Pressure-induced Myogenic Response Research Articles

The Functional Availability of Arterial Kv7 Channels Is Suppressed Considerably by Large-Conductance Calcium-Activated Potassium Channels in 2- to 3-Month Old but Not in 10- to 15-Day Old Rats

BackgroundVoltage-gated potassium (Kv) channels, especially Kv7 channels, are major potassium channels identified in vascular smooth muscle cells with a great, albeit differential functional impact in various vessels. Vascular smooth muscle Kv7 channels always coexist with other K channels, in particular with BK channels. BK channels differ in the extent to which they influence vascular contractility. Whether this difference also causes the variability in the functional impact of Kv7 channels is unknown. Therefore, this study addressed the hypothesis that the functional impact of Kv7 channels depends on BK channels.Experimental ApproachExperiments were performed on young and adult rat gracilis and saphenous arteries using real-time PCR as well as pressure and wire myography.Key ResultsSeveral subfamily members of Kv7 (KCNQ) and BK channels were expressed in saphenous and gracilis arteries: the highest expression was observed for BKα, BKβ1 and KCNQ4. Arterial contractility was assessed with methoxamine-induced contractions and pressure-induced myogenic responses. In vessels of adult rats, inhibition of Kv7 channels or BK channels by XE991 or IBTX, respectively enhanced arterial contractility to a similar degree, whereas activation of Kv7 channels or BK channels by retigabine or NS19504, respectively reduced arterial contractility to a similar degree. Further, IBTX increased both the contractile effect of XE991 and the anticontractile effect of retigabine, whereas NS19504 reduced the effect of retigabine and impaired the effect of XE991. In vessels of young rats, inhibition of Kv7 channels by XE991 enhanced arterial contractility much stronger than inhibition of BK channels by IBTX, whereas activation of Kv7 by retigabine reduced arterial contractility to a greater extent than activation of BK channels by NS19504. Further, IBTX increased the anticontractile effect of retigabine but not the contractile effect of XE991, whereas NS19504 reduced the effect of retigabine and impaired the effect of XE991.ConclusionKv7 and BK channels are expressed in young and adult rat arteries and function as negative feedback modulators in the regulation of contractility of these arteries. Importantly, BK channels govern the extent of functional impact of Kv7 channels. This effect depends on the relationship between the functional activities of BK and Kv7 channels.

Maternal High-Sucrose Diet Accelerates Vascular Stiffness in Aged Offspring via Suppressing Cav 1.2 and Contractile Phenotype of Vascular Smooth Muscle Cells.

The fetal programming in response to over-nutrition during pregnancy is involved in pathogenesis of cardiovascular diseases later in life. The authors' previous work reported that prenatal high-sucrose (HS) diet impaired functions of large-conductance Ca2+ -activated K+ channels (BK) in mesenteric arteries in the adolescent offspring rats. This study determines whether prenatal HS has a long-term impact on resistance vasculature in the aged offspring rats. Pregnant rats are fed with a high-sucrose diet until delivery. Aged offspring from prenatal HS exhibit elevated fasting insulin level, insulin resistance index, and diastolic pressure. Both pressure-induced myogenic responses and phenylephrine-stimulated contraction of mesenteric arteries in HS are weakened. Electrophysiological tests and western blot indicate that BK and L-type calcium channels (Cav 1.2) are impaired in HS group. On the other hand, expression of matrix metalloproteinase 2 of mesenteric arteries is reduced in HS group while expression of tissue inhibitors of metalloproteinase is increased, indicating that extra cellular matrix (ECM) is remodeled. Furthermore, expression of α-smooth muscle actin is decreased, and insulin/insulin receptor/phosphoinositide3-kinase (PI3K) signaling pathway is downregulated. The results suggest that prenatal HS induced stiffness of mesenteric arteries in aged offspring by inhibiting Cav 1.2 function and PI3K-associated contractile phenotype of VSMCs.

