The inhibitory effect of curcumin on voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells

We investigated the effects of curcumin, the principal active compound of turmeric, on voltage-dependent K⁺ (Kv) channels in freshly isolated rabbit coronary arterial smooth muscle cells using the voltage-clamp technique. Curcumin reduced the Kv current in a dose-dependent manner with an apparent K_d value of 1.07 ± 0.03 μM. Although curcumin did not alter the kinetics of Kv current activation, it predominantly accelerated the decay rate of channel inactivation. The association and dissociation rate constants of curcumin were 1.35 ± 0.05 μM^?1 s^?1 and 1.47 ± 0.17 s^?1, respectively. Curcumin did not alter the steady-state activation or inactivation curves. Application of train pulses (1 or 2 Hz) increased curcumin-induced blockade of the Kv current, and the recovery time constant also increased in the presence of curcumin suggesting, that the inhibitory action of Kv currents by curcumin was use-dependent. From these results, we concluded that curcumin inhibited vascular Kv current in a state-, time-, and use-dependent manner.     

The calmodulin inhibitor and antipsychotic drug trifluoperazine inhibits voltage-dependent K channels in rabbit coronary arterial smooth muscle cells

We investigated the effect of the calmodulin inhibitor and antipsychotic drug trifluoperazine on voltage-dependent K⁺ (Kv) channels. Kv currents were recorded by whole-cell configuration of patch clamp in freshly isolated rabbit coronary arterial smooth muscle cells. The amplitudes of Kv currents were reduced by trifluoperazine in a concentration-dependent manner, with an apparent IC₅₀ value of 1.58 ± 0.48 μM. The rate constants of association and dissociation by trifluoperazine were 3.73 ± 0.33 μM⁻¹ s⁻¹ and 5.84 ± 1.41 s⁻¹, respectively. Application of trifluoperazine caused a positive shift in the activation curve but had no significant effect on the inactivation curve. Furthermore, trifluoperazine provoked use-dependent inhibition of the Kv current under train pulses (1 or 2 Hz). These findings suggest that trifluoperazine interacts with Kv current in a closed state and inhibits Kv current in the open state in a time- and use-dependent manner, regardless of its function as a calmodulin inhibitor and antipsychotic drug.     

The protein kinase C inhibitor, bisindolylmaleimide (I), inhibits voltage-dependent K+ channels in coronary arterial smooth muscle cells

We examined the effects of the protein kinase C (PKC) inhibitor, bisindolylmaleimide (BIM) (I), on voltage-dependent K+ (K(V)) channels in rabbit coronary arterial smooth muscle cells using whole-cell patch clamp technique. BIM (I) reversibly and dose-dependently inhibited the K(V) currents with an apparent Kd value of 0.27 microM. The inhibition of the K(V) current by BIM (I) was highly voltage-dependent between -30 and +10 mV (voltage range of channel activation), and the additive inhibition of the K(V) current by BIM (I) was voltage-dependence in the full activation voltage range. The rate constants of association and dissociation for BIM (I) were 18.4 microM(-1) s(-1) and 4.7 s(-1), respectively. BIM (I) had no effect on the steady-state activation and inactivation of K(V) channels. BIM (I) caused use-dependent inhibition of K(V) current, which was consistent with the slow recovery from inactivation in the presence of BIM (I) (recovery time constants were 856.95 +/- 282.6 ms for control, and 1806.38 +/- 110.0 ms for 300 nM BIM (I)). ATP-sensitive K+ (K(ATP)), inward rectifier K+ (K(IR)), Ca2+-activated K+ (BK(Ca)) channels, which regulate the membrane potential and arterial tone, were not affected by BIM (I). The PKC inhibitor, chelerythrine, and protein kinase A (PKA) inhibitor, PKA-IP, had little effect on the K(V) current and did not significantly alter the inhibitory effects of BIM (I) on the K(V) current. These results suggest that BIM (I) inhibits K(V) channels in a phosphorylation-independent, and voltage-, time- and use-dependent manner.     

