Sham Arteries Research Articles

Paricalcitol Has a Potent Anti-Inflammatory Effect in Rat Endothelial Denudation-Induced Intimal Hyperplasia.

Neointimal hyperplasia is the main cause of vascular graft failure in the medium term. Vitamin D receptor activation modulates the biology of vascular smooth muscle cells and has been reported to protect from neointimal hyperplasia following endothelial injury. However, the molecular mechanisms are poorly understood. We have now explored the impact of the selective vitamin D receptor activator, paricalcitol, on neointimal hyperplasia, following guidewire-induced endothelial cell injury in rats, and we have assessed the impact of paricalcitol or vehicle on the expression of key cell stress factors. Guidewire-induced endothelial cell injury caused neointimal hyperplasia and luminal stenosis and upregulated the expression of the growth factor growth/differentiation factor-15 (GDF-15), the cytokine receptor CD74, NFκB-inducing kinase (NIK, an upstream regulator of the proinflammatory transcription factor NFκB) and the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Immunohistochemistry confirmed the increased expression of the cellular proteins CD74 and NIK. Paricalcitol (administered in doses of 750 ng/kg of body weight, every other day) had a non-significant impact on neointimal hyperplasia and luminal stenosis. However, it significantly decreased GDF-15, CD74, NIK and MCP-1/CCL2 mRNA expression, which in paricalcitol-injured arteries remained within the levels found in control vehicle sham arteries. In conclusion, paricalcitol had a dramatic effect, suppressing the stress response to guidewire-induced endothelial cell injury, despite a limited impact on neointimal hyperplasia and luminal stenosis. This observation identifies novel molecular targets of paricalcitol in the vascular system, whose differential expression cannot be justified as a consequence of improved tissue injury.

Phosphodiesterases type 2, 3 and 4 promote vascular tone in mesenteric arteries from rats with heart failure

Phosphodiesterases (PDE) type 3 and 4 promote vasoconstriction by hydrolysing cAMP. In experimental heart failure (HF), PDE3 makes PDE4 redundant in aorta, but it is not known if this occurs in resistance vessels, such as mesenteric artery. As PDE2 is increased in the failing myocardium, its possible role in the vasculature also needs to be addressed. Here, the function of PDE2, PDE3 and PDE4 in rat mesenteric arteries was characterized in experimental HF.Mesenteric arteries were isolated from rats sacrificed 22 weeks after surgical stenosis of the ascending aorta (HF), or Sham surgery. PDE inhibitors were used to probe isoenzyme contributions in enzymatic and isometric tension assays. PDE2 and PDE4 activities, but not PDE3 activity, facilitate contraction produced by the thromboxane analogue U46619 in Sham arteries, while in HF all three isoenzymes contribute to this response. NO synthase inhibition by L-NAME abolished the action of the PDE2 inhibitor. L-NAME eliminated the contribution of PDE4 in HF, but unmasked a contribution for PDE3 in Sham. PDE3 and PDE4 activities attenuated relaxant response to β-adrenergic stimulation in Sham and HF. PDE2 did not participate in cAMP or cGMP-mediated relaxant responses. PDE3 and PDE4 cAMP-hydrolysing activities were smaller in HF mesenteric arteries, while PDE2 activity was scarce in both groups. Endothelial cells and arterial myocytes displayed PDE2 immunolabelling.We highlight that, by contrast with previous observations in aorta, PDE4 participates equally as PDE3 in contracting mesenteric artery in HF. PDE2 activity emerges as a promoter of contractile response that is preserved in HF.

Abstract 21106: The Matrikine Proline-Glycine-Proline is Locally Generated Following Acute Arterial Vascular Injury