AdipoRon, adiponectin receptor agonist, improves vascular function in the mesenteric arteries of type 2 diabetic mice

BackgroundAdiponectin is one of the most abundant adipokines secreted from adipose tissue. An orally active synthetic adiponectin receptor agonist, adipoRon has been suggested to ameliorate insulin resistance, myocardial apoptosis, and pancreatic tumor. It has been reported that adiponectin directly induces vascular relaxation however, the chronic effect of adipoRon in the vascular dysfunction in type 2 diabetes has not been studied yet. Thus, in this study, we examined whether adipoRon improves vascular function in type 2 diabetes and what mechanism is involved.MethodsTen to 12‐week old male type 2 diabetic (db−/db−) mice were treated with adiponectin receptor agonist (adipoRon, 10 mg/kg/everyday, by oral gavage) for 2 weeks. Isolated mesenteric arteries were mounted in the arteriography and arterial diameter was measured. And western blot analysis was assessed.ResultsPressure‐induced myogenic response was significantly increased, whereas endothelium‐dependent relaxation was significantly reduced in the mesenteric arteries from type 2 diabetic mice. Interestingly, treatment of adipoRon normalized potentiated myogenic response. However, endothelium‐dependent relaxation was not affected by treatment of adipoRon. The expression levels of adiponectin receptor 1, 2 and adaptor protein phosphotyrosine interacting with PH domain and leucine zipper 1, 2 (APPL 1, 2) were increased in the mesenteric arteries from Type 2 diabetic mice and treatment of adipoRon did not affect them. Interestingly, adipoRon treatment increased the phosphorylation level of AMP‐activated protein kinase (AMPK) and decreased phosphorylation of myosin phosphatase target subunit 1 (MYPT1) in the type 2 diabetic mice while there was no change in the level of eNOS phosphorylation.ConclusionThe treatment of adipoRon improves vascular function in the mesenteric arteries from type 2 diabetic mice through endothelium‐independent mechanism. We suggest that myosin light chain phosphatase (MLCP) activation through reduced phosphorylation of MYPT1 might be the dominant mechanism in the adipoRon‐induced vascular effect.Support or Funding InformationThis work was supported by the Basic Science Research Program of the National Research Foundation (NRF) of Korea funded by the Ministry of Education, Science and Technology (NRF‐2018R1D1A1B07041820).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

AdipoRon, adiponectin receptor agonist improves vascular function in the mesenteric arteries of type 2 diabetic mice

AdipoRon, adiponectin receptor agonist improves vascular function in the mesenteric arteries of type 2 diabetic mice

Abstract 027: Hyperglycemia and Oxidative Stress Contribute to Impaired CerebralAutoregulation in Association With Dementia in Diabetes

Diabetes mellitus is characterized by hyperglycemia and is well established to associate with oxidative stress. The present study examines whether impaired myogenic response of middle cerebral artery MCA and autoregulation of cerebral blood flow (CBF) in our T2DN diabetic rats is due to hyperglycemia and oxidative stress induced actin cytoskeleton reorganization, and if this contributes to dementia. We identified that both young (4-month) and older (18-month) T2DN diabetic rats exhibit impaired pressure-induced myogenic response in isolated MCAs. Forced dilatation occurred at pressures above 140 mmHg in MCAs isolated from elderly T2DN rats with mild hypertension but not controls. Cortical blood flow measured by laser Doppler flowmetry rose by 137 ± 15% and 36 ± 5%, respectively, in T2DN and SD rats when MAP was increased from 100 to 180 mmHg. Autoregulation of CBF was shifted to lower pressures in elderly T2DN rats and they exhibited breakthrough at pressures above 140 mmHg. The F-Actin distribution area was significantly reduced in the vascular smooth muscle cells (VSMCs) freshly isolated from MCAs of T2DN rats compared with normal Sprague Dawley (SD) rats. The F-actin was disrupted similarly in VSMCs treated with high glucose or H 2 O 2 . Superoxide production is elevated in fresh isolated middle cerebral arteries (MCA) in T2DN diabetic rats compared to normal SD rats. We also found increased production of superoxide in primary VSMCs isolated from normal SD rats cultured in high glucose condition or freshly isolated from diabetic rats. T2DN rats exhibited neurons death in the hippocampus and cortex. Elderly T2DN rats showed memory disabilities. These results indicate that hyperglycemia induced elevated superoxide production causes actin cytoskeleton reorganization in cerebral VSMCs of diabetic T2DN rats, which may contribute to impaired cerebral vascular function, neurodegeneration and cognitive impairment.

Hypertension-Induced Enhanced Myogenic Constriction of Cerebral Arteries Is Preserved after Traumatic Brain Injury.