Angiotensin II inhibits inward rectifier K+ channels in rabbit coronary arterial smooth muscle cells through protein kinase Calpha

We investigated the effects of the vasoconstrictor angiotensin (Ang) II on the whole cell inward rectifier K(+) (Kir) current enzymatically isolated from small-diameter (<100 microm) coronary arterial smooth muscle cells (CASMCs). Ang II inhibited the Kir current in a dose-dependent manner (half inhibition value: 154 nM). Pretreatment with phospholipase C inhibitor and protein kinase C (PKC) inhibitors prevented the Ang II-induced inhibition of the Kir current. The PKC activator reduced the Kir currents. The inhibitory effect of Ang II was reduced by intracellular and extracellular Ca(2+) free condition and by G?6976, which inhibits Ca(2+)-dependent PKC isoforms alpha and beta. However, the inhibitory effect of Ang II was unaffected by a peptide that selectively inhibits the translocation of the epsilon isoform of PKC. Western blot analysis confirmed that PKCalpha, and not PKCbeta, was expressed in small-diameter CASMCs. The Ang II type 1 (AT(1))-receptor antagonist CV-11974 prevented the Ang II-induced inhibition of the Kir current. From these results, we conclude that Ang II inhibits Kir channels through AT(1) receptors by the activation of PKCalpha.     

Activation of inward rectifier K+ channels by hypoxia in rabbit coronary arterial smooth muscle cells

We examined the effects of acute hypoxia on Ba2+-sensitive inward rectifier K+ (K(IR)) current in rabbit coronary arterial smooth muscle cells. The amplitudes of K(IR) current was definitely higher in the cells from small-diameter (<100 microm) coronary arterial smooth muscle cells (SCASMC, -12.8 +/- 1.3 pA/pF at -140 mV) than those in large-diameter coronary arterial smooth muscle cells (>200 microm, LCASMC, -1.5 +/- 0.1 pA pF(-1)). Western blot analysis confirmed that Kir2.1 protein was expressed in SCASMC but not LCASMC. Hypoxia activated much more KIR currents in symmetrical 140 K+. This effect was blocked by the adenylyl cyclase inhibitor SQ-22536 (10 microM) and mimicked by forskolin (10 microM) and dibutyryl-cAMP (500 microM). The production of cAMP in SCASMC increased 5.7-fold after 6 min of hypoxia. Hypoxia-induced increase in KIR currents was abolished by the PKA inhibitors, Rp-8-(4-chlorophenylthio)-cAMPs (10 microM) and KT-5720 (1 microM). The inhibition of G protein with GDPbetaS (1 mM) partially reduced (approximately 50%) the hypoxia-induced increase in KIR currents. In Langendorff-perfused rabbit hearts, hypoxia increased coronary blood flow, an effect that was inhibited by Ba2+. In summary, hypoxia augments the KIR currents in SCASMC via cAMP- and PKA-dependent signaling cascades, which might, at least partly, explain the hypoxia-induced coronary vasodilation.     

Exploring the specificity of the PI3K family inhibitor LY294002

The PI3Ks (phosphatidylinositol 3-kinases) regulate cellular signalling networks that are involved in processes linked to the survival, growth, proliferation, metabolism and specialized differentiated functions of cells. The subversion of this network is common in cancer and has also been linked to disorders of inflammation. The elucidation of the physiological function of PI3K has come from pharmacological studies, which use the enzyme inhibitors Wortmannin and LY294002, and from PI3K genetic knockout models of the effects of loss of PI3K function. Several reports have shown that LY294002 is not exclusively selective for the PI3Ks, and could in fact act on other lipid kinases and additional apparently unrelated proteins. Since this inhibitor still remains a drug of choice in numerous PI3K studies (over 500 in the last year), it is important to establish the precise specificity of this compound. We report here the use of a chemical proteomic strategy in which an analogue of LY294002, PI828, was immobilized onto epoxy-activated Sepharose beads. This affinity material was then used as a bait to fish-out potential protein targets from cellular extracts. Proteins with high affinity for immobilized PI828 were separated by one-dimensional gel electrophoresis and identified by liquid chromatography-tandem MS. The present study reveals that LY294002 not only binds to class I PI3Ks and other PI3K-related kinases, but also to novel targets seemingly unrelated to the PI3K family.     

