indomethacin and 30 microM N(G)-nitro-L-arginine (L-NOARG), both bradykinin and substance P induced a transient decrease in [Ca(2+)]i and tension in arterial strips contracted with 100 nM U46619 (thromboxane A2 analogue). A 6 h pre-treatment with 20 microg ml(-1) brefeldin A abolished the bradykinin-induced relaxation, while it had no effect on the substance P-induced relaxation. 3. In the absence of indomethacin and L-NOARG, brefeldin A had no effect on the bradykinin-induced relaxation during the contraction induced by U46619 or 118 mM K(+). 4. The indomethacin/L-NOARG-resistant relaxation induced by bradykinin was completely inhibited by 3 mM tetrabutylammonium (non-specific Ca(2+)-activated K(+) channel blocker), while that induced by substance P was not inhibited by 3 mM tetrabutylammonium or 1 mM 4-aminopyridine (voltage-dependent K(+) channels blocker) alone, but completely inhibited by their combination. 5. Brefeldin A had no effect on the [Ca(2+)]i elevation in endothelial cells induced by bradykinin or substance P. 6. In conclusion, bradykinin produce EDHF in a brefeldin A-sensitive mechanism in the porcine coronary artery. However, this mechanism is not active in a substance P-induced production of EDHF, which thus suggests EDHF to be more than a single entity.

Oxidized-LDL enhances coronary vasoconstriction by increasing the activity of protein kinase C isoforms alpha and epsilon.

Oxidized low-density lipoprotein (ox-LDL) plays a critical role in the development of atherosclerotic coronary vasospasm; however, the cellular mechanisms involved are not fully understood. We tested the hypothesis that ox-LDL enhances coronary vasoconstriction by increasing the activity of specific protein kinase C (PKC) isoforms in coronary smooth muscle. Active stress was measured in de-endothelialized porcine coronary artery strips; cell contraction and [Ca(2+)](i) were monitored in single coronary smooth muscle cells loaded with fura-2; and the cytosolic and particulate fractions were examined for PKC activity and reactivity with isoform-specific anti-PKC antibodies with Western blots. Ox-LDL (100 microgram/mL) caused slow but significant increases in active stress to 1.3+/-0.4x10(3) N/m(2) and cell contraction (10%) that were completely inhibited by GF109203X (10(-6) mol/L), an inhibitor of Ca(2+)-dependent and -independent PKC isoforms, with no significant change in [Ca(2+)](i). 5-Hydroxytryptamine (5-HT; 10(-7) mol/L) and KCl (24 mmol/L) caused increases in cell contraction and [Ca(2+)](i) that were inhibited by the Ca(2+) channel blocker verapamil (10(-6) mol/L). Ox-LDL enhanced coronary contraction to 5-HT and KCl with no additional increases in [Ca(2+)](i). Direct activation of PKC by phorbol 12-myristate13-acetate (PMA; 10(-7) mol/L) caused a contraction similar in magnitude and time course to ox-LDL-induced contraction and enhanced 5-HT- and KCl-induced contraction with no additional increases in [Ca(2+)](i). The ox-LDL-induced enhancement of 5-HT and KCl contraction was inhibited by Gö6976 (10(-6) mol/L), an inhibitor of Ca(2+)-dependent PKC isoforms. Both ox-LDL and PMA caused an increase in PKC activity in the particulate fraction, a decrease in the cytosolic fraction, and an increase in the particulate/cytosolic PKC activity ratio. Western blots revealed the Ca(2+)-dependent PKC-alpha and the Ca(2+)-independent PKC-delta, -epsilon, and -zeta isoforms. In unstimulated tissues, PKC-alpha- and -epsilon were mainly cytosolic, PKC-delta was mainly in the particulate fraction, and PKC-zeta was equally distributed in the cytosolic and particulate fractions. Ox-LDL alone or PMA alone caused translocation of PKC-epsilon from the cytosolic to particulate fraction, whereas the distribution pattern of PKC-alpha, -delta, and -zeta remained unchanged. 5-HT (10(-7) mol/L) alone and KCl alone did not change PKC activity. In tissues pretreated with ox-LDL or PMA, 5-HT and KCl caused additional increases in PKC-alpha activity. Native LDL did not significantly affect coronary contraction, [Ca(2+)](i), or PKC activity. These results suggest that ox-LDL causes coronary contraction via activation of the Ca(2+)-independent PKC-epsilon and enhances the contraction to [Ca(2+)](i)-increasing agonists by activating the Ca(2+)-dependent PKC-alpha. Activation of PKC-alpha and -epsilon may represent a possible cellular mechanism by which ox-LDL could enhance coronary vasospasm.