Traumatic brain injury (TBI) was shown to impair pressure-induced myogenic response of cerebral arteries, which is associated with vascular and neural dysfunction and increased mortality of TBI patients. Hypertension was shown to enhance myogenic tone of cerebral arteries via increased vascular production of 20-hydroxyeicosatrienoic acid (HETE). This adaptive mechanism protects brain tissue from pressure/volume overload; however, it can also lead to increased susceptibility to cerebral ischemia. Although both effects may potentiate the detrimental vascular consequences of TBI, it is not known how hypertension modulates the effect of TBI on myogenic responses of cerebral vessels. We hypothesized that in hypertensive rats, the enhanced myogenic cerebrovascular response is preserved after TBI. Therefore, we investigated the myogenic responses of isolated middle cerebral arteries (MCA) of normotensive and spontaneously hypertensive rats (SHR) after severe impact acceleration diffuse brain injury. TBI diminished myogenic constriction of MCAs isolated from normotensive rats, whereas the 20-HETE-mediated enhanced myogenic response of MCAs isolated from SHRs was not affected by TBI. These results suggest that the optimal cerebral perfusion pressure values and vascular signaling pathways can be different and, therefore, should be targeted differently in normotensive and hypertensive patients following TBI.

Abstract 223: Zinc-finger Nuclease Knockout Of Dual-specificity Protein Phosphatase-5 Enhances Myogenic Response In Autoregulation Of Cerebral Blood Flow In Fhh.1bn Rats

We recently reported that the pressure-induced myogenic responses of afferent arteries (Af-Art) and middle cerebral arteries (MCAs) were impaired in the fawn hooded hypertensive (FHH) rats and were restored in FHH.1BN congenic strain in which chromosome 1 from the Brown Norway (BN) rats containing 11 genes including dual-specificity protein phosphatase-5 (Dusp5) was transferred into FHH genetic background. There are 4 single nucleotide polymorphisms (SNP) in Dusp5 in FHH as compared with BN rats, one of which causes G155R mutation. To determine whether Dusp5 contributes to the impaired vascular myogenic response in FHH rats, we created a Dusp5 knockout (KO) rats in the FHH.1BN genetic background using zinc-finger nuclease (ZFN) that introduced a premature stop codon at amino acid (AA) 121. The expression of Dusp5 in KO rats were significantly decreased and the level of phosphorylated ERK2 (p-ERK2) was significantly increased in multiple organs including liver, spleen and white blood cells (WBCs). The luminal diameter of the MCAs in FHH.1BN rats (n=12) decreased 20 ± 2 % when the perfusion pressure was increased from 40 to 140 mmHg, whereas it decreased 34 ± 7 % in Dusp5 KO rats (n=6) and increased 10 ± 4% in FHH strain (n=8). Autoregulation was markedly impaired and CBF increased by 54 ± 6% in FHH rats when MAP was increased from 100 to 160 mmHg. CBF was better autoregulated in FHH.1BN strain and Dusp5 KO rats increased by only 26 ± 3% and 12 ± 3% when MAP was increased over the same range. However, the range of autoregulation of CBF was extended in the FHH rats (n=7) in that CBF rose to 107 ± 6% in FHH.1BN rats (n=7) when pressure was increased to 190 mmHg versus 33 ± 4% in the Dusp5 KO animals (n=6). These results suggest that Dusp5 plays an important role in modulating of myogenic tone in the cerebral circulation. Unless the G155R mutation activates Dusp5 in FHH rats, it is unlikely that Dusp5 is responsible for the impaired myogenic response in FHH rats.

Role of Endogenous ENaC and TRP Channels in the Myogenic Response of Rat Posterior Cerebral Arteries

AimsMechanogated ion channels are predicted to mediate pressure-induced myogenic vasoconstriction in small resistance arteries. Recent findings have indicated that transient receptor potential (TRP) channels and epithelial sodium channels (ENaC) are involved in mechanotransduction. The purpose of this study was to investigate the role of TRP channels and ENaC in the myogenic response. Our previous study suggested that ENaC could be a component of the mechanosensitive ion channels in rat posterior cerebral arteries (PCA). However, the specific ion channel proteins mediating myogenic constriction are unknown. Here we found, for the first time, that ENaC interacted with TRPM4 but not with TRPC6 using immunoprecipitation and confocal microscopy.Methods and ResultsTreatment with a specific βENaC inhibitor, amiloride, a specific TRPM4 inhibitor, 9-phenanthrol, and a TRPC6 inhibitor, SKF96365, resulted in inhibition of the pressure-induced myogenic response. Moreover, the myogenic response was inhibited in rat PCA transfected with small interfering RNA of βENaC, TRPM4, and TRPC6. Co-treatment with amiloride and 9-phenanthrol showed a similar inhibitory effect on myogenic contraction compared to single treatment with amiloride or 9-phenanthrol. The myogenic response was not affected by 9-phenanthrol or amiloride treatment in PCA transfected with βENaC or TRPM4 siRNA, respectively. However, pressure-induced myogenic response was fully inhibited by co-treatment with amiloride, 9-phenanthrol, and SKF96365, and by treatment with SKF96365 in PCA transfected with βENaC siRNA.ConclusionOur results suggest that ENaC, TRPM4, and TRPC6 play important roles in the pressure-induced myogenic response, and that ENaC and TRPM4 interact in rat PCA.

