Electrophysiological characterization of the activating action of a novel liposomal nitric oxide carrier on Maxi-K channels in pulmonary artery smooth muscle cells.

The aim of this study was to establish the mechanisms of action of a novel liposomal nitric oxide (NO) carrier on large-conductance Ca2+-activated channels (BKCa or Maxi-K) expressed in vascular smooth muscle cells (VSMCs) isolated from the rat main pulmonary artery (MPA). Experimental design comprised of both whole-cell and cell-attached single-channel recordings using the patch-clamp techniques. The liposomal form of NO, Lip(NO), increased whole-cell outward K+ currents in a dose dependent manner while shifting the activation curve negatively by about 50 mV with respect to unstimulated cells with the EC50 value of 0.55 ± 0.17 µM. At the single channel level, Lip(NO) increased the probability of the open state (Po) of Maxi-K channels from 0.0020 ± 0.0008 to 0.74 ± 0.02 with half-maximal activation occurring at 4.91 ± 0.01 µM, while sub-maximal activation was achieved at 10-5 M Lip(NO). Channel activation was mainly due to significant decrease in the mean closed dwell time (about 500-fold), rather than an increase in the mean open dwell time, which was comparatively modest (about twofold). There was also a slight decrease in the amplitude of the elementary Maxi-K currents (approximately 15%) accompanied by an increase in current noise, which might indicate some non-specific effects of Lip(NO) on the plasma membrane itself and/or on the phospholipids environment of the channels. In conclusion, the activating action of Lip(NO) on the Maxi-K channel is due to the destabilization of the closed conformation of the channel protein, which causes its more frequent openings and, accordingly, increases the probability of channel transition to its open state.

C60 fullerenes selectively inhibit BKCa but not Kv channels in pulmonary artery smooth muscle cells.

Possessing unique physical and chemical properties, C60 fullerenes are arising as a potential nanotechnological tool that can strongly affect various biological processes. Recent molecular modeling studies have shown that C60 fullerenes can interact with ion channels, but there is lack of data about possible effects of C60 molecule on ion channels expressed in smooth muscle cells (SMC). Here we show both computationally and experimentally that water-soluble pristine C60 fullerene strongly inhibits the large conductance Ca2+-dependent K+ (BKCa), but not voltage-gated K+ (Kv) channels in pulmonary artery SMC. Both molecular docking simulations and analysis of single channel activity indicate that C60 fullerene blocks BKCa channel pore in its open state. In functional tests, C60 fullerene enhanced phenylephrine-induced contraction of pulmonary artery rings by about 25% and reduced endothelium-dependent acetylcholine-induced relaxation by up to 40%. These findings suggest a novel strategy for biomedical application of water-soluble pristine C60 fullerene in vascular dysfunction.

Liposomal quercetin potentiates maxi-K channel openings in smooth muscles and restores its activity after oxidative stress.

The effects of quercetin-loaded liposomes (PCL-Q) and their constituents, that is, free quercetin (Q) and 'empty' phosphatidylcholine vesicles (PCL), on maxi-K channel activity were studied in single mouse ileal myocytes before and after H2O2-induced oxidative stress. Macroscopic Maxi-K channel currents were recorded using whole-cell patch clamp techniques, while single BKCa channel currents were recorded in the cell-attached configuration. Bath application of PCL-Q (100 µg/ml of lipid and 3 µg/ml of quercetin) increased single Maxi-K channel activity more than threefold, from 0.010 ± 0.003 to 0.034 ± 0.004 (n = 5; p < 0.05), whereas single-channel conductance increased non-significantly from 138 to 146 pS. In the presence of PCL-Q multiple simultaneous channel openings were observed, with up to eight active channels in the membrane patch. Surprisingly, 'empty' PCL (100 µg/ml) also produced some channel activation, although it was less potent compared to PCL-Q, that is, these increased NPo from 0.010 ± 0.003 to 0.019 ± 0.003 (n = 5; p < 0.05) and did not affect single-channel conductance (139 pS). Application of PCL-Q restored macroscopic Maxi-K currents suppressed by H2O2-induced oxidative stress in ileal smooth muscle cells. We conclude that PCL-Q can activate Maxi-K channels in ileal myocytes mainly by increasing channel open probability, as well as maintain Maxi-K-mediated whole-cell current under the conditions of oxidative stress. While fusion of the 'pure' liposomes with the plasma membrane may indirectly activate Maxi-K channels by altering channel's phospholipids environment, the additional potentiating action of quercetin may be due to its better bioavailability.