Introduction: Neointimal formation following vascular injury remains a clinically significant obstacle. Endovascular procedures, including percutaneous coronary intervention, disrupt the endothelium leading to inflammation and cellular proliferation within the vessel wall. Extracellular matrix (ECM) degradation by matrix metalloproteases (MMPs) has increasingly been understood to generate bioactive chemokines (i.e. matrikines) capable of causing cellular activation. Despite this knowledge, the contribution of matrikine signaling to vascular injury remains poorly understood. Hypothesis: We examined the hypothesis that the pro-inflammatory matrikines Proline-Glycine-Proline (PGP) and acetylated-PGP (AcPGP) are generated following acute arterial vascular injury. Methods: 20 male, c57bl/6 mice underwent unilateral carotid ligation of the left common carotid artery. Suture was passed, but not tied, under the contralateral carotid artery to serve as a sham procedure. Mice were sacrificed at Days 0, 1, 3 and 14 (n = 5 mice per day). Carotids and serum were harvested and processed for biochemical analysis. PGP and AcPGP were measured using liquid chromatography-tandem mass spectrometry, while total and pro-MMP9 was measured by ELISA. Results: Sham arteries had no significant induction of total or pro-MMP9 at any time. In contrast, ligated arteries showed a robust induction of both total and pro-MMP9 by Day 1 that was sustained through Day 14 (P < 0.0001 compared to mean sham arteries at all days). Importantly, PGP concentrations from ligated arteries showed a significant and sustained increase compared to Sham arteries by Day 3 (mean 150 vs 20 pg/mg carotid, P < 0.0001), while no difference was observed in AcPGP concentrations. Serum PGP and AcPGP concentrations remained low and unchanged throughout the 14-day experiment (mean ~100 and 20 pg/mL respectively). Conclusions: The current results identify PGP as a novel matrikine associated with acute vascular injury. AcPGP has previously been shown to activate endothelial cells to increase permeability and endothelin-1 synthesis via CXC Chemokine Receptor 2 (CXCR2). These results implicate PGP, a similar CXCR2 ligand, as a potentially novel mediator of inflammation following vascular injury.

Intermittent hypoxia in rats reduces activation of Ca2+ sparks in mesenteric arteries.

Ca(+) sparks are vascular smooth muscle cell (VSMC) Ca(2+)-release events that are mediated by ryanodine receptors (RyR) and promote vasodilation by activating large-conductance Ca(2+)-activated potassium channels and inhibiting myogenic tone. We have previously reported that exposing rats to intermittent hypoxia (IH) to simulate sleep apnea augments myogenic tone in mesenteric arteries through loss of hydrogen sulfide (H2S)-induced dilation. Because we also observed that H2S can increase Ca(2+) spark activity, we hypothesized that loss of H2S after IH exposure reduces Ca(2+) spark activity and that blocking Ca(2+) spark generation reduces H2S-induced dilation. Ca(2+) spark activity was lower in VSMC of arteries from IH compared with sham-exposed rats. Furthermore, depolarizing VSMC by increasing luminal pressure (from 20 to 100 mmHg) or by elevating extracellular [K(+)] increased spark activity in VSMC of arteries from sham rats but had no effect in arteries from IH rats. Inhibiting endogenous H2S production in sham arteries prevented these increases. NaHS or phosphodiesterase inhibition increased spark activity to the same extent in sham and IH arteries. Depolarization-induced increases in Ca(2+) spark activity were due to increased sparks per site, whereas H2S increases in spark activity were due to increased spark sites per cell. Finally, inhibiting Ca(2+) spark activity with ryanodine (10 μM) enhanced myogenic tone in arteries from sham but not IH rats and blocked dilation to exogenous H2S in arteries from both sham and IH rats. Our results suggest that H2S regulates RyR activation and that H2S-induced dilation requires Ca(2+) spark activation. IH exposure decreases endogenous H2S-dependent Ca(2+) spark activation to cause membrane depolarization and enhance myogenic tone in mesenteric arteries.

Loss of Hydrogen Sulfide‐Stimulated Ca 2+ Spark Activity in Arteries from Rats with Intermittent Hypoxia‐Induced Hypertension

Obstructive sleep apnea (OSA) is a risk factor for hypertension. Previous studies from our laboratory demonstrate increased blood pressure and increased myogenic vascular tone in mesenteric arteries from rats exposed to intermittent hypoxia (IH), a model of OSA. We have also observed that hydrogen sulfide (H2S) activates Ca2+ sparks in mesenteric arteries, and that IH exposure in rats decreases H2S-dependent dilation in mesenteric arteries. Therefore, we hypothesized that IH exposure would decrease H2S-activated Ca2+spark activity in mesenteric arteries to augment vasoconstriction. Mesenteric arteries collected from male Sprague-Dawley rats exposed to IH (20 exposures/hr of 5% O2/5% CO2 7 hr/day for 14 days) were loaded with the Ca2+indicators fluo-4 AM and Oregon Green BAPTA-1 and then cannulated and pressurized to 75 mmHg. Basal Ca2+spark activity was decreased in arteries from IH rats (0.31±0.04 vs. 0.43±0.02 sparks/cell/sec in Sham). The inhibitor of H2S synthesis (beta cyanoalanine, BCA) decreased spark activity activity in Sham arteries te abolish this diffference. Vascular smooth muscle cell (VSMC) depolarization induced by elevating extracellular [K+] or by increasing luminal pressure) increased Ca2+ spark activity in arteries from Sham but not IH rats and this increase in Ca2+ spark activity was prevented by removing the endothelium or by incubating with BCA. Ryanodine (10 µM) eliminated basal Ca2+ spark activity and prevented both H2S-induced dilation and H2S-induced spark activation. Our results provide evidence of impaired H2S-induced Ca2+ spark regulation of vascular reactivity in a rodent model of OSA and point to this pathway as a target for blood pressure control in hypertensives.