Remanent cell traction force in renal vascular smooth muscle cells induced by integrin-mediated mechanotransduction

It was previously demonstrated in isolated renal vascular smooth muscle cells (VSMCs) that integrin-mediated mechanotransduction triggers intracellular Ca(2+) mobilization, which is the hallmark of myogenic response in VSMCs. To test directly whether integrin-mediated mechanotransduction results in the myogenic response-like behavior in renal VSMCs, cell traction force microscopy was used to monitor cell traction force when the cells were pulled with fibronectin-coated or low density lipoprotein (LDL)-coated paramagnetic beads. LDL-coated beads were used as a control for nonintegrin-mediated mechanotransduction. Pulling with LDL-coated beads increased the cell traction force by 61 ± 12% (9 cells), which returned to the prepull level after the pulling process was terminated. Pulling with noncoated beads had a minimal increase in the cell traction force (12 ± 9%, 8 cells). Pulling with fibronectin-coated beads increased the cell traction force by 56 ± 20% (7 cells). However, the cell traction force was still elevated by 23 ± 14% after the pulling process was terminated. This behavior is analogous to the changes of vascular resistance in pressure-induced myogenic response, in which vascular resistance remains elevated after myogenic constriction. Fibronectin is a native ligand for α(5)β(1)-integrins in VSMCs. Similar remanent cell traction force was found when cells were pulled with beads coated with β(1)-integrin antibody (Ha2/5). Activation of β(1)-integrin with soluble antibody also triggered variations of cell traction force and Ca(2+) mobilization, which were abolished by the Src inhibitor. In conclusion, mechanical force transduced by α(5)β(1)-integrins triggered a myogenic response-like behavior in isolated renal VSMCs.

Hydrogen peroxide via thromboxane A2 receptors mediates myogenic response of small skeletal muscle veins in rats

We aimed to test two hypotheses: (1) isolated small veins develop substantial myogenic tone in response to elevation of intraluminal pressure, (2) H2O2 contributes to the mediation of myogenic response via activation of arachidonic acid (AA) cascade and release constrictor prostaglandins. Small veins were isolated from gracilis muscle of male rats, then cannulated. Changes of diameter to increases in intraluminal pressure, H2O2 and arachidonic acid in the presence and absence of various inhibitors were measured by videomicroscope and microangiometer. At the end of experiments the passive diameter were obtained in Ca2+ -free PSS solution. Isolated rat gracilis muscle small veins developed a substantial myogenic tone in response to increases in intraluminal pressure (1-12 mmHg). Calculated maximum myogenic tone was 70 ± 5% at 10 mmHg. Presence of catalase or indomethacin or SQ 29,548 reduced significantly the pressure-induced myogenic response. Also, H2O2 (10-9-10-5 M) and arachidonic acid (10-7-10-4 M) elicited concentration dependent constrictions, which were inhibited by the presence of indomethacin or SQ 29,548. We propose that both myogenic response and pressure-induced release of H2O2 play important roles in regulating the vasomotor function of venules both in physiological and pathological conditions.

Role of dual-specificity protein phosphatase-5 in modulating the myogenic response in rat cerebral arteries

