Bidirectional TRP/L Type Ca2+ Channel/RyR/BKCa Molecular and Functional Signaloplex in Vascular Smooth Muscles.

TRP channels are expressed both in vascular myocytes and endothelial cells, but knowledge of their operational mechanisms in vascular tissue is particularly limited. Here, we show for the first time the biphasic contractile reaction with relaxation followed by a contraction in response to TRPV4 agonist, GSK1016790A, in a rat pulmonary artery preconstricted with phenylephrine. Similar responses were observed both with and without endothelium, and these were abolished by the TRPV4 selective blocker, HC067047, confirming the specific role of TRPV4 in vascular myocytes. Using selective blockers of BKCa and L-type voltage-gated Ca2+ channels (CaL), we found that the relaxation phase was inducted by BKCa activation generating STOCs, while subsequent slowly developing TRPV4-mediated depolarisation activated CaL, producing the second contraction phase. These results are compared to TRPM8 activation using menthol in rat tail artery. Activation of both types of TRP channels produces highly similar changes in membrane potential, namely slow depolarisation with concurrent brief hyperpolarisations due to STOCs. We thus propose a general concept of bidirectional TRP-CaL-RyR-BKCa molecular and functional signaloplex in vascular smooth muscles. Accordingly, both TRPV4 and TRPM8 channels enhance local Ca2+ signals producing STOCs via TRP-RyR-BKCa coupling while simultaneously globally engaging BKCa and CaL channels by altering membrane potential.

CRISPR-Mediated Single Nucleotide Polymorphism Modeling in Rats Reveals Insight Into Reduced Cardiovascular Risk Associated With Mediterranean G6PD Variant.

Epidemiological studies suggest that individuals in the Mediterranean region with a loss-of-function, nonsynonymous single nucleotide polymorphism (S188F), in glucose-6-phosphate dehydrogenase (G6pd) are less susceptible to vascular diseases. However, this association has not yet been experimentally proven. Here, we set out to determine whether the Mediterranean mutation confers protection from vascular diseases and to discover the underlying protective mechanism. We generated a rat model with the Mediterranean single nucleotide polymorphism (G6PDS188F) using CRISPR-Cas9 genome editing. In rats carrying the mutation, G6PD activity, but not expression, was reduced to 20% of wild-type (WT) littermates. Additionally, unbiased metabolomics analysis revealed that the pentose phosphate pathway and other ancillary metabolic pathways connected to the pentose phosphate pathway were reduced (P<0.05) in the arteries of G6PDS188F versus WT rats. Intriguingly, G6PDS188F mutants, as compared with WT rats, developed less large arterial stiffness and hypertension evoked by high-fat diet and nitric oxide synthase inhibition with L-NG-nitroarginine methyl ester. Intravenous injection of a voltage-gated L-type Ca2+ channel agonist (methyl 2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-1,4-dihydropyridine-3-carboxylate; Bay K8644) acutely increased blood pressure in WT but not in G6PDS188F rats. Finally, our results suggested that (1) lower resting membrane potential of smooth muscle caused by increased expression of K+ channel proteins and (2) decreased voltage-gated Ca2+ channel activity in smooth muscle contributed to reduced hypertension and arterial stiffness evoked by L-NG-nitroarginine methyl ester and high-fat diet to G6PDS188F mutants as compared with WT rats. In summary, a mutation resulting in the replacement of a single amino acid (S188F) in G6PD, the rate-limiting enzyme in the pentose phosphate pathway, ascribed properties to the vascular smooth muscle that shields the organism from risk factors associated with vascular diseases.

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.

Dehydroepiandrosterone inhibits ICa,L and its window current in voltage-dependent and -independent mechanisms in arterial smooth muscle cells.

Dehydroepiandrosterone (DHEA) is an adrenal steroid hormone, which has the highest serum concentration among steroid hormones with DHEA sulfate (DHEAS). DHEA possesses an inhibitory action on glucose-6-phosphate dehydrogenase (G6PD), the first pentose-phosphate pathway enzyme that reduces NADP+ to NADPH. DHEA induced relaxation of high K+-induced contraction in rat arterial strips, whereas DHEAS barely induced it. We studied the effects of DHEA on L-type Ca2+ current ( ICa,L) of A7r5 arterial smooth muscle cells and compared the mechanism of inhibition with that produced by the 6-aminonicotinamide (6-AN) competitive inhibitor of G6PD. DHEA moderately inhibited ICa,L that was elicited from a holding potential (HP) of -80 mV [voltage-independent inhibition (VIDI)] and accelerated decay of ICa,L during the depolarization pulse [voltage-dependent inhibition (VDI)]. DHEA-induced VDI decreased peak ICa,L at depolarized HPs. By applying repetitive depolarization pulses from multiple HPs, novel HP-dependent steady-state inactivation curves ( f∞-HP) were constructed. DHEA shifted f∞-HP to the left and inhibited the window current, which was recorded at depolarized HPs and obtained as a product of current-voltage relationship and f∞-HP. The IC50 value of ICa,L inhibition was much higher than serum concentration. DHEA-induced VDI was downregulated by the dialysis of guanosine 5'- O-(2-thiodiphosphate), which shifted f∞-voltage to the right before the application of DHEA. 6-AN gradually and irreversibly inhibited ICa,L by VIDI, suggesting that the inhibition of G6PD is involved in DHEA-induced VIDI. In 6-AN-pretreated cells, DHEA induced additional inhibition by increasing VIDI and generating VDI. The inhibition of G6PD underlies DHEA-induced VIDI, and DHEA additionally induces VDI as described for Ca2+ channel blockers. NEW & NOTEWORTHY Dehydroepiandrosterone, the most abundantly released adrenal steroid hormone with dehydroepiandrosterone sulfate, inhibited L-type Ca2+ current and its window current in aortic smooth muscle cells. The IC50 value of inhibition decreased with the depolarization of holding potential to 15 µM at -20 mV. The inhibition occurred in a voltage-dependent manner as described for Ca2+ channel blockers and in a voltage-independent manner because of the inhibition of glucose-6-phosphate dehydrogenase.