TRPV4‐associated alterations of Ca2+ pulsars in a murine model of pulmonary hypertension secondary to heart failure (1090.11)

Heart failure is the most frequent cause of pulmonary hypertension (PH) (group 2). Pathophysiological mechanisms modulating pulmonary vascular tone and leading to group 2‐PH are poorly defined. Since Ca2+ homeostasis is crucial to endothelial control of vascular tone, mechanisms involved, including TRPV4 channels, may be altered. This study aimed to characterize intracellular Ca2+ dynamics in pulmonary endothelium and their alterations in group 2 PH. A mouse model of group 2 PH was developed and 3rd order resistance arteries were isolated to investigate endothelial intracellular Ca2+. TRPV4 involvement was studied using a high‐speed confocal microscope. Characterization of endothelial localized calcium transients, similar to Ca2+ pulsars, was carried out in pulmonary arteries from control mice. Exposure of Sham arteries to GSK1016790A (100 nM), a TRPV4‐agonist, increased the frequency (1.5 fold) of Ca2+ pulsars. However, exposure to GSK1016790A had little if any effect on Ca2+ dynamics in PH arteries. TRPV4 channel expression at both mRNA and protein levels within pulmonary endothelium was confirmed by qPCR, IHC and WB approaches. Our data suggest a potential role for endothelial TRPV4 in vasoregulatory alterations involving local Ca2+ dynamics. This study strengthens our understanding of endothelial Ca2+ dyshomeostasis occurring in a clinically relevant model of PH.Grant Funding Source: FRQS, HSFC, FICM, CIHR

The 5‐HT1A receptor and sympathetic neurotransmission to mesenteric blood vessels in salt‐sensitive hypertension

Salt‐sensitive hypertension is prevalent U.S. and elsewhere. Salt acts in the brain to activate sympathetic nerves which supply the heart and blood vessels. Increases in sympathetic nerve activity cause high blood pressure (hypertension). Serotonin modulates blood vessel tone in the mesentery, a major contributor to peripheral resistance and venous return. Previous studies showed that sympathetic α2‐adrenergic autoreceptor function is impaired leading to increased norepinephrine release and vasoconstriction in deoxycorticosterone acetate (DOCA)‐salt rats. We tested the hypothesis that there is also impaired function of 5‐HT1A receptors on sympathetic nerve terminals in the mesentery leading to increased vasoconstriction. We used computer assisted video microscopy to measure in vitro changes in mesenteric blood vessel diameter caused by transmural nerve stimulation in the presence of 8‐OH‐DPAT, a 5‐HT1A receptor agonist. In sham (normotensive) arteries and veins, 8‐OH‐DPAT produced a dose‐dependent inhibition of constriction. There was a significant rightward shift in response in DOCA‐salt arteries and veins (p<.05). These findings suggest that impaired 5‐HT1A receptor function in the mesentery may contribute to a loss in regulation of sympathetic activity. This may be a mechanism by which pathology is maintained in salt‐sensitive hypertension. (Supported by P01HL070687)

Intermittent Hypoxia in Rats Increases Myogenic Tone Through Loss of Hydrogen Sulfide Activation of Large-Conductance Ca 2+ -Activated Potassium Channels

Myogenic tone, an important regulator of vascular resistance, is dependent on vascular smooth muscle (VSM) depolarization, can be modulated by endothelial factors, and is increased in several models of hypertension. Intermittent hypoxia (IH) elevates blood pressure and causes endothelial dysfunction. Hydrogen sulfide (H(2)S), a recently described endothelium-derived vasodilator, is produced by the enzyme cystathionine γ-lyase (CSE) and acts by hyperpolarizing VSM. Determine whether IH decreases endothelial H(2)S production to increase myogenic tone in small mesenteric arteries. Myogenic tone was greater in mesenteric arteries from IH than sham control rat arteries, and VSM membrane potential was depolarized in IH in comparison with sham arteries. Endothelium inactivation or scavenging of H(2)S enhanced myogenic tone in sham arteries to the level of IH. Inhibiting CSE also enhanced myogenic tone and depolarized VSM in sham but not IH arteries. Similar results were seen in cerebral arteries. Exogenous H(2)S dilated and hyperpolarized sham and IH arteries, and this dilation was blocked by iberiotoxin, paxilline, and KCl preconstriction but not glibenclamide or 3-isobutyl-1-methylxanthine. Iberiotoxin enhanced myogenic tone in both groups but more in sham than IH. CSE immunofluorescence was less in the endothelium of IH than in sham mesenteric arteries. Endogenouse H(2)S dilation was reduced in IH arteries. IH appears to decrease endothelial CSE expression to reduce H(2)S production, depolarize VSM, and enhance myogenic tone. H(2)S dilatation and hyperpolarization of VSM in small mesenteric arteries requires BK(Ca) channels.