The present study examined the role of the dual-specificity protein phosphatase-5 (DUSP-5) in the pressure-induced myogenic responses of organ-cultured cerebral arterial segments. In these studies, we initially compared freshly isolated and organ-cultured cerebral arterial segments with respect to responses to step increases in intravascular pressure, vasodilator and vasoconstrictor stimuli, activities of the large-conductance arterial Ca(2+)-activated K(+) (K(Ca)) single-channel current, and stable protein expression of DUSP-5 enzyme. The results demonstrate maintained pressure-dependent myogenic vasoconstriction, DUSP-5 protein expression, endothelium-dependent and -independent dilations, agonist-induced constriction, and unitary K(Ca) channel conductance in organ-cultured cerebral arterial segments similar to that in freshly isolated cerebral arteries. Furthermore, using a permeabilization transfection technique in organ-cultured cerebral arterial segments, gene-specific small interfering RNA (siRNA) induced knockdown of DUSP-5 mRNA and protein, which were associated with enhanced pressure-dependent cerebral arterial myogenic constriction and increased phosphorylation of PKC-βII. In addition, siRNA knockdown of DUSP-5 reduced levels of phosphorylated ROCK and ERK1 with no change in the level of phosphorylated ERK2. Pharmacological inhibition of ERK1/2 phosphorylation significantly attenuated pressure-induced myogenic constriction in cerebral arteries. The findings within the present studies illustrate that DUSP-5, native in cerebral arterial muscle cells, appears to regulate signaling of pressure-dependent myogenic cerebral arterial constriction, which is crucial for the maintenance of constant cerebral blood flow to the brain.

The Role of Sphingosine Kinase 1/Sphingosine-1-Phosphate Pathway in the Myogenic Tone of Posterior Cerebral Arteries

AimsThe goal of the current study was to determine whether the sphingosine kinase 1 (SK1)/sphingosine-1-phosphate (S1P) pathway is involved in myogenic vasoconstriction under normal physiological conditions. In the present study, we assessed whether endogenous S1P generated by pressure participates in myogenic vasoconstriction and which signaling pathways are involved in SK1/S1P-induced myogenic response under normal physiological conditions.Methods and ResultsWe measured pressure-induced myogenic response, Ca2+ concentration, and 20 kDa myosin light chain phosphorylation (MLC20) in rabbit posterior cerebral arteries (PCAs). SK1 was expressed and activated by elevated transmural pressure in rabbit PCAs. Translocation of SK1 by pressure elevation was blocked in the absence of external Ca2+ and in the presence of mechanosensitive ion channel and voltage-sensitive Ca2+ channel blockers. Pressure-induced myogenic tone was inhibited in rabbit PCAs treated with sphingosine kinase inhibitor (SKI), but was augmented by treatment with NaF, which is an inhibitor of sphingosine-1-phosphate phosphohydrolase. Exogenous S1P further augmented pressure-induced myogenic responses. Pressure induced an increase in Ca2+ concentration leading to the development of myogenic tone, which was inhibited by SKI. Exogenous S1P further increased the pressure-induced increased Ca2+ concentration and myogenic tone, but SKI had no effect. Pressure- and exogenous S1P-induced myogenic tone was inhibited by pre-treatment with the Rho kinase inhibitor and NADPH oxidase inhibitors. Pressure- and exogenous S1P-induced myogenic tone were inhibited by pre-treatment with S1P receptor blockers, W146 (S1P1), JTE013 (S1P2), and CAY10444 (S1P3). MLC20 phosphorylation was increased when the transmural pressure was raised from 40 to 80 mmHg and exogenous S1P further increased MLC20 phosphorylation. The pressure-induced increase of MLC20 phosphorylation was inhibited by pre-treatment of arteries with SKI.ConclusionsOur results suggest that the SK1/S1P pathway may play an important role in pressure-induced myogenic responses in rabbit PCAs under normal physiological conditions.

Organ culture as an in vitro model for the study of dual‐specificity phosphatase‐5 (DUSP‐5) and myogenic response in rat cerebral arterioles

Increase in intravascular pressure causes depolarization and constriction of cerebral arteries. The molecular mechanism of pressure‐induced myogenic response remains obscure. Our goal was to provide an optimal time window and preserved vascular function for an organ culture of cerebral arteries for the study of mechanisms involved in the regulation of cerebral arterial function. We initiated a comparative study of cultured and freshly isolated cerebral arteries in response to elevated intravascular pressure, vasodilator and vasoconstrictor stimuli, activity of Ca2+‐activated K+ (KCa) single‐channel current and protein expression of CYP 4 and DUSP‐5 enzymes. The results indicate that 72 h time frame of organ culture of cerebral arteries maintains cellular integrity, pressure dependent constriction, CYP4A2 and 4F protein expression, endothelium‐dependent and independent dilations, KCa channel current activity and vasoconstrictor response to serotonin. Furthermore, using a permeabilization, transfection technique the 72 h cultured arteries elicited a gene specific siRNA induced knockdown of DUSP‐5 protein expression. Subsequent pressure myograph studies and inhibitor studies revealed role of DUSP‐5 in regulation of myogenic tone. We propose a possible mechanism by which DUSP‐5 and ERK regulate myogenic response in cerebral arterioles. RO1 HL033833, RO1 HL092105, T32 HL007792.