Mechanisms of vascular dysfunction evoked by ionizing radiation and possible targets for its pharmacological correction.

Ionizing radiation (IR) leads to a variety of the cardiovascular diseases, including the arterial hypertension. A number of studies have demonstrated that blood vessels represent important target for IR, and the endothelium is one of the most vulnerable components of the vascular wall. IR causes an inhibition of nitric oxide (NO)-mediated endothelium-dependent vasodilatation and generation of reactive oxygen (ROS) and nitrogen (RNS) species trigger this process. Inhibition of NO-mediated vasodilatation could be due to endothelial NO synthase (eNOS) down-regulation, inactivation of endothelium-derived NO, and abnormalities in diffusion of NO from the endothelial cells (ECs) leading to a decrease in NO bioavailability. Beside this, IR suppresses endothelial large conductance Ca2+-activated K+ channels (BKCa) activity, which control NO synthesis. IR also leads to inhibition of the BKCa current in vascular smooth muscle cells (SMCs) which is mediated by protein kinase C (PKC). On the other hand, IR-evoked enhanced vascular contractility may result from PKC-mediated increase in SMCs myofilament Ca2+ sensitivity. Also, IR evokes vascular wall inflammation and atherosclerosis development. Vascular function damaged by IR can be effectively restored by quercetin-filled phosphatidylcholine liposomes and mesenchymal stem cells injection. Using RNA-interference technique targeted to different PKC isoforms can also be a perspective approach for pharmacological treatment of IR-induced vascular dysfunction.

Ionizing non-fatal whole-body irradiation inhibits Ca2+-dependent K+ channels in endothelial cells of rat coronary artery: possible contribution to depression of endothelium-dependent vascular relaxation.

PURPOSE: The goal of this study was to evaluate the influence of ionizing irradiation on large conductance Ca2+-dependent potassium (BKCa) channels in rat coronary endothelial cells. MATERIALS AND METHODS: Rats were exposed to a 6 Gy dose from a cobalt60 source. Experimental design of this study comprised recording of contractile force using isolated rat aortic rings and whole-cell patch clamp techniques to study whole-cell potassium currents in isolated rat coronary artery endothelial cells. RESULTS: It has been shown that outward potassium currents in endothelial cells 9 days after irradiation appear to be suppressed or even totally abolished. The reversal potential for these currents in irradiated cells was shifted to more positive values. Paxilline (500 nM), an inhibitor of BKCa channels, had no or only a negligible effect on irradiated cells. The experiments using isolated aortic rings demonstrated that both paxilline and irradiation significantly shifted the acetylcholine dependent concentration-relaxation response curve to the right. Irradiated tissues were insensitive to paxilline. CONCLUSION: The results suggest that non-fatal, whole-body gamma-irradiation suppresses large conductance, calcium-activated potassium channels, which control the driving force for Ca2+ entry and therefore Ca2+ dependent nitric oxide (NO) synthesis in endothelial cells. This may contribute, in part, to radiation-induced endothelium dysfunction and an increase in arterial blood pressure.

Mechanisms of endothelial dysfunction after ionized radiation: selective impairment of the nitric oxide component of endothelium-dependent vasodilation.

(1) Gamma radiation impairs vascular function, leading to the depression of endothelium-dependent vasodilatation. Loss of the nitric oxide (NO) pathway has been implicated, but little is known about radiation effects on other endothelial mediators. (2) This study investigated the mechanisms of endothelial dysfunction in rabbits subjected to whole-body irradiation from a cobalt(60) source. (3) The endothelium-dependent relaxation of rabbit aorta evoked by acetylcholine (ACh) or A23187 was impaired in a dose-dependent manner by irradiation at 2 Gy or above. Inhibition was evident 9 days post-irradiation and persisted over the 30 day experimental period. (4) Endothelium-independent responses to glyceryl trinitrate (GTN), sodium nitroprusside (SNP) and 3-morpholino-sydnonimine (SIN-1) were suppressed over a similar dose range at 7-9 days post-irradiation, but recovered fully by 30 days post-irradiation. (5) In healthy vessels, ACh-induced relaxation was inhibited by L-N(omega)-nitroarginine (L-NA; 3 x 10(-4) M) and charybdotoxin (10(-8) M) plus apamin (10(-6) M) but resistant to indomethacin, indicating the involvement of NO and endothelium-derived hyperpolarizing factor (EDHF). Supporting this, ACh caused smooth muscle hyperpolarization that was reduced by L-NA and charybdotoxin plus apamin. (6) In irradiated vessels, responses to ACh were insensitive to L-NA but abolished by charybdotoxin plus apamin, indicating selective loss of NO-mediated relaxation. (7) In animals treated shortly after irradiation with the antioxidant, alpha-tocopherol acetate, the NO-dependent relaxation was restored without effect on the EDHF-dependent component. (8) The results imply that radiation selectively impairs the NO pathway as a consequence of oxidative stress, while EDHF is able to maintain endothelium-dependent relaxation at a reduced level.