Antioxidant treatment restores prejunctional regulation of purinergic transmission in mesenteric arteries of deoxycorticosterone acetate-salt hypertensive rats

Norepinephrine (NE) and ATP are co-released by periarterial sympathetic nerves. In mesenteric arteries (MA) from deoxycorticosterone-acetate (DOCA)-salt hypertensive rats, ATP, but not norepinephrine, release is impaired suggesting that their release may be regulated differently. We tested the hypothesis that different calcium channels contribute to ATP and norepinephrine release from sympathetic nerves in vitro in MA from normotensive and DOCA-salt hypertensive rats and that oxidative stress disrupts prejunctional regulation of co-transmission. Excitatory junction potentials (EJPs) were used to measure ATP release. Norepinephrine release was measured amperometrically with carbon-fiber microelectrodes. CdCl 2 (30 μM) inhibited norepinephrine release in sham and DOCA-salt arteries by 78% and 85%, respectively. The N-type calcium channel antagonist, ω-conotoxin GVIA (CTX, 0.1 μM) inhibited norepinephrine release by 50% and 67% in normotensive and DOCA-salt arteries, respectively while CTX blocked EJPs. The P/Q-type calcium channel antagonist ω-agatoxin IVA (ATX; 0.03 μM) reduced norepinephrine release in sham but not DOCA-salt arteries and increased EJPs in sham but not DOCA-salt arteries. ATX did not increase EJPs in sham arteries in the presence of the α 2-adrenergic receptor antagonist, yohimbine (1 μM). α 2-Autoreceptor-sensitive EJP facilitation is impaired in DOCA-salt hypertension but this response is restored in DOCA-salt rats treated chronically with the antioxidant, apocynin. Apocynin restored α 2-autoreceptor regulation of norepinephrine release. We conclude that ATP released from periarterial sympathetic nerves is controlled directly by N-type calcium channels. Norepinephrine release is controlled by N and P/Q type calcium channels. Norepinephrine release controlled by P/Q channels acts at α 2-adrenergic receptors to inhibit norepinephrine release suggesting that there may be multiple pools of norepinephrine in periarterial sympathetic nerves. Regulation of norepinephrine release by α 2-autoreceptors and P/Q-type channels is impaired in DOCA-salt hypertension and α 2-autoreceptor function is disrupted by oxidative stress.

Intermittent Hypoxia in Rats Reduces Hydrogen Sulfide Dependent Dilation to Enhance Myogenic Tone in Mesenteric Arteries

Sleep apnea is an independent risk factor for hypertension and rats exposed to intermittent hypoxia (IH) develop hypertension. We have shown that exposing rats for 14 days to IH enhances myogenic tone (MT) in small mesenteric arteries (MA) compared to MA from control (Sham) rats. Endothelial disruption enhanced MT in Sham arteries but had no effect in IH arteries. H2S is an endogenous vasodilator produced by cystathionine‐γ‐lyase (CSE) in the vasculature. We hypothesized that H2S produced by CSE in the endothelium reduces MT in MA from Sham rats and that H2S production in MA is impaired after 14 days of IH. Vessel wall [Ca2+]i and inner diameter were recorded in pressurized, fura‐2 loaded MA from IH and Sham rats. The CSE inhibitor β cyano L‐alanine (BCA) enhanced MT in Sham arteries compared to vehicle (20.2 ± 6.5% vs. 8.6 ± 1.0) and had no effect in IH arteries (24.0 ± 5.2 vs. 23.6 ± 7.9%). The H2S scavenger Bi(III) subsalicylate enhanced MT in Sham arteries similarly to BCA (20.4 ± 3.4%). NaHS (H2S donor) dilated both Sham and IH endothelium disrupted arteries (42.7 ± 5.7 vs. 57.5 ± 14.4%) with MT. These results suggest that H2S produced by CSE reduces MT in small endothelium intact MA and IH abolishes this effect of H2S. Dilation to NaHS is unaffected in IH endothelium disrupted arteries suggesting that enhanced MT is due to lower levels of endogenous H2S in MA.This work was supported by NIH grants HL82799 (NLK) and HL07736 (OJW).