Oxidized-Ldl Enhances Coronary Vasoconstriction by Increasing the Activity of α and ε Protein Kinase C Isoforms

31 Oxidized low density lipoprotein (ox-LDL) plays a critical role in the development of atherosclerotic coronary vasospasm; however, the cellular mechanisms involved are not fully understood. We tested the hypothesis that ox-LDL enhances coronary vasoconstriction by increasing the activity of specific protein kinase C (PKC) isoforms in coronary smooth muscle. Active stress was measured in de-endothelialized porcine coronary artery strips, [Ca 2+ ] i was monitored in single coronary smooth muscle cells loaded with fura-2, and the whole tissue, cytosolic and particulate fractions were examined for PKC activity and reactivity with isoform-specific anti-PKC antibodies using Western blot analysis. Ox-LDL (100 μg/ml) caused a slow but significant increase in active stress to 1.2±0.2 x10 3 N/m 2 , that was completely inhibited by the PKC inhibitors staurosporine and calphostin C, with no significant increase in [Ca 2+ ] i . Ox-LDL enhanced coronary contraction to increasing concentrations of 5-hydroxytryptamine (5-HT) and extracellular KCl with no additional increases in [Ca 2+ ] i . Direct activation of PKC by phorbol 12,13-dibutyrate (PDBu, 0.1 μM) caused a contraction similar in magnitude and time course to ox-LDL induced contraction. PDBu also enhanced 5-HT and KCl-induced contraction with no additional increases in [Ca 2+ ] i . Both ox-LDL and PDBu caused an increase in PKC activity in the particulate fraction and a decrease in the cytosolic fraction and increased the particulate/cytosolic PKC activity ratio. Western blot analysis revealed α-, δ-, ε- and ζ-PKC isoforms. In unstimulated tissue, α- and ε-PKC were mainly cytosolic, δ-PKC was mainly in the particulate fraction, while ζ-PKC was equally distributed in the cytosolic and particulate fractions. Ox-LDL and PDBu caused translocation of α- and ε-PKC from the cytosolic to particulate fraction, with minimal effect on the distribution of δ-PKC and ζ-PKC. Native LDL did not significantly affect coronary contraction, [Ca 2+ ] i or PKC activity. These results suggest that ox-LDL enhances coronary vasoconstriction via activation and translocation of α- and ε-PKC isoforms in coronary smooth muscle.

Mechanisms of the thapsigargin‐induced Ca2+ entry in in situ endothelial cells of the porcine aortic valve and the endothelium‐dependent relaxation in the porcine coronary artery

The mechanisms of the thapsigargin (TG)-induced capacitative Ca(2+) entry in in situ endothelial cells and its role in the regulation of arterial tone were investigated using front-surface fluorimetry and fura-2-loaded strips of porcine aortic valve and coronary artery. In the presence of extracellular Ca(2+), TG induced an initial rapid and a subsequent sustained elevation of cytosolic Ca(2+) concentration ([Ca(2+)](i)) in valvular strips. In the absence of extracellular Ca(2+), TG induced only a transient increase in [Ca(2+)](i). The TG-induced sustained elevation of [Ca(2+)](i) in endothelial cells was inhibited completely by 1 mM Ni(2+) and partly by 10 microM econazole and 30 microM ML-9, but not by 900 ng ml(-1) pertussis toxin or 100 microM wortmannin. Therefore, cytochrome P450 and protein phosphorylation are suggested to be involved in the TG-induced Ca(2+) influx in in situ endothelial cells. TG induced an endothelium-dependent large relaxation consisting of an initial and a late sustained relaxation in coronary arterial strip precontracted with U46619 (a thromboxane A2 analogue). Indomethacin alone had no effect, while indomethacin plus N(omega)-nitro-L-arginine (L-NOARG) markedly inhibited the sustained phase and slightly inhibited the initial phase of the TG-induced relaxation. TG induced a smaller but sustained relaxation during the 40 mM K(+)-induced precontraction than that seen during the U46619-induced precontraction. This relaxation was completely abolished by the pretreatment with indomethacin plus L-NOARG. In conclusion, both nitric oxide (NO) and endothelium-derived hyperpolarizing factor were suggested to mediate the TG-induced relaxation, while NO plays a major role in the sustained relaxation. The TG-induced sustained [Ca(2+)](i) elevation in endothelial cells was thus suggested to be mainly linked to the sustained production of NO.

