Effect Of Ca～(2+) Release Via IP₃R And DG-PKC Pathway On Regulation Of UTP In Relaxation And Contraction Of Porcine Coronary Artery

objective: Contraction and relaxation intimately correlates to intracellular Ca²⁺ concentration ([Ca²⁺]i) in smooth muscle, elevation in [Ca²⁺]i leads to concentration in smooth muscle and reduction in [Ca²⁺]i leads to relaxation in smooth muscle. Elevation in [Ca²⁺]i result from Ca²⁺ release from intracellular Ca²⁺ store and extracellular Ca²⁺ influxs via Ca²⁺ channels in plasma membrane. In vascular smooth muscle cells, sarcoplasmic reticulum is known as most important Ca²⁺ store in the process of Ca²⁺ mobilization. Ryanodine receptor in SR is one Ca²⁺ release channel, Ca²⁺ sparks are one of important calcium signaling events caused by the opening of ryanodine-sensitive Ca²⁺-release (RyR) channels. They result in a rapid, highly localized increase in [Ca²⁺]i and activate nearby BKCa channels to cause spontaneous transient outward currents(STOCs). These BKCa currents cause membrane potential hyperpolarization of arterial myocytes, which would lead to vasodilation through decreasing Ca²⁺ entry via voltage-dependent Ca²⁺ channels. The effect of Ca²⁺ sparks on global intracellular concentration of Ca²⁺ is very little, therefore STOCs which result from activation of BKCa channels are very important in regulating on vasoconstriction and vasorelaxation .Inositol 1,4,5- triphosphate receptor in SR is another Ca²⁺ release channel, Ca²⁺ puffs caused by the opening of inositol 1,4,5- triphosphate -sensitive Ca²⁺-release (IP3R ) channels participates regulation of vasoconstriction and vasorelaxation. Recent investigations have suggested that inositol 1,4,5- triphosphate ( IP3) and diacylglycel (DG) also play an important role in regulation of vasoconstriction and vasorelaxation. However, the precise mechanisms have been unclear. So far, it has been discovered that a wide variety of external factors stimulates phospholipase C (PLC) through activation of an associated G protein (Gq). PLC cleaves phosphatidylinositol bisphosphate to DG and IP3. These stimuni regulate cellular functions through IP3 and DG pathway. So, it is necessary to research the mechanism in vasoconstriction and vasorelaxation regulated by two messengers pathway. In the present study, we would study the effect of uridine 5,-triphosphate (UTP) on tension of porcine coronary artery vascular rings and effect of phorbol-12-myristate-13-acetate (PMA) on STOCs in porcine coronary artery smooth muscle cells applied with isolated pig coronary vascular rings combining with patch clamp methods, designed to expound the mechanisms of regulation of Ca²⁺ release via IP3Rs activated by IP3 productions and of DG-PKC pathway in the process of UTP induced contraction of isolated coronary artery vascular rings. Methods: (1) Vascular rings of coronary smooth muscle ex vivo experiment: The anterior descending branch of coronary artery was dissected free quickly from the heart of porcine subsequently placed into solution bubbled with O2, the free artery will be cut into 0.3-0.4㎝-long vascular ring. Vascular rings were cut through with two silver wires and holded in 95% O2 and 5% CO2, 37℃permanent solution I (pH 4.0). We can observe the effects of UTP, 2APB(IP3Rs channel inhibitor), BisI(DG-PKC pathway inhibitor) on the tension of coronary smooth muscle vascular rings. (2) Patch clamp experiment: The coronary artery was excised from the fresh porcine heart and cut into small segments and then transferred to enzymatic dissociation solution for incubation. Single smooth muscle cells were obtained by two-step enzyme digestion. STOCs were recorded by a whole-cell, amphotericin-perforated configuration of patch-clamp techniques. The currents were amplified and filtered by patch clamp amplifier (EPC10), and then the digitized data were recorded by pClamp9.0 software and further analyzed by MiniAnalysis6.0 program. The followings were studied: the effect of PMA, an agonist of PKC, on elementary STOCs in coronary smooth muscle cells ,and the effect of caffeine on STOCs induced by PMA. Results: (1) The effect of UTP on coronary artery smooth muscle vascular ring ex vivo :â‘ 300μM UTP had little direct effect on basal tension of coronary artery unpretreated with KCl(60min) (n=6, P>0.05);â‘¡300μM UTP had little direct effect on contraction produced by 45mM KCl (n=6, P>0.05);â‘¢300μM UTP increased basal tension of coronary artery pretreated with 45mM KCl for 3 times, Basal tention was increased by 0.36±0.07g (n=30,P<0.01). (2) The effect of 2APB on contraction evoked by UTP:â‘ 300μM 2APB initially augmented contraction induced by UTP of the same vascular ring by 0.11±0.04g (n=6, P<0.05), but eventually reduced tension evoked by UTP by 0.18±0.06g (n=6, P<0.05);â‘¡600μM 2APB significantly strengthed contraction induced by UTP of the same vascular ring by 0.26±0.10g (n=6, P<0.05) at the beginning , but eventually decreased contraction induced by UTP by 0.35±0.11g (n=6, P<0.05). (3) 300μM 2APB significantly contracted the same vascular ring in basal tension by 0.31±0.08g (n=6, P<0.05 ). (4) 7.5μM BisI significantly reduced tension evoked by UTP of the same vascular ring in basal tension by 0.25±0.09g (n=6, P<0.05). (5) STOCs under basal conditions were significantly inhibited after application of PMA (10nM). Amplitude and frequency was reduced by 11.74±2.74pA (n=10, P<0.01), 0.30±0.07Hz(n=10,P<0.01)respectively; caffeine (1mM) has little directly effect on decreasing of amplitude and frequence evoked by PMA(10nM) (n=6, P>0.05). Conclusions: (1) UTP has little direct action on basal tension of porcine coronary artery smooth muscle ; and UTP has little direct effect on contraction evoked by 45mM KCl ; however, UTP induces contraction of vascular rings of coronary artery ex vivo, SR Ca²⁺ over loaded maybe play an important role in the effect of UTP. (2) 2APB eventually reduces contraction evoked by UTP, but initially augments contraction evoked by UTP, which suggests UTP via PLC- IP3 activating Ca²⁺ release from IP3R channels activates STOCs, and negatively regulates tension of smooth muscle; however, Ca²⁺ release via IP3R channels eventually elevates global intracellular Ca²⁺ concentration leading to contraction. (3) Ca²⁺ release via IP3R channels negatively regulates the tension of coronary artery smooth muscle in basical condition (free agonists) (i.e, sponstaneous Ca²⁺ release via IP3R channels) maintains basal tension of smooth muscle cells, promoting relaxation of smooth muscle, Ca²⁺ release via IP3R channels activating STOCs maybe participates the relaxation. (4) DG-PKC pathway positively regulates tension in process of contraction of coronary artery smooth muscle induced by UTP, PKC inhibits STOCs of isolated coronary artery smooth muscle cells, which participates positively regulation of DG-PKC pathway.