Protein kinase C in enhanced vascular tone in diabetes mellitus.

Diabetes mellitus (DM) is a complex syndrome which leads to multiple dysfunctions including vascular disorders. Hyperglycemia is considered to be a key factor responsible for the development of diabetic vascular complications and can mediate their adverse effects through multiple pathways. One of those mechanisms is the activation of protein kinase C (PKC). This important regulatory enzyme is involved in a signal transduction of several vascular functions including vascular smooth muscle contractility. Many studies have shown that hyperglycemia in DM results in oxidative stress. Overproduction of reactive oxygen species (ROS) by different oxidases and the mitochondrial electron transport chain (ETC), advanced glycation end products, polyol pathway flux, and hyperglicemia-induced rising in diacylglycerol (DAG) contribute to the activation of PKC. Activation of endothelial PKC in DM leads to endothelium-dependent vasodilator dysfunction. The main manifestations of this are inhibition of vasodilatation mediated by nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF) and prostacyclin, and activation of vasoconstriction mediated by endothelin-1 (ET-1), prostaglandin E2 (PGE2) and thromboxane A2 (TXA2). Activated PKC in DM also increases vascular endothelial growth factor (VEGF) expression and activates NADPH oxidases leading to raised ROS production. On the other hand, PKC in DM is involved in enhancement of vascular contractility in an endothelium-independent manner by inactivation of K(+) channels and Ca(2+) sensitization of myofilaments in vascular smooth muscle cells. This shows that PKC is a potential therapeutic target for treating vascular diabetic complications.

Gap junctions support the sustained phase of hypoxic pulmonary vasoconstriction by facilitating calcium sensitization.

AIMS: To determine the role of gap junctions (GJs) in hypoxic pulmonary vasoconstriction (HPV). METHODS AND RESULTS: Studies were performed in rat isolated intrapulmonary arteries (IPAs) mounted on a myograph and in anaesthetized rats. Hypoxia induced a biphasic HPV response in IPAs preconstricted with prostaglandin F2α (PGF2α, 3 µM) or 20 mM K⁺. The GJ inhibitors 18ß-glycyrrhetinic acid (18ß-GA, 30 µM), heptanol (3.5 mM), or 2-aminoethoxydiphenyl borate (2-APB) (75 µM) had little effect on the transient Phase 1 of HPV, but abolished the sustained Phase 2 which is associated with Ca²âº sensitization. The voltage-dependent Ca²âº channel blocker diltiazem (10 µM) had no effect on HPV, and did not alter the inhibitory action of 18ß-GA. Sustained HPV is enhanced by high glucose (15 mM) via potentiation of Ca²âº sensitization, in the presence of high glucose 18ß-GA still abolished sustained HPV. Simultaneous measurement of tension and intracellular Ca²âº using Fura PE-3 demonstrated that whilst 18ß-GA abolished tension development during sustained HPV, it did not affect the elevation of intracellular Ca²âº. Consistent with this, 18ß-GA abolished hypoxia-induced phosphorylation of the Rho kinase target MYPT-1. In anaesthetized rats hypoxia caused a biphasic increase in systolic right ventricular pressure. Treatment with oral 18ß-GA (25 mg/kg) abolished the sustained component of the hypoxic pressor response. CONCLUSION: These results imply that GJs are critically involved in the signalling pathways leading to Rho kinase-dependent Ca²âº sensitization during sustained HPV, but not elevation of intracellular Ca²âº, and may explain the dependence of the former on an intact endothelium.

Selective glycolysis blockade in guinea pig pulmonary artery and aorta reverses contractile and electrical responses to acute hypoxia.