Preconditioning of coronary artery against vasoconstriction by endothelin-1 and prostaglandin F2alpha during repeated downregulation of epsilon-protein kinase C.

The cellular mechanisms of coronary vasospasm are unclear, and a role for protein kinase C (PKC) activation by the endogenous vasoconstrictors endothelin-1 (ET-1) and prostaglandin F2alpha (PGF2alpha) has been suggested. In this study, we developed a phorbol ester-induced PKC downregulation protocol to investigate the relation between the amount and activity of specific PKC isoforms in coronary arterial smooth muscle and coronary vasoconstriction by ET-1 and PGF2alpha. Isometric tension was measured in deendothelialized porcine coronary artery strips, [Ca2+]i was monitored in single coronary smooth muscle cells loaded with fura-2, and the whole tissue, cytosolic, and particulate fractions were examined for PKC activity and reactivity with isoform-specific anti-PKC antibodies using Western blot analysis. In Ca(2+)-free (2 mM EGTA) Krebs solution, ET-1 (10(-7) M), PGF2alpha (10(-5) M) and PKC activator phorbol 12,13-dibutyrate (PDBu) (10(-6) M) caused significant contractions that were completely inhibited by the PKC inhibitors staurosporine and calphostin C, no significant change in [Ca2+]i, and significant activation and translocation of the Ca(2+)-independent epsilon-PKC but not the Ca(2+)-dependent alpha-PKC. In Ca(2+)-free Krebs, a single application of PDBu produced maximal contraction and PKC activity after 30 min, which declined to basal levels in 3 h and remained steady for 24 h, but did not prevent subsequent increases in contraction and PKC activity with a new addition of PDBu and did not significantly decrease the amount of alpha- or epsilon-PKC. Repeated (five to eight) applications of PDBu in Ca(2+)-free Krebs at 3-h intervals completely inhibited subsequent increases in contraction and PKC activity to PDBu, ET-1, or PGF2alpha, and significantly decreased the amount of epsilon-PKC but not that of alpha-PKC. These results provide evidence that a Ca(2+)-independent coronary vasoconstriction induced by ET-1 and PGF2alpha is associated with activation of the epsilon-PKC isoform. The results suggest that, in coronary artery smooth muscle, downregulation of PKC is isoform specific and is more dependent on the frequency rather than the duration of PKC activation. The results also suggest that repeated downregulation of epsilon-PKC might play a role in preconditioning of the coronary artery against vasoconstriction by ET-1 and PGF2alpha.

Vasorelaxation and inhibition of the voltage-operated Ca2+ channels by FK506 in the porcine coronary artery.

Using fura-2 fluorometry, the effects of FK506, an immunosuppressant, on changes in cytosolic Ca2+ concentrations ([Ca2+]i) and tension were investigated in porcine coronary arterial strips. The effects of FK506 on the activity of voltage-operated Ca2+ channels were examined by applying a whole cell patch clamp to the isolated smooth muscle cells of porcine coronary artery. FK506 inhibited the sustained increases in both [Ca2+]i and tension induced by 118 mM K+ depolarization and 100 nM U46619 in a concentration-dependent manner (1-30 microM). The extent of inhibition of the K+-induced contraction was greater than that of the U46619-induced contraction. The increases in [Ca2+]i and tension induced by histamine and endothelin- in the presence of extracellular Ca2+ were also inhibited by 10 microM FK506. FK506 (10 microM) had no effect on Ca2+ release induced by caffeine or by histamine in the Ca2+-free solution. FK506 (10 microM) had no effect on the [Ca2+]i-tension relationships of the contractions induced by cumulative increases of extracellular Ca2+ during K+ depolarization or stimulation with U46619. In the patch clamp experiments, FK506 (30 microM) partially inhibited the inward current induced by depolarization pulse from -80 mV to 0 mV. In conclusion, FK506 induces arterial relaxation by decreasing [Ca2+]i mainly due to the inhibition of the L-type Ca2+ channels, with no effect on the Ca2+ sensitivity of the contractile apparatus.