Abstract 3142: Exaggerated K Ca Channel Current Activity Accounts for Reduced Myogenic Constriction in the Cerebral Vasculature of the Fawn Hooded Hypertensive (FHH) rats

The ionic mechanism by which pressure induces myogenic arterial constriction is not completely understood. In the present study we compared the Fawn Hooded Hypertensive (FHH) rat strain bred that does not autoregulate CBF and the FHH.1 BN rat strain created by transfer of a 2.6 Mb region of a Brown Norway (BN) rat chromosome 1 and introgressed into the FHH genetic background and recovered the myogenic phenotype, with respect to K Ca channel activity and pressure-induced myogenic constriction. We investigated the functional role of Ca 2+- activated K + channel in the impaired pressure-induced myogenic response in cannulated and pressurized middle cerebral arterial segments of FHH and compared with that of the FHH.1 BN rat strain. The cannulated arterial segments of FHH rat strain elicited significantly reduced pressure-induced myogenic constriction, whereas those of the FHH.1 BN exhibited more than 40-50 % constriction or reduction in diameter during step increases in intravascular pressure from 40 to 140 mmHg. Pretreatment of the he FHH rat cerebral arterial segments with the K Ca channel inhibitor iberiotoxin (100 nM) rendered the FHH rat cannulated arterial segments elicit near equal level of myogenic constriction as that of the FHH.1 BN . The magnitude and density (pA/pF) of a macroscopic whole-cell K Ca channel current recorded from FHH and FHH.1 BN rat cerebral arterial muscle cells during a 10 mV step depolarization from -70 mV to +80 mV was 2- to 3-fold greater in the FHH than that in the FHH.1 BN rat cerebral arterial muscle cells. Similarly, the NPo of the K Ca single-channel currents recorded at +40 mV using symmetrical KCl solution was significantly higher in the FHH (0.0035 ± 0.0002) than in the FHH.1BN (0.0013 ± 0.0003, n =5), albeit the unitary conductances were not different. These findings indicate that exaggerated K Ca channel current activity in the FHH rat cerebral arterial muscle cell membrane could account for the impaired pressure-induced myogenic constriction in the FHH rat strain and may have implications for stroke susceptibility in the FHH rat model.

Effect of heterozygous deletion of WNK1 on the WNK-OSR1/ SPAK-NCC/NKCC1/NKCC2 signal cascade in the kidney and blood vessels.

We found that a mechanism of hypertension in pseudohypoaldosteronism type II (PHAII) caused by a WNK4 missense mutation (D561A) was activation of the WNK-OSR1/SPAK-NCC signal cascade. However, the pathogenic effect of intronic deletions in WNK1 genes also observed in PHAII patients remains unclear. To understand the pathophysiological roles of WNK1 in vivo, WNK1(+/-)mice have been analyzed, because homozygous WNK1 knockout is embryonic lethal. Although WNK1(+/-) mice have been reported to have hypotension, detailed analyses of the WNK signal cascade in the kidney and other organs of WNK1(+/-) mice have not been performed. We assess the effect of heterozygous deletion of WNK1 on the WNK-OSR1/SPAK-NCC/NKCC1/NKCC2 signal cascade in the kidney and blood vessels. Contrary to the previous report, the blood pressure of WNK1(+/-) mice was not decreased, even under a low-salt diet. Under a WNK4(D561A/+) background, the heterozygous deletion of the WNK1 gene did not reduce the high blood pressure either. We then evaluated the phosphorylation status of OSR1, SPAK, NCC, NKCC1, and NKCC2 in the kidney, but no significant decrease in the phosphorylation was observed in WNK1(+/-) mice or WNK1(+/-)WNK4(D561A/+) mice. In contrast, a significant decrease in NKCC1 phosphorylation in the aorta and a decreased pressure-induced myogenic response in the mesenteric arteries were observed in WNK1(+/-) mice. The contribution of WNK1 to total WNK kinase activity in the kidney may be small, but that WNK1 may play a substantial role in the regulation of blood pressure in the arteries.