HIV-1 infection can infect and replicate in human macrophages undetected by the host innate immune system

Abstract Introduction: A key element in the management of septic shock is maintenance of a blood pressure adequate for organ perfusion. Patients in prolonged septic shock have impaired pressor responses to norepinephrine (NE) yet enhanced sensitivity to vasopressin (VP). As both cause vasoconstriction through G protein-coupled receptors, activating the inositol phosphate cascade to increase vascular smooth muscle Ca2+ levels, the reason for this disparity is uncertain. We postulated that these drugs may have diverse effects on different Ca2+ mobilisation pathways during sepsis. We investigated this hypothesis using specific modulators of Ca2+ release and influx on the contractile responses to VP and NE in mesenteric arteries taken from septic and sham-operated rats. Methods: Sepsis was induced in 6 awake, fluid-resuscitated Wistar rats by i.p. injection of faecal slurry. Paired sham controls received no injection. Rats were sacrificed after 24 hours, and mesenteric arteries mounted on a wire myograph to measure isometric tension responses to VP and NE. The contributions of sarcoplasmic reticulum Ca2+ release and Ca2+ entry through the store-operated channel (SOC) were assessed by removing and returning extracellular Ca2+ respectively. The contribution of the voltage-gated Ca2+ channel (VGCC) was assessed by applying VP/NE in the presence of nifedipine. Results: Mimicking the clinical scenario, contractions were significantly enhanced to VP but depressed to NE in septic vessels (p<0.05). In all arteries, constriction to both agonists relied predominantly on extracellular Ca2+ influx rather than Ca2+ release from sarcoplasmic reticulum. The Ca2+ influx in response to NE was almost entirely VGCC-mediated, with a negligible contribution from SOCs in both sham and septic arteries. On the other hand, SOCs contributed significantly to VP-induced contraction, and SOCrather than VGCC-mediated influx of Ca2+ predominated in septic arteries. Discussion: Despite coupling to similar 2nd messenger systems, VP and NE appear to differentially activate SOCand VGCC-mediated Ca2+ entry. In sepsis, hyperpolarisation of the vascular smooth muscle membrane may impair VGCC activity. The ability of VP to preferentially activate SOCs may explain its preserved vasoconstrictor function in septic shock. Ca2+ channel modulation in this context is a potential new therapeutic paradigm.

Temperature‐dependent differences in sympathetic neuroeffector transmission in mesenteric arteries and veins in hypertension

Norepinephrine (NE) is released from sympathetic nerves supplying arteries and veins. We studied temperature‐related (28–37 ºC) differences in NE release and clearance using amperometry to measure NE in vitro. Tissues from sham and DOCA‐salt hypertensive rats were studied. Peak NE currents in sham veins declined more with temperature compared to arteries. Peak currents in DOCA‐salt arteries also showed greater temperature sensitivity than those in sham arteries. Currents in sham veins and DOCA‐salt arteries increased faster, decayed more slowly and were more temperature sensitive than those in sham arteries. Idazoxan (blocks α2 adrenergic receptors, α2ARs) and cocaine (blocks the NE transporter, NET) accelerated current rise time in sham arteries only. NE current decay was accelerated by idazoxan in veins but not arteries at 37 ºC and in arteries but not veins at 28 ºC, with a smaller increase in DOCA‐salt arteries at 28 ºC than sham arteries. We conclude that control of NE release from peri‐arterial nerves is more complex and therefore more temperature‐sensitive than in peri‐venous nerves. DOCA‐salt hypertension impairs NE release and clearance in arteries but not veins. α2ARs functional as autoreceptors and possibly as a “clearance receptor” holding NE in the neuroeffector junction. Clearance of NE in sham arteries is NET‐dependent and in sham veins and DOCA‐salt arteries is α2 NE clearance receptor dependent.