Mechanisms of vasorelaxation induced by troglitazone, a novel antidiabetic drug, in the porcine coronary artery.

Troglitazone (TRO), a novel antidiabetic drug, has been reported to decrease blood pressure and relax vascular strips. The mechanism of relaxation induced by TRO was determined in terms of Ca2+ signaling in smooth muscle cells. Front-surface fluorometry and fura 2-loaded medial strips of porcine coronary artery were used to examine the effects of TRO on cytosolic Ca2+ concentrations ([Ca2+]i) and contractions. The sustained contraction induced by 100 nmol/L U46619 was similar to that induced by 60 mmol/L K+ depolarization (60K+). TRO concentration dependently decreased [Ca2+]i and the force of these contractions. The concentration of TRO required to induce 50% inhibition of U46619-induced force (2.9 micromol/L) was significantly lower than that required in the case of 60K+-induced force (7.3 micromol/L). Replacing extracellular Ca2+ with Mn2+ gradually quenched fluorescence at 360 nm excitation. This decline was accelerated by 100 nmol/L U46619 and 30K+ to a similar extent, indicating a similar activation of Ca2+ influx. TRO completely inhibited U46619-activated influx but partly inhibited depolarization-activated influx. Cumulative applications of extracellular Ca2+ during stimulations with U46619 or 118K+ induced stepwise increases in [Ca2+]i and force. TRO shifted the [Ca2+]i-force relation to the right during both stimulations. TRO relaxes coronary artery by decreasing [Ca2+]i and Ca2+ sensitivity of contractile apparatus. Inhibition of Ca2+ influx was important in decreasing [Ca2+]i. TRO more effectively inhibits receptor-operated Ca2+ influx than voltage-operated Ca2+ channels.

Isoform-specific protein kinase C activity at variable Ca2+ entry during coronary artery contraction by vasoactive eicosanoids

Vasoactive eicosanoids have been implicated in the pathogenesis of coronary vasospasms. The signaling mechanisms of eicosanoid-induced coronary vasoconstriction are unclear, and a role for protein kinase C (PKC) has been suggested. Activated PKC undergoes translocation to the surface membrane in the vicinity of Ca2+ channels; however, the effect of Ca2+ entry on the activity of the specific PKC isoforms in coronary smooth muscle is unknown. In the present study, 45Ca2+ influx and isometric contraction were measured in porcine coronary artery strips incubated at increasing extracellular calcium concentrations ([Ca2+]e) and stimulated with prostaglandin F2α (PGF2α) or the stable thromboxane A2 analog U46619, while in parallel, the cytosolic (C) and particulate (P) fractions were examined for PKC activity and reactivity with anti-PKC antibodies using Western blot analysis. At 0-300 µM [Ca2+]e, both PGF2α and U46619 (10-5 M) significantly increased PKC activity and contraction in the absence of a significant increase in 45Ca2+ influx. At 600 µM [Ca2+]e, PGF2α and U46619 increased P/C PKC activity ratio to a peak of 9.52 and 14.58, respectively, with a significant increase in 45Ca2+ influx and contraction. The 45Ca2+ influx - PKC activity - contraction relationship showed a 45Ca2+-influx threshold of ~7 µmol·kg-1·min-1 for maximal PKC activation by PGF2α and U46619. 45Ca2+ influx > 10 µmol·kg-1·min-1 was associated with further increases in contraction despite a significant decrease in PKC activity. Western blotting analysis revealed α-, δ-, ε-, and ζ-PKC in porcine coronary artery. In unstimulated tissues, α- and ε-PKC were mostly distributed in the cytosolic fraction. Significant eicosanoid-induced translocation of ε-PKC from the cytosolic to the particulate fraction was observed at 0 [Ca2+]e, while translocation of α-PKC was observed at 600 µM [Ca2+]e. Thus, a significant component of eicosanoid-induced coronary contraction is associated with significant PKC activity in the absence of significant increase in Ca2+ entry and may involve activation and translocation of the Ca2+-independent ε-PKC. An additional Ca2+-dependent component of eicosanoid-induced coronary contraction is associated with a peak PKC activity at submaximal Ca2+ entry and may involve activation and translocation of the Ca2+-dependent α-PKC. The results also suggest that a smaller PKC activity at supramaximal Ca2+ entry may be sufficient during eicosanoid-induced contraction of coronary smooth muscle.Key words: protein kinase C, isoform, calcium influx, eicosanoids, vascular smooth muscle, contraction.