Epithelial Na+ channel proteins are mechanotransducers of myogenic constriction in rat posterior cerebral arteries

It has been suggested that mechanosensitive ion channels initiate myogenic responses in vessels; however, the molecular identity of the mechanosensitive ion channel complex is unknown. Although previous reports have suggested that epithelial Na(+) channel (ENaC) proteins are mechanotransducers in arteries, experimental evidence demonstrating that ENaC proteins are mechanotransducers are not fully elucidated. The goal of the present study was to determine whether the ENaC is a mechanotransducer for the myogenic response by providing supporting evidence in the rat posterior cerebral artery (PCA). We measured the effect of ENaC inhibition on the pressure-induced myogenic response, Ca(2+) concentration and 20 kDa myosin light chain (MLC(20)) phosphorylation. We detected expression of βENaC and γENaC subunits in rat PCA by Western blots and immunofluorescence. Inhibition of ENaCs with amiloride, ethyl isopropyl amiloride or benzamil blocked the myogenic response. Moreover, the myogenic response was inhibited in rat PCA transfected with βENaC and γENaC small interfering RNA. The myogenic response was inhibited by elimination of external Na(+), which was replaced with N-methyl-d-glucamine. Amiloride and nifedipine inhibited the pressure-induced increase in Ca(2+) concentration. Finally, MLC(20) increased when the intraluminal pressure was raised, and the pressure-induced increase in MLC(20) phosphorylation was inhibited by pretreatment with amiloride, and in arteries transfected with βENaC or γENaC small interfering RNA. Our results suggest that ENaCs may play an important role as mechanosensitive ion channels initiating pressure-induced myogenic responses in rat PCA.

Raloxifene improves vascular reactivity in pressurized septal coronary arteries of ovariectomized hamsters fed cholesterol diet

Although vascular effects of selective estrogen receptor modulators (SERMs) have been extensively examined in conduit arteries, whether SERMs could favorably modulate myogenic response in resistance arteries is unknown. The impact of raloxifene therapy and cholesterol diet on myogenic constriction during estrogen deficiency is unresolved. This study investigated changes in vascular reactivity and myogenic responses in female ovariectomized (Ovx) hamsters fed high-cholesterol diet (HCD) with and without chronic treatment of raloxifene. Functional studies were performed on hamster septal coronary arteries cannulated in a pressure myograph. Acetylcholine (ACh)-induced dilatation was reduced in arteries from cholesterol-fed Ovx hamsters, but not in those from cholesterol-fed hamsters, while pressure-induced myogenic constriction was unaffected. Chronic treatment with raloxifene restored ACh-induced dilatation in cholesterol-fed Ovx hamsters. U46619-induced constriction was increased in arteries from cholesterol-fed Ovx hamsters but not from cholesterol-fed control hamsters, which was normalized by chronic raloxifene treatment. The pressure–diameter relationship is presented as normalized diameter versus intraluminal pressure, while the effect of ACh or U46619 is expressed as percentage of tone at 80mmHg. Two-way analysis of variance (ANOVA) followed by Bonferroni post-tests were used for statistical evaluation among different treatment groups. P<0.05 was taken as statistically significant. The present results show that chronic treatment with raloxifene could benefit myogenically active coronary arteries by (i) restoring ACh-induced dilatation and (ii) reducing U46619-induced constriction without affecting pressure-induced myogenic responses in cholesterol-fed hamsters during estrogen deficiency. If such benefit can be observed in humans, raloxifene and other SERMs may be useful to preserve endothelial function and curtail vascular hypersensitivity in resistance coronary arteries in post-menopausal women with hypercholesterolemia or hyperlipidemia, a lipid condition implicated in the pathogenesis of myocardial infarction.

Structural and functional responses of extremity veins to long-term gravitational loading or unloading—lessons from animal systems

Long, transparent tubular tilt-cages were developed to maintain experimental rats either in 45 ∘ head-up (orthostasis model), or in 45 ∘ head-down body position (antiorthostasis model) for several weeks. In order to study the functional and structural changes in extremity blood vessels, also novel pressure angiograph systems, as well as special quantitative electron microscopic methods were applied. It was found that several adaptive mechanisms are activated in the lower limb superficial veins and microvessels of muscles when an organism is exposed to long-term (1–2 weeks) orthostatic-type gravitational load including a reversible amplification of the pressure-dependent myogenic response, tuning of the myogenic tone by Ca ++ - and voltage-sensitive K + channels in humans, augmentation of the intramural sympathetic innervation involving an increased nerve terminal density and synaptic vesicle count with functional remodeling, reorganization of vascular network properties (microvascular rarefaction in muscles, decreased branching angles in superficial veins), and responses of an endothelin and platelet-derived growth factor (PDGF) containing vesicle system in the endothelium. On the other hand, when applying long-term head-down tilting, the effects are dichotomous, e.g. it suppresses significantly the pressure-induced myogenic response, however does not diminish the adventitial sympathetic innervation density.