Ketamine blocks Ca2+-activated K+ channels in rabbit cerebral arterial smooth muscle cells

Although ketamine and Ca2+-activated K+ (KCa) channels have been implicated in the contractile activity regulation of cerebral arteries, no studies have addressed the specific interactions between ketamine and the KCa channels in cerebral arteries. The purpose of this study was to examine the direct effects of ketamine on KCa channel activities using the patch-clamp technique in single-cell preparations of rabbit middle cerebral arterial smooth muscle. We tested the hypothesis that ketamine modulates the KCa channel activity of the cerebral arterial smooth muscle cells of the rabbit. Vascular myocytes were isolated from rabbit middle cerebral arteries using enzymatic dissociation. Single KCa channel activities of smooth muscle cells from rabbit cerebral arteries were recorded using the patch-clamp technique. In the inside-out patches, ketamine in the micromolar range inhibited channel activity with a half-maximal inhibition of the ketamine concentration value of 83.8 +/- 12.9 microM. The Hill coefficient was 1.2 +/- 0.3. The slope conductance of the current-voltage relationship was 320.1 +/- 2.0 pS between 0 and +60 mV in the presence of ketamine and symmetrical 145 mM K+. Ketamine had little effect on either the voltage-dependency or open- and closed-time histograms of KCa channel. The present study clearly demonstrates that ketamine inhibits KCa channel activities in rabbit middle cerebral arterial smooth muscle cells. This inhibition of KCa channels may represent a mechanism for ketamine-induced cerebral vasoconstriction.     

The protein kinase A inhibitor, H-89, directly inhibits KATP and Kir channels in rabbit coronary arterial smooth muscle cells

The effects of the protein kinase A (PKA) inhibitor H-89 on ATP-sensitive K+ (KATP) and inward rectifier K+ (Kir) currents were examined in rabbit coronary arterial smooth muscle cells using the patch clamp technique. The H-89, in a dose-dependent manner, inhibited KATP and Kir currents with apparent Kd values of 1.19+/-0.18 and 3.78+/-0.37 microM, respectively. H-85, which is considered as an inactive form of H-89, inhibited KATP and Kir currents, similar to the result of H-89. KATP and Kir currents were not affected by either Rp-8-CPT-cAMPs, which is a membrane-permeable selective PKA inhibitor, or KT 5720, which is also known as a PKA inhibitor. Also, these two drugs did not significantly alter the effects of H-89 on the KATP and Kir currents. These results suggest that H-89 directly inhibits the KATP and Kir currents of rabbit coronary arterial smooth muscle cells independently of PKA inhibition.     

PDE2 inhibition by the PI3 kinase inhibitor LY294002 and analogues

Synthetic 2-morpholinochromones, including the known PI3-kinase inhibitor LY294002, have been evaluated in vitro as inhibitors of isolated human platelet phosphodiesterases. Inhibition of the cAMP-phosphodiesterases, PDE2 and PDE3 by LY294002 is reported for the first time. Preliminary screening across a range of 2-morpholinochromones has revealed structural features for optimised PDE2 inhibition.     

Selective serotonin reuptake inhibitor sertraline inhibits voltage-dependent K<Superscript>+</Superscript> channels in rabbit coronary arterial smooth muscle cells

We examined the effects of the selective serotonin reuptake inhibitor (SSRI) sertraline on voltage-dependent K⁺ (Kv) channels in freshly isolated rabbit coronary arterial smooth muscle cells using the voltage-clamp technique. Sertraline decreased the Kv channel current in a dose-dependent manner, with an IC₅₀ value of 0.18 μM and a slope value (Hill coefficient) of 0.61. Although the application of 1 μM sertraline did not affect the steady-state activation curves, sertraline caused a significant, negative shift in the inactivation curves. Pretreatment with another SSRI, paroxetine, had no significant effect on Kv currents and did not alter the inhibitory effects of sertraline on Kv currents. From these results, we concluded that sertraline dose-dependently inhibited Kv currents independently of serotonin reuptake inhibition by shifting inactivation curves to a more negative potential.     