Isoform-specific protein kinase C activity at variable Ca2+ entry during coronary artery contraction by vasoactive eicosanoids.

Vasoactive eicosanoids have been implicated in the pathogenesis of coronary vasospasms. The signaling mechanisms of eicosanoid-induced coronary vasoconstriction are unclear, and a role for protein kinase C (PKC) has been suggested. Activated PKC undergoes translocation to the surface membrane in the vicinity of Ca2+ channels; however, the effect of Ca2+ entry on the activity of the specific PKC isoforms in coronary smooth muscle is unknown. In the present study, 45Ca2+ influx and isometric contraction were measured in porcine coronary artery strips incubated at increasing extracellular calcium concentrations ([Ca2+]e) and stimulated with prostaglandin F2alpha (PGF2alpha) or the stable thromboxane A2 analog U46619, while in parallel, the cytosolic (C) and particulate (P) fractions were examined for PKC activity and reactivity with anti-PKC antibodies using Western blot analysis. At 0-300 microM [Ca2+]e, both PGF2alpha and U46619 (10(-5) M) significantly increased PKC activity and contraction in the absence of a significant increase in 45Ca2+ influx. At 600 microM [Ca2+]e, PGF2alpha and U46619 increased P/C PKC activity ratio to a peak of 9.52 and 14.58, respectively, with a significant increase in 45Ca2+ influx and contraction. The 45Ca2+ influx--PKC activity--contraction relationship showed a 45Ca2+-influx threshold of approximately 7 micromol x kg(-1) x min(-1) for maximal PKC activation by PGF2alpha and U46619. 45Ca2+ influx > 10 micromol x kg(-1) x min(-1) was associated with further increases in contraction despite a significant decrease in PKC activity. Western blotting analysis revealed alpha-, delta-, epsilon-, and zeta-PKC in porcine coronary artery. In unstimulated tissues, alpha- and epsilon-PKC were mostly distributed in the cytosolic fraction. Significant eicosanoid-induced translocation of epsilon-PKC from the cytosolic to the particulate fraction was observed at 0 [Ca2+]e, while translocation of alpha-PKC was observed at 600 microM [Ca2+]e. Thus, a significant component of eicosanoid-induced coronary contraction is associated with significant PKC activity in the absence of significant increase in Ca2+ entry and may involve activation and translocation of the Ca2+-independent epsilon-PKC. An additional Ca2+-dependent component of eicosanoid-induced coronary contraction is associated with a peak PKC activity at submaximal Ca2+ entry and may involve activation and translocation of the Ca2+-dependent alpha-PKC. The results also suggest that a smaller PKC activity at supramaximal Ca2+ entry may be sufficient during eicosanoid-induced contraction of coronary smooth muscle.

Effects of pimobendan and its active metabolite, UD-CG 212 Cl, on Ca2+-activated K+ channels in vascular smooth-muscle cells.

The aim of this study was to clarify the mechanisms of activation of Ca2+-dependent K+ (K(Ca)) channels by pimobendan, a new cardiotonic and vasodilator agent with phosphodiesterase inhibition properties, and its main metabolite, UD-CG 212 Cl, in vascular smooth-muscle cells from porcine coronary arteries. Both pimobendan and UD-CG 212 Cl induced relaxation of porcine coronary artery strips. However, in the presence of 100 microM Rp-cAMPS (Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate), a membrane-permeable antagonist of cAMP, the effects of pimobendan decreased significantly. Application of 1 microM pimobendan activated K(Ca) channels in cell-attached patches, and this increase in activity was suppressed by 100 nM H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide), a cAMP-dependent protein-kinase inhibitor. Pimobendan was ineffective in altering the activity of K(Ca) channels in inside-out patches. In contrast, UD-CG 212 Cl, at a concentration of 1 microM, activated K(Ca) channels not only in cell-attached patches but also in inside-out patches. Application of 100 nM H-89 also inhibited UD-CG 212 Cl-induced K(Ca) channels activity but to a lesser degree than that induced by pimobendan in cell-attached patches. In conclusion, there are two mechanisms of activation of K(Ca) channels by pimobendan and UD-CG 212 Cl. Activation by pimobendan occurs primary through the cAMP pathway, whereas UD-CG 212 Cl activates K(Ca) channels directly as well as through the cyclic AMP pathway.