Involvement of gap junctions between smooth muscle cells in sustained hypoxic pulmonary vasoconstriction development: a potential role for 15-HETE and 20-HETE.

In response to hypoxia, the pulmonary artery normally constricts to maintain optimal ventilation-perfusion matching in the lung, but chronic hypoxia leads to the development of pulmonary hypertension. The mechanisms of sustained hypoxic pulmonary vasoconstriction (HPV) remain unclear. The aim of this study was to determine the role of gap junctions (GJs) between smooth muscle cells (SMCs) in the sustained HPV development and involvement of arachidonic acid (AA) metabolites in GJ-mediated signaling. Vascular tone was measured in bovine intrapulmonary arteries (BIPAs) using isometric force measurement technique. Expression of contractile proteins was determined by Western blot. AA metabolites in the bath fluid were analyzed by mass spectrometry. Prolonged hypoxia elicited endothelium-independent sustained HPV in BIPAs. Inhibition of GJs by 18ß-glycyrrhetinic acid (18ß-GA) and heptanol, nonspecific blockers, and Gap-27, a specific blocker, decreased HPV in deendothelized BIPAs. The sustained HPV was not dependent on Ca(2+) entry but decreased by removal of Ca(2+) and by Rho-kinase inhibition with Y-27632. Furthermore, inhibition of GJs decreased smooth muscle myosin heavy chain (SM-MHC) expression and myosin light chain phosphorylation in BIPAs. Interestingly, inhibition of 15- and 20-hydroxyeicosatetraenoic acid (HETE) synthesis decreased HPV in deendothelized BIPAs. 15-HETE- and 20-HETE-stimulated constriction of BIPAs was inhibited by 18ß-GA and Gap-27. Application of 15-HETE and 20-HETE to BIPAs increased SM-MHC expression, which was also suppressed by 18ß-GA and by inhibitors of lipoxygenase and cytochrome P450 monooxygenases. More interestingly, 15,20-dihydroxyeicosatetraenoic acid and 20-OH-prostaglandin E2, novel derivatives of 20-HETE, were detected in tissue bath fluid and synthesis of these derivatives was almost completely abolished by 18ß-GA. Taken together, our novel findings show that GJs between SMCs are involved in the sustained HPV in BIPAs, and 15-HETE and 20-HETE, through GJs, appear to mediate SM-MHC expression and contribute to the sustained HPV development.

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.

PKC-δ isozyme gene silencing restores vascular function in diabetic rat.

Abstract Background: Endothelium and K+ channel functionality in smooth muscle cells (SMCs) regulates vascular function and is exposed to damage in diabetes. The regulatory enzyme protein kinase C (PKC) is known to play a key role in vascular tone regulation in health and disease. In this study, we evaluated the effect of PKC-δ gene silencing using small interfering RNAs (siRNAs) on endothelial dysfunction and acquired potassium channelopathy in vascular SMCs in diabetes. Methods: The experimental design comprised diabetes induction by streptozotocin (65 mg/kg) in rats, RNA interference, isolated aortic ring contractile recordings, whole-cell patch-clamp technique, measurements of reactive oxygen species (ROS), and real-time polymerase chain reaction technique. Animals were killed by cervical dislocation following ketamine (45 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) anesthesia administration on the third month of diabetes and on the seventh day after intravenous injection of siRNAs. Results: The aortas of diabetic rats demonstrated depressed endothelium-dependent relaxation and integral SMCs outward K+ currents as compared with those of controls. On the seventh day, PKC-δ gene silencing effectively restored K+ currents and increased the amplitude of vascular relaxation up to control levels. An increased level of PKC-δ mRNA in diabetic aortas appeared to be reduced after targeted PKC-δ gene silencing. Similarly, the level of ROS production that was increased in diabetes came back to control values after siRNAs administration. Conclusions: The silencing of PKC-δ gene expression using siRNAs led to restoration of vasodilator potential in rats with diabetes mellitus. It is likely that the siRNA technique can be a good therapeutic tool to normalize vascular function in diabetes.

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.