INTERMITTENT HYPOXIA IN RATS INHIBITS ENDOTHELIUM‐DEPENDENT VASODILATION

Rats exposed to eucapnic intermittent hypoxia (E‐IH) to mimic sleep apnea have elevated mean arterial pressure (MAP) and increased vascular production of superoxide anion (O2−). We hypothesized that increased arterial O2− in rats exposed to E‐IH quenches NO to inhibit acetylcholine‐induced (ACh) dilation. To test this hypothesis, aortas were dissected from rats exposed to E‐IH or air cycling (Sham) for 14 days. Aortic segments were suspended in tissue baths containing physiological saline solution in the presence of the superoxide dismutase mimetic, tiron (10 μM), the eNOS inhibitor, nitro‐L‐arginine (L‐NNA, 100 μM) or vehicle. Increasing concentrations of ACh were added to endothelium‐intact aortic rings constricted to 50% of maximal tension with phenylephrine. ACh‐induced dilation was diminished in E‐IH aortas compared to Sham (75.5±4.0 vs 97.1±1.5, p&lt;0.05). L‐NNA diminished dilation in both groups but dilation in E‐IH arteries was still less than that in Sham arteries (16.06±7.7 vs. 41.5±11.4, p &lt;0.05). Tiron treatment appeared to increase ACh‐induced dilation in E‐IH aortic segments but had no effect on dilation in Sham segments. Consistent with our hypothesis, E‐IH appears to cause endothelial dysfunction that may be caused in part by loss of a non‐NOS dilator and in part by increased O2− scavenging of NO.

Eucapnic intermittent hypoxia augments endothelin-1 vasoconstriction in rats: role of PKCδ

We reported previously that simulating sleep apnea by exposing rats to eucapnic intermittent hypoxia (E-IH) causes endothelin-dependent hypertension and increases constrictor sensitivity to endothelin-1 (ET-1). In addition, augmented ET-1-induced constriction in small mesenteric arteries (sMA) is mediated by increased Ca(2+) sensitization independent of Rho-associated kinase. We hypothesized that exposing rats to E-IH augments ET-1-mediated vasoconstriction by increasing protein kinase C (PKC)-dependent Ca(2+) sensitization. In sMA, the nonselective PKC inhibitor GF-109203x (3 microM) significantly inhibited ET-1-stimulated constriction in E-IH arteries but did not affect ET-1-stimulated constriction in sham arteries. Phospholipase C inhibitor U-73122 (1 microM) also inhibited constriction by ET-1 in E-IH but not sham sMA. In contrast, the classical PKC (cPKC) inhibitor Gö-6976 (1 microM) had no effect on ET-1-mediated vasoconstriction in either group, but a PKCdelta-selective inhibitor (rottlerin, 3 microM) significantly decreased ET-1-mediated constriction in E-IH but not in sham sMA. ET-1 increased PKCdelta phosphorylation in E-IH but not sham sMA. In contrast, ET-1 constriction in thoracic aorta from both sham and E-IH rats was inhibited by Gö-6976 but not by rottlerin. These observations support our hypothesis that E-IH exposure significantly increases ET-1-mediated constriction of sMA through PKCdelta activation and modestly augments ET-1 contraction in thoracic aorta through activation of one or more cPKC isoforms. Therefore, upregulation of a PKC pathway may contribute to elevated ET-1-dependent vascular resistance in this model of hypertension.

ROK contribution to endothelin-mediated contraction in aorta and mesenteric arteries following intermittent hypoxia/hypercapnia in rats

We reported previously that intermittent hypoxia with CO(2) to maintain eucapnia (IH-C) elevates plasma endothelin-1 (ET-1) and arterial pressure. In small mesenteric arteries (sMA; inner diameter = 150 microm), IH-C augments ET-1 constrictor sensitivity but diminishes ET-1-induced increases in intracellular Ca(2+) concentration, suggesting IH-C exposure increases both ET-1 levels and ET-1-stimulated Ca(2+) sensitization. Because Rho-associated kinase (ROK) can mediate Ca(2+) sensitization, we hypothesized that augmented vasoconstrictor sensitivity to ET-1 in arteries from IH-C-exposed rats is dependent on ROK activation. In thoracic aortic rings, ET-1 contraction was not different between groups, but ROK inhibition (Y-27632, 3 and 10 microM) attenuated ET-1 contraction more in IH-C than in sham arteries (50 +/- 11 and 78 +/- 7% vs. 41 +/- 12 and 48 +/- 9% inhibition, respectively). Therefore, ROK appears to contribute more to ET-1 contraction in IH-C than in sham aorta. In sMA, ROK inhibitors did not affect ET-1-mediated constriction in sham arteries and only modestly inhibited it in IH-C arteries. In ionomycin-permeabilized sMA with intracellular Ca(2+) concentration held at basal levels, Y-27632 did not affect ET-1-mediated constriction in either IH-C or sham sMA and ET-1 did not stimulate ROK translocation. In contrast, inhibition of myosin light-chain kinase (ML-9, 100 microM) prevented ET-1-mediated constriction in sMA from both groups. Therefore, IH-C exposure increases ET-1 vasoconstrictor sensitivity in sMA but not in aorta. Furthermore, ET-1 constriction is myosin light-chain kinase dependent and mediated by Ca(2+) sensitization that is independent of ROK activation in sMA but not aorta. Thus ET-1-mediated signaling in aorta and sMA is altered by IH-C but is dependent on different second messenger systems in small vs. large arteries.