Tranilast inhibits contraction and Ca 2+ movement of porcine coronary arteries

In a recent clinical study, tranilast, an anti-allergic agent, was shown to reduce the rate of coronary restenosis after percutaneous transluminal coronary angioplasty, although the mechanism of this effect is unclear. The present study was undertaken to investigate the effects of tranilast on contraction and Ca 2+ movement of the coronary arteries. We characterized the effects of tranilast on isometric force and aequorin-estimated intracellular Ca 2+ concentrations ([Ca 2+] i) of porcine coronary artery strips. Tranilast concentration-dependently (10–500 μM) inhibited histamine (3×10 −5 M)-induced contraction of the coronary arteries. A similar tendency was observed in the response to high K + (30 mM) stimulation. Histamine caused phasic and tonic increases in [Ca 2+] i, and high K + caused a tonic increase in [Ca 2+] i of smooth muscle, both of which were significantly suppressed in the presence of tranilast. These results suggest that tranilast inhibits the contraction of coronary arteries by inhibiting both Ca 2+ influx from extracellular environment and Ca 2+ release from intracellular Ca 2+ stores, which might be related to its preventive effect on restenosis after coronary angioplasty.

Studies on endothelium-dependent vasorelaxation by hydralazine in porcine coronary artery

Hydralazine relaxed porcine coronary artery strips in a concentration-dependent manner by distinct endothelium-dependent and endothelium-independent actions. With lower doses (≤10 −6 M), the hydralazine-induced relaxation appears to be completely endothelium-dependent and we designed the present study to elucidate the mechanisms of this endothelium-dependent relaxation. Hydralazine (10 −6 M)-induced endothelium-dependent relaxation was not affected by the presence of 10 −5 M indomethacin, 10 −3 M l- N G-nitro-arginine ( l-NOARG) or 10 −5 M haemoglobin, and was accompanied by accumulation of cGMP but not of cAMP in artery strips. There was a close time-dependent parallel relationship between endothelium-dependent relaxation and accumulation of cGMP induced by hydralazine (10 −6 M). The endothelium-dependent relaxation and accumulation of cGMP induced by hydralazine showed much slower kinetics than those induced by ionomycin (10 −7 M). Pretreatment of the strips with actinomycin D (10 μg/ml) significantly inhibited not the endothelium-dependent relaxation and accumulation of cGMP induced by ionomycin (10 −7 M) but those induced by hydralazine (10 −6 M). These results suggest that hydralazine induces endothelium-dependent vasorelaxation via the slow accumulation of cGMP in the strips. This does not occur through the release of nitric oxide or prostaglandin I 2 but through immediate transcription and probably expression of a molecule in the endothelium.

CPA induces a sustained increase in [Ca2+]i of endothelial cells in situ and relaxes porcine coronary artery.

Using fura 2 fluorometry, we investigated the effect of cyclopiazonic acid (CPA), an inhibitor of Ca2+ pump adenosinetriphosphatase of the endoplasmic reticulum, on cystosolic Ca2+ concentration ([Ca2+]i) and tension in porcine aortic valvular endothelial cells and coronary arterial strips with endothelium. In normal physiological salt solution, CPA induced a sustained increase in [Ca2+]i in the valvular strips, whereas in Ca(2+)-free physiological salt solution, CPA elicited a transient elevation of [Ca2+]i. CPA(30 microM) relaxed coronary strips with endothelium precontracted by 100 nM U-46619; this relaxation was partially inhibited by N omega-nitro-L-arginine (100 microM). These results indicate that the CPA-induced increase in [Ca2+]i depends on the Ca2+ release and the Ca2+ influx in the endothelial cells in situ and that the CPA-induced endothelium-dependent decreases in [Ca2+]i and tension in the smooth muscle are due to the combined effect of N omega-nitro-L-arginine-sensitive and -resistant factors.

Detection of sarcoma virus family tyrosine kinase activity in coronary arterial tissue.