Direct inhibition of a PKA inhibitor, H-89 on KV channels in rabbit coronary arterial smooth muscle cells

We examined the effects of the protein kinase A (PKA) inhibitor H-89 on voltage-dependent K(+) (K(V)) currents in freshly isolated rabbit coronary arterial smooth muscle cells, using a whole-cell patch clamp technique. H-89 inhibited the K(V) current in a concentration-dependent manner, with a K(d) value of 1.02 microM. However, the PKA inhibitors KT 5720 and Rp-8-CPT-cAMPS did not significantly alter the K(V) current or the inhibitory effects of H-89 on the K(V) current. Moreover, H-85, a structurally similar but inactive analog of H-89, showed similar inhibitory effects on the K(V) channel. H-89 had no effect on the voltage-dependency of activation or inactivation, or on recovery kinetics. These results suggest that in rabbit coronary arterial smooth muscle cells, H-89 inhibits the K(V) current directly by blocking the pore cavity, an effect independent of PKA inhibition.     

尼古丁调节大鼠冠状动脉平滑肌细胞大电导钙激活钾通道

目的:研究尼古丁对Wistar大鼠冠状动脉平滑肌大电导钙激活钾通道(BKca)活性的抑制作用及其细胞信号转导机制。方法:8周雄性Wistar大鼠随机分为两组:生理盐水组和尼古丁组;分别予以生理盐水和尼古丁2mg/(kg.d)注射21 d,蛋白酶法分离冠状动脉血管平滑肌细胞,将两组平滑肌细胞分别以对氯苯硫基环腺苷酸(CPT-cAMP,100μmol/L)和佛司可林(forskolin,10μmol/L)干预,单通道膜片钳记录干预前后平滑肌细胞单通道电流的平均开放时间(To)、平均关闭时间(Tc)、平均开放概率(Po)。结果:CPT-cAMP和Forskolin均能显著延长生理盐水组大鼠BKca的平均开放时间,缩短平均关闭时间,增加通道开放概率(P均<0.01)。对尼古丁组BKca的To、Tc、Po均无明显影响。结论:尼古丁促使冠状动脉血管收缩的生理机制是通过抑制cAMP/PKA途径诱导的大电导钙激活钾通道活性增加实现的。     

Vasoconstrictors inhibit ATP-sensitive K+ channels in arterial smooth muscle through protein kinase C

The effects of vasoconstrictor-receptor (neuropeptide Y, alpha- adrenergic, serotonergic, histaminergic) stimulation on currents through ATP-sensitive potassium (KATP) channels in arterial smooth muscle cells were examined. Whole-cell KATP currents, activated by the synthetic KATP channel opener pinacidil or by the endogenous vasodilator, calcitonin gene-related peptide, which acts through protein kinase A, were measured in smooth muscle cells isolated from mesenteric arteries of rabbit. Stimulation of NPY-, alpha 1-, serotonin (5-HT2)-, and histamine (H1)-receptors inhibited KATP currents by 40- 56%. The signal transduction pathway that links these receptors to KATP channels was investigated. An inhibitor of phospholipase C (D609) and of protein kinase C (GF 109203X) reduced the inhibitory effect of these vasoconstrictors on KATP currents from 40-56% to 11-23%. Activators of protein kinase C, a diacylglycerol analogue and phorbol 12-myristate 13- acetate (PMA), inhibited KATP currents by 87.3 and 84.2%, respectively. KATP currents, activated by calcitonin gene-related peptide, were also inhibited (47-87%) by serotonin, phenylephrine, and PMA. We propose that KATP channels in these arterial myocytes are subject to dual modulation by protein kinase C (inhibition) and protein kinase A (activation).     

Increased sensitivity of serotonin on the voltage-dependent K channels in mesenteric arterial smooth muscle cells of OLETF rats