Intermittent hypoxia increases ET‐1 vasoconstrictor sensitivity through increased PKC‐dependent Ca‐sensitization

We previously observed that in mesenteric arteries from intermittent hypoxia-induced (IH) hypertensive rats endothelin-1 (ET-1) constriction is augmented compared to constriction in Sham arteries. Furthermore, in Sham arteries, ET-1 constriction was accompanied by increases in vessel wall [Ca2+] but in IH arteries, constriction occurred without increases in [Ca2+]. Thus IH appears to augment ET-1 constriction by increasing vascular smooth muscle Ca-sensitizing pathways. We hypothesized that IH increased ET-1 activation of either protein kinase C (PKC) or Rho associated kinase (ROK) to augment Ca-sensitivity. We observed that ROK inhibition (Y-27632, 3 μM) attenuated ET-1 constriction slightly more in IH arteries than in Sham arteries (IH 80±3, Sham 89±4 % control ET-1 constriction). However, PKC inhibition (GF-109203x, 3 μM) greatly attenuated ET-1 mediated constriction in IH arteries but did not affect ET-1 constriction in Sham arteries (IH 50±3*, Sham 98±4 % control ET-1 constriction, *P<0.05). This suggests that IH greatly increases ET-1 activation of PKC-mediated constriction and slightly increases ET-1 activation of ROK-dependent constriction. Western analysis suggested that there is a tendency for PKC-α expression to be increased in IH mesenteric arteries compared to Sham but the difference was not significant (P = 0.33). Therefore augmented ET-1 vasoconstriction in IH arteries is mediated almost exclusively through Ca-sensitization with a large contribution by PKC activation that appears to be independent of IH-induced increases PKC-α expression. (Supported by AHA Established Investigator award and EPA RD-83186001, NLK)

Impaired alpha‐adrenergic autoreceptor modulation of purinergic transmission in mesenteric arteries of DOCA‐salt rats

Hypertension is associated with an increase in NE and ATP release from sympathetic nerves associated with mesenteric arteries where ATP is the principal constrictor transmitter. Prejunctional α 2-adrenoreceptors (ARs), regulate ATP and NE release. It is hypothesized that impairment of α 2-AR function may cause increased ATP release contributing to arterial constriction and hypertension. Our goal was to determine whether impaired α 2-AR function contributes to increased ATP release in mesenteric arteries of DOCA-salt hypertensive rats. Mesenteric preparations were maintained in vitro and intracellular recordings of excitatory junction potentials (EJPs) from smooth muscle cells were recorded. EJPs, mediated by ATP acting at P2X1 receptors, were evoked using single electrical stimuli (10–100V). There were no differences in peak EJP amplitude recorded at any voltage from sham or DOCA-salt arteries, nor were there differences in resting membrane potential or EJP rise/decay time. Clonidine (α 2-AR agonist) caused a concentration dependent inhibition of EJPs in sham and DOCA-salt arteries but EJPs in DOCA-salt arteries was less sensitive to inhibition by clonidine (P < 0.05). Clonidine did not change ATP-induced constrictions of sham arteries. However, clonidine caused direct constrictions of DOCA-salt arteries. We conclude that DOCA-salt hypertension does not modify the properties of arterial P2X1 receptors. In addition, α 2-ARs modulate ATP release and their function is impaired in DOCA-salt hypertension. DOCA-salt arteries are sensitized to the contractile effects of clonidine through an unidentified mechanism.