Nitric oxide modulates the interaction of pressure-induced wall mechanics and myogenic response of rat intramural coronary arterioles

Interactions between the biomechanical characteristics and pressure-induced active response of coronary microvessels are still not well known. We tested the hypothesis that pressure-dependent biomechanical characteristics of the coronary vascular wall are modulated by the active myogenic response and local vasodilators. We have utilized data obtained previously in isolated rat intramural coronary arterioles (approximately 100 microm in diameter), in which the diameter was investigated as a function of intraluminal pressure (Szekeres et al.: J. Cardiovasc. Pharmacol., 43, 242-249, 2004). To characterize the magnitude of myogenic response, diameter was expressed as percent of passive diameter as a function of pressure (normalized diameter; ND). In addition, circumferential wall stress (WS) and incremental distensibility (ID) were calculated. In control conditions, after an initial increase between 0-30 mm Hg, ND decreased substantially as pressure increased from 30 to 150 mm Hg. Correspondingly, WS gradually increased as a function of pressure (from 0.3 +/- 0.03 to 34.7 +/- 4.4 kPa) exhibiting a plateau phase between 40-80 mm Hg. In contrast, ID decreased and reached negative values (min: -104.9 +/- 21.9 10(-6) m2/N at 50 mm Hg). Inhibition of nitric oxide (NO) synthase by L-NNA decreased basal diameter (approximately 35% at 2 mm Hg), eliminated pressure-induced changes in ND, reduced the slope of pressure-WS curve, and decreased ID at lower pressures. Simultaneous administration of L-NNA and adenosine (which restored initial diameter, i.e. length of smooth muscle) restored--in part--the pressure-induced reduction in ND, reversed the pressure-induced behavior of WS to control, but not that of ID. These results not only confirm that in coronary arterioles wall stress is regulated by the myogenic response, but also suggest that there is interplay between the mechanical behavior of the wall and the myogenic response. Furthermore, the presence of NO seems to be necessary for maintaining a higher distensibility of intramural coronary arterioles allowing increases in diameter to lower pressures, which then activate the myogenic mechanism resulting in constrictions and full development of myogenic tone, as indicated by the presence of negative slope of pressure-diameter curve in the presence of NO.

Pregnancy attenuates uterine artery pressure-dependent vascular tone: role of PKC/ERK pathway

The mechanisms of adaptation of uterine artery vascular tone to pregnancy are not fully understood. The present study tested the hypothesis that pregnancy decreases the PKC-mediated Ca(2+) sensitivity of the contractile process and attenuates myogenic tone in resistance-sized uterine arteries. In pressurized uterine arteries from nonpregnant (NPUA) and near-term pregnant (PUA) sheep, we measured, simultaneously in the same tissue, vascular diameter and vessel wall intracellular Ca(2+) concentration ([Ca(2+)](i)) as a function of intraluminal pressure. In both NPUA and PUA, membrane depolarization with KCl caused a rapid increase in [Ca(2+)](i) and a decrease in diameter. A pressure increase from 20 to 100 mmHg resulted in a transient increase in diameter that was associated with an increase in [Ca(2+)](i), followed by myogenic contractions in the absence of further changes in [Ca(2+)](i). In addition, activation of PKC by phorbol 12,13-dibutyrate induced a decrease in diameter in the absence of changes in [Ca(2+)](i). Pressure-dependent myogenic responses were significantly decreased in PUA compared with NPUA. However, pressure-induced increases in [Ca(2+)](i) were not significantly different between PUA and NPUA. The ratio of changes in diameter to changes in [Ca(2+)](i) was significantly greater for pressure-induced contraction of NPUA than that of PUA. Inhibition of PKC by calphostin C significantly attenuated the pressure-induced vascular tone and eliminated the difference of myogenic responses between NPUA and PUA. In contrast, the MAPKK (MEK) inhibitor PD-098059 had no effect on NPUA but significantly enhanced myogenic responses of PUA. In the presence of PD-098059, there was no difference in pressure-induced myogenic responses between NPUA and PUA. The results suggest that pregnancy downregulates pressure-dependent myogenic tone of the uterine artery, which is partly due to increased MEK/ERK activity and decreased PKC signal pathway leading to a decrease in Ca(2+) sensitivity of myogenic mechanism in the uterine artery during pregnancy.