The goal of this study was to clarify the mechanisms of hypoxic pulmonary vasoconstriction (HPV) reversal following selective glycolysis blockade and to assess possible contribution of endothelial electrogenesis to this phenomenon as a trigger mechanism. We compared smooth muscle (SM) contractility and endothelial cell (EC) membrane potential (MP) during acute hypoxia before and after glycolysis blockade. MPs were recorded from the endothelium of guinea pig pulmonary artery (GPPA) and thoracic aorta (GPTA) using the patch-clamp technique. Acute hypoxia caused hyperpolarization in GPTA EC, while EC from GPPA were depolarized. Also, acute hypoxia elicited constriction in isolated GPPA and dilatation in GPTA. Selective glycolysis inhibition always reversed both electrical and contractile responses in GPPA to hypoxia, but in GPTA this only occurred in 30% of experiments. It is likely that an unknown glycolysis-driven mechanism in EC mediates vascular tone regulation under hypoxia and underlies the paradoxical difference in the response of pulmonary and systemic arterial SM to hypoxia. Our data suggest that HPV development in GPPA might, at least partially, be driven by EC depolarization spreading to the underlying SM cells.

Protein kinase C-dependent inhibition of BK(Ca) current in rat aorta smooth muscle cells following gamma-irradiation.

PURPOSE: The aim of this study was to estimate the effects of non-fatal whole-body gamma-irradiation on outward potassium plasma membrane conductivity in rat vascular smooth muscle cells (VSMC), and to identify underlying mechanisms. MATERIALS AND METHODS: Rats were exposed to a 6 Gy dose irradiation from a cobalt(60) source. Whole-cell potassium current was measured in freshly isolated rat aorta smooth muscle cells using standard patch-clamp technique. RESULTS: We have determined that whole-body ionising irradiation significantly inhibits whole-cell outward K(+) current in rat aortic VSMC obtained from irradiated rats 9 and 30 days after irradiation, and this inhibition appears to be increased throughout post-irradiation period. Using selective inhibitors of small conductance Ca(2+)-activated K(+) channels (SK(Ca)), apamin (1 microM), intermediate conductance Ca(2+)-activated K(+) channels (IK(Ca,)), charybdotoxin (1 microM) and a large conductance Ca(2+)-activated K(+) channels (BK(Ca)), paxilline (500 nM), we established that the main component of whole-cell outward K(+) current in rat aortic VSMC is due to BK(Ca). It is clear that on the 9th day after irradiation paxilline had only a small effect on whole-cell outward K(+) current in VSMC, and was without effect on the 30th day post-irradiation, suggesting complete suppression of the BK(Ca) current. The PKC inhibitor, chelerythrine (100 nM), effectively reversed the suppression of whole-cell outward K(+) current induced by ionising irradiation in the post-irradiation period of 9 and 30 days. CONCLUSIONS: The results suggest that irradiation-evoked inhibition of the BK(Ca) current in aortic VSMC is mediated by PKC. Taken together, our data indicate that one of the mechanisms leading to elevation of vascular tone and related arterial hypertension development under ionising irradiation impact is a PKC-mediated inhibition of BK(Ca) channels in VSMC.

Ionizing radiation alters myofilament calcium sensitivity in vascular smooth muscle: potential role of protein kinase C.

Radiation exposure increases vascular responsiveness, and this change involves endothelial damage, as well as direct effects on vascular smooth muscle. In this study, we tested the hypothesis that myofilament Ca(2+) sensitivity in vascular smooth muscle is increased from single whole body gamma irradiation (6 Gy). We measured contractile responses from intact and permeabilized rat thoracic aortic rings combined with cytosolic Ca(2+) ([Ca(2+)](i)) measurements. The sensitivity to KCl and phenylephrine increased significantly in tissues from animals on the 9th and 30th days postirradiation compared with control. Irradiation also significantly increased Ca(2+) sensitivity in beta-escin permeabilized smooth muscle on the 9th and 30th days postirradiation. Inhibitors of protein kinase C, chelerythrine, and staurosporine, had no effect on the pCa-tension curves in control permeabilized tissues but significantly decreased Ca(2+) sensitivity in permeabilized tissues on the 9th and 30th days postirradiation. Phorbol dibutyrate (PDBu, 10(-7) M) increased Ca(2+) sensitivity in control skinned smooth muscle but was without effect in irradiated vascular rings. Simultaneous measurement of contractile force and [Ca(2+)](i) showed that myofilament Ca(2+) sensitivity defined as the ratio of force change to [Ca(2+)](i) significantly increased following gamma-irradiation. PDBu (10(-6) M) stimulation of intact aorta produced a sustained contraction, while the increase in [Ca(2+)](i) was transient. In irradiated tissues, PDBu-induced contractions were greater than those seen in control tissues but there was no elevation in [Ca(2+)](i). Taken together, these data strongly support the hypothesis that irradiation increases the sensitivity of vascular smooth muscle myofilaments to Ca(2+) and this effect is dependent on activation of protein kinase C.