This study examined the mechanisms of hypertension in diabetes. We investigated the effects of serotonin (5-HT) on voltage-dependent K⁺ (Kv) channel activity, vasoconstriction, 5-HT receptor expression levels, and the involvement of protein kinase C (PKC) in mesenteric arteries of Otsuka Long-Evans Tokushima fatty (OLETF) rats compared with Long-Evans Tokushima Otsuka (LETO) rats. Blood pressure, body weight, blood glucose level, and mesenteric arterial wall thickness were greater in OLETF rats. The 5-HT-induced vasoconstriction of mesenteric arteries was greater in OLETF rats than in LETO rats and inhibited by the 5-HT_2A inhibitor inhibitor, ketanserin. The Kv currents in mesenteric arterial smooth muscle cells (MASMCs), determined using a perforated patch clamp technique, was inhibited by 1 mM 4-AP (42.5 ± 4.1% vs. 63.5 ± 2.3% in LETO vs. OLETF rats at +40 mV), but was insensitive to 1 mM TEA and 100 nM iberiotoxin. The inhibition of Kv current by 1 μM 5-HT in MASMCs was greater in OLETF rats than in LETO rats (17.1 ± 2.2% vs. 33.2 ± 2.7% in LETO vs. OLETF rats at +40 mV), and the inhibition was prevented by treatment with the PKCα- and β- selective inhibitor, Gö6976. The expression level of 5-HT_2A, but not 5-HT_2B, receptor and the expression levels of total PKC, PKCβ, and PKCε, but not PKCα, were higher in the mesenteric arteries of OLETF rats compared with LETO rats. The enhanced expression of 5-HT_2A receptor together with PKCβ may promote mesenteric vasoconstriction and increase vascular resistance in OLETF rats.     

Hyposmotic challenge inhibits inward rectifying K+ channels in cerebral arterial smooth muscle cells

This study sought to define whether inward rectifying K(+) (K(IR)) channels were modulated by vasoactive stimuli known to depolarize and constrict intact cerebral arteries. Using pressure myography and patch-clamp electrophysiology, initial experiments revealed a Ba(2+)-sensitive K(IR) current in cerebral arterial smooth muscle cells that was active over a physiological range of membrane potentials and whose inhibition led to arterial depolarization and constriction. Real-time PCR, Western blot, and immunohistochemical analyses established the expression of both K(IR)2.1 and K(IR)2.2 in cerebral arterial smooth muscle cells. Vasoconstrictor agonists known to depolarize and constrict rat cerebral arteries, including uridine triphosphate, U46619, and 5-HT, had no discernable effect on whole cell K(IR) activity. Control experiments confirmed that vasoconstrictor agonists could inhibit the voltage-dependent delayed rectifier K(+) (K(DR)) current. In contrast to these observations, a hyposmotic challenge that activates mechanosensitive ion channels elicited a rapid and sustained inhibition of the K(IR) but not the K(DR) current. The hyposmotic-induced inhibition of K(IR) was 1) mimicked by phorbol-12-myristate-13-acetate, a PKC agonist; and 2) inhibited by calphostin C, a PKC inhibitor. These findings suggest that, by modulating PKC, mechanical stimuli can regulate K(IR) activity and consequently the electrical and mechanical state of intact cerebral arteries. We propose that the mechanoregulation of K(IR) channels plays a role in the development of myogenic tone.     

The PI3K inhibitor LY294002 prevents p53 induction by DNA damage and attenuates chemotherapy-induced apoptosis

The p53 tumor suppressor plays a key role in the natural protection against cancer. Activation of p53 by DNA-damaging agents can contribute to successful elimination of cancer cells via chemotherapy-induced apoptosis. The phosphatidylinositol-3 kinase (PI3K) pathway, triggered in normal cells upon exposure to growth factors, regulates a cascade of proliferation and survival signals. The PI3K pathway is abnormally active in many cancers, thus making it an attractive target for inactivation in an attempt to achieve better cancer therapy. We report here that exposure to LY294002, a potent PI3K inhibitor, aborts the activation of p53 by several drugs commonly used in cancer chemotherapy. Concomitantly, LY294002 attenuates p53-dependent, chemotherapy-induced apoptosis of cancer cells. These findings invoke an unexpected positive role for PI3K in p53 activation by anticancer agents, and suggest that the efficacy of PI3K inhibitors in cancer therapy may be greatly affected by the tumor p53 status.     

LY294002-geldanamycin heterodimers as selective inhibitors of the PI3K and PI3K-related family

Several LY294002-GM heterodimers were synthesized with the intent of modulating their activity in the presence of hsp90 and thereby creating selective inhibitors of PI3K and PI3K-related family.