We have observed that the tyrosine kinase inhibitors genistein and tyrphostin can selectively block angiotensin II mediated and vasopressin-mediated contractions in porcine coronary arterial strips, without affecting the action of acetylcholine. Therefore, we assessed the presence of tyrosine kinase activity in the porcine coronary artery tissue, using an assay specific for sarcoma virus (src) related tyrosine kinases. In both membrane and cytosolic fractions of porcine coronary artery, we detected src-related tyrosine kinase activity that could be inhibited by both genistein and tyrphostin. The tyrosine kinase activity in membrane extracts was separated into two peaks by sequential chromatography on hydroxylapatite and Mono-Q columns. Protein in both peaks exhibited Western blot cross-reactivity with anti-src antibodies and contained tyrosine kinase activity that was inhibited by genistein and tyrphostin. We conclude that porcine coronary artery tissue contains src-related tyrosine kinase activity. However, because of the comparatively low sensitivity of the isolated src kinase activity towards genistein and tyrphostin, compared with the much higher sensitivity of the contractile response to these inhibitors, a direct role for c-rsc in the regulation of contractions elicited by agonists such as angiotensin II and arginine vasopressin cannot yet be assigned.

Mechanism of endothelium‐dependent relaxation induced by substance P in the coronary artery of the pig

1. Using front-surface fluorometry of fura-2-loaded porcine coronary arterial strips with the endothelium intact, we investigated the mechanisms of vasorelaxation induced by substance P (SP). Fura-2 fluorescence signals which indicated the cytosolic Ca2+-concentration ([Ca2+]i), were observed to arise exclusively from teh smooth muscle cells in these strips. 2. During the contractions induced by U46619 (100 nM), a thromboxane A2 analogue, an SP-induced endothelium-dependent, biphasic vasorelaxation was observed, which consisted of an initial rapid relaxation phase followed by a sustained phase, with a transient decrease in [Ca2+]i. Pretreatment with indomethacin (Ind) had no effect on the SP-induced relaxation; however, pretreatment with NG-nitro-L-arginine (L-NOARG) partially, but significantly inhibited the decrease in both the [Ca2+]i and tension abolished. Thus, part of the relaxation was considered to be mediated by L-NOARG-sensitive relaxing factor (endothelium-derived relaxing factor: EDRF). 3. During the 40 mM K+-depolarization-induced contraction which may eliminate the effects of endothelium-derived hyperpolarizing factor (EDRF), the vasorelaxation reduced by SP was completely inhibited by L-NOARG. 4. During the vasorelaxation induced SP, the [Ca2+]i-tension relationships shifted to the right of the contractions induced by either U46619 or high K+-depolarization. 5. Using front-surface fluorometry of fura-2 loaded porcine aortic valvular strips, we examined the effects of SP on [Ca2+]i in endothelial cells in situ. SP induced a rapid increase in [Ca2+]i of endothelial cells in situ followed by a small sustained phase in normal PSS (5.9 mM K+). The increase in extracellular K+ had no apparent effect on the SP-induced [Ca2+]i elevation of endothelial cells.

17 beta-Estradiol inhibits Ca2+ influx and Ca2+ release induced by thromboxane A2 in porcine coronary artery.

We wished to investigate the possible mechanism of the protective effect of estrogen replacement on coronary atherosclerosis observed in postmenopausal women. Cytosolic Ca2+ concentration ([Ca2+]i) and contraction were measured simultaneously in fura 2-loaded porcine coronary arterial strips stimulated by the thromboxane A2 analogue U46619 and high-K+ depolarization in the presence and absence of 17 beta-estradiol. Pretreatment with 17 beta-estradiol (30 nmol/L to 30 mumol/L) inhibited the sustained elevation of [Ca2+]i and the sustained contraction induced by 300 nmol/L U46619. Higher concentrations of 17 beta-estradiol (1 to 100 mumol/L) also inhibited the U46619-induced transient increase in [Ca2+]i and contraction in the absence of extracellular Ca2+. In the strips precontracted by 90 mmol/L K+, 17 beta-estradiol (30 mumol/L) inhibited the increases in [Ca2+]i and contraction to resting levels. In contrast, 30 mumol/L 17 beta-estradiol only partially inhibited the U46619-induced sustained contraction, despite complete inhibition of the sustained increase in [Ca2+]i. Verapamil (10 mumol/L) also strongly inhibited the sustained increase in [Ca2+]i induced by 300 nmol/L U46619, with a partial inhibition of the U46619-induced sustained contraction. A subsequent addition of 30 mumol/L 17 beta-estradiol did not show an additional inhibitory effect on either the [Ca2+]i or the tension after the addition of verapamil. 17 beta-Estradiol (10 mumol/L) also inhibited the increase in [Ca2+]i and the contraction induced by cumulative addition of Ca2+ in the strips pretreated with 90 mmol/L K+. However, 17 beta-estradiol did not change the slope of the [Ca2+]i-tension curves. 17 beta-Estradiol (10 mumol/L) had no effect on the levels of cAMP and cGMP in the coronary strips. 17 beta-Estradiol inhibits the contraction of coronary vascular smooth muscle mainly inhibiting Ca2+ influx without changing Ca2+ sensitivity of contractile elements. The Ca2+ channel blocker-like action of 17 beta-estradiol may explain at least a part of the antiatherosclerotic effect of estrogen.