Activation of Vascular BK Channel by Tempol in DOCA-Salt Hypertensive Rats

Large-conductance Ca2+-activated potassium (BK) channels modulate vascular smooth muscle tone. Tempol, a superoxide dismutase (SOD) mimetic, lowers blood pressure and inhibits sympathetic nerve activity in normotensive and hypertensive rats. In the present study, we tested the hypotheses depressor responses caused by tempol are partly mediated by vasodilation. It was found that tempol, but not tiron (a superoxide scavenger), dose-dependently relaxed mesenteric arteries (MA) in anesthetized sham and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Tempol also reduced perfusion pressure in isolated, norepinephrine (NE) preconstricted MA from sham and DOCA-salt hypertensive rats. Maximal responses in DOCA-salt rats were twice as large as those in sham rats. The vasodilation caused by tempol was blocked by iberiotoxin (IBTX, BK channel antagonist, 0.1 micromol/L) and tetraethylammonium chloride (TEA) (1 mmol/L). Tempol did not relax KCl preconstricted arteries in sham or DOCA-salt rats, and Nomega-nitro-L-arginine methyl ester (L-NAME), apamin, or glibenclamide did not alter tempol-induced vasodilation. IBTX constricted MA and this response was larger in DOCA-salt compared with sham rats. Western blots and immunohistochemical analysis revealed increased expression of BK channel alpha subunit protein in DOCA-salt arteries compared with sham arteries. Whole-cell patch clamp studies revealed that tempol enhanced BK channel currents in HEK-293 cells transiently transfected with mslo, the murine BK channel a subunit. These currents were blocked by IBTX. The data indicate that tempol activates BK channels and this effect contributes to depressor responses caused by tempol. Upregulation of the BK channel alpha subunit contributes to the enhanced depressor response caused by tempol in DOCA-salt hypertension.

Alpha-1B adrenoceptors mediate neurogenic constriction in mesenteric arteries of normotensive and DOCA-salt hypertensive mice

The contribution of alpha-1 adrenergic receptor (α 1-AR) subtypes to neurogenic constrictions of mesenteric resistance arteries from SHAM and deoxycorticosterone acetate-salt (DOCA-salt) hypertensive mice was assessed. Frequency-response curves (0.5–30 Hz) for transmural stimulation-evoked contractions were examined in SHAM and DOCA-salt arteries in vitro in the absence (control) and presence of prazosin (0.1 μM), PPADS (10 μM), yohimbine (1 μM), 5-methylurapidil (5-MU; 0.1 μM), L-765,314 (1 μM) and BMY-7378 (0.3 μM); selective antagonists at α 1-, P2X, α 2-, α 1A-, α 1B-, and α 1D-AR, respectively. In SHAM arteries, prazosin but not PPADS inhibited neurogenic responses. L-765,314 substantially inhibited neurogenic responses while 5-MU had a small inhibitory effect. BMY-7378 did not alter contractile responses at all. In DOCA-salt arteries, prazosin reduced neurogenic responses with no further significant inhibition seen with PPADS. L-765,314 antagonized neurogenic constrictions to a level similar to that seen in SHAM arteries. Furthermore, 5-MU and BMY-7378 did not affect these responses. The density of noradrenergic nerves (assessed using glyoxylic acid-induced fluorescence) or norepinephrine (NE) content was not altered by DOCA-salt hypertension. These results indicate that NE is the primary mediator of neurogenic constriction of murine mesenteric arteries. Nerve-released NE acts primarily at α 1B-and to a lesser extent at α 1A-ARs in SHAM arteries whereas NE mediates neurogenic constrictions in DOCA-salt arteries by acting at α 1B-ARs.

Vascular reactivity of mesenteric arteries and veins to endothelin-1 in a murine model of high blood pressure

We characterized vascular reactivity to endothelin-1 (ET-1) in mesenteric vessels from DOCA-salt hypertensive and SHAM control mice and assessed the effect that endothelial-derived vasodilators have on ET-1-induced vasoconstriction. Changes in the diameter of unpressurized small mesenteric arteries and veins (100- to 300-μm outside diameter) were measured in vitro using computer-assisted video microscopy. Veins were more sensitive than arteries to the contractile effects of ET-1. There was a decrease in arterial maximal responses ( E max) compared to veins, this effect was larger in DOCA-salt arteries. The selective ET B receptor agonist, sarafotoxin 6c (S6c), contracted DOCA-salt and SHAM veins but did not contract arteries. The ET B receptor antagonist, BQ-788 (100 nM), but not the ET A receptor antagonist, BQ-610 (100 nM), blocked S6c responses. BQ-610 partially inhibited responses to ET-1 in mesenteric veins from DOCA-salt and SHAM mice while BQ-788 did not affect responses to ET-1. Co-administration of both antagonists inhibited responses to ET-1 to a greater extent than BQ-610 alone suggesting a possible functional interaction between ET A and ET B receptors. Responses to ET-1 in mesenteric arteries were completely inhibited by BQ-610 while BQ-788 did not affect arterial responses. Nitric oxide synthase inhibition potentiated ET-1 responses in veins from SHAM but not DOCA-salt mice. There was a prominent role for ET-mediated nitric oxide release in DOCA-salt but not SHAM arteries. In summary, these studies showed a differential regulation of ET-1 contractile mechanisms between murine mesenteric arteries and veins.