Loss of contractile activity of endothelin-1 induced by electrical field stimulation-generated free radicals.

1. Electrical field stimulation (EFS; 10 V, 10 Hz, 2 ms) of porcine coronary artery strips precontracted with 10 nM endothelin-1 (ET-1) for 5 min caused a biphasic response, consisting of a slight contraction during EFS and a marked and irreversible relaxation just after EFS. This irreversible relaxation after EFS has never been investigated. In the present study, we have investigated the mechanism of the relaxation after EFS. 2. The EFS-induced response was not affected by the presence or absence of endothelium and was insensitive to 10 microM tetrodotoxin (TTX). 3. In the presence of free radical scavengers (40 u ml-1 superoxide dismutase (SOD), 1200 u ml-1 catalase or 80 mM D-mannitol), the relaxation after EFS was significantly inhibited. Moreover, relaxation after EFS was not observed in porcine coronary artery strips precontracted with 20 mM KCl. 4. In a cascade experiment, EFS of Krebs-Ringer solution containing 10 nM ET-1 induced marked suppression of the contractile activity of ET-1 in porcine coronary artery strips, which was in accord with the observed decrease in release of immunoreactive ET-1 (ir-ET-1). This effect of EFS was significantly inhibited by each of the free radical scavengers, 3 mM vitamin C, 40 u ml-1 SOD, 1200 u ml-1 catalase and 80 mM D-mannitol. 5. The exchange of 95% O2/5% CO2 gas for 95% N2/5% CO2 gas significantly inhibited the EFS-induced decrease in release of ir-ET-1. 6. Neither superoxide anions generated by xanthine (10 JM) plus xanthine oxidase (0.1 micro ml-1) nor hydrogen peroxide (10 microM) exogenously added to Krebs-Ringer solution containing 10 nM ET-1 affected the level of ir-ET-1.7. Generation of hydroxyl radicals was detected in the EFS-applied Krebs-Ringer solution. The EFS-induced generation of hydroxyl radicals was dependent on the period of stimulation and 02-bubbling, and significant generation of hydroxyl radicals was detectable with stimulation of over 5 min.Moreover, hydroxyl radicals generated in 50 mM NaCl solution containing 10 nM ET-1 by H202 plus Fe2 , i.e. the Fenton reaction, significantly decreased the level of ir-ET-l.8. These findings suggest that oxygen-derived hydroxyl radicals generated by EFS of porcine coronary artery strips inactivate ET-1, probably by structural modification. Thus, porcine coronary artery strips precontracted with ET-1 are potently relaxed by EFS.

Cytosolic Ca 2+ transients in endothelium-dependent relaxation of pig coronary artery, and effects of captopril

To determine the mechanism of endothelium-dependent relaxation by bradykinin, we simultaneously measured changes in cytosolic calcium concentrations ([Ca 2+] i) and force of fura-2-loaded strips of porcine coronary artery. We also examined effects of captopril, an angiotensin converting enzyme inhibitor, on bradykinin-induced relaxation. Bradykinin, in a concentration-dependent manner (10 −10 to 10 −7 M), decreased both [Ca 2+] i and force to resting levels, during 10 −5 M prostaglandin F 2α-induced contractions, only when endothelium was intact. Treatment with 10 −5 M captopril enhanced the bradykinin-induced decreases in [Ca 2+] i and force and shifted the concentration-response bradykinin induced a greater relaxation than that expected from the reduction in [Ca 2+] i. Captopril had no effects on the relationship between reduction in [Ca 2+] i and relaxation induced by bradykinin. Bradykinin relaxes porcine coronary artery in an endothelium-dependent manner, by decreasing [Ca 2+] i and also by controlling the Ca 2+ sensitivity of the contractile apparatus of smooth muscle. Captopril enhanced the bradykinin-induced relaxation, with no apparent direct effect on Ca 2+ sensitivity of the contractile apparatus.