EDHF and NO: different pathways for production--similar actions.
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Animals↗
Biomedical subjects
W M Chilian
Publications and source records attributed to W M Chilian.
The elastin-laminin receptor functions as a mechanotransducer in vascular smooth muscle.
Laminin and elastin, two major constituents of the extracellular matrix, bind to cells via the elastin-laminin receptor (ELR), a receptor distinct from integrins. Despite the ubiquitous nature of elastin and laminin in the matrix, the consequences of activation of the ELR are unknown. Because integrins are capable of mechanosensitive transduction, we hypothesized that the ELR would exert a similar function. Accordingly, we examined the effects of cyclical stretch on canine coronary smooth muscle gene expression and proliferation that are mediated by the ELR. Northern blot analyses showed a 31% decrease in serum-induced expression of c-fos when cells were stretched for 30 min on elastin, but no change in expression was observed on collagen. Serum-induced proliferation of stretched cells was markedly attenuated on elastin when compared with collagen. Both the molecular (decreased c-fos expression) and biological (decreased proliferation) responses on elastin were restored after blockade of the ELR with the elastin fragment hexapeptide (valine-glycine-valine-alanine-proline-glycine, VGVAPG). The inhibition was specific for this peptide, as another hydrophobic hexapeptide (valine-serine-leucine-serine-proline-glycine, VSLSPG) did not inhibit the responses. These results demonstrate that cyclic stretch inhibits c-fos expression and proliferation of coronary vascular smooth muscle cells grown on elastin matrixes, a mechanosensitive response that is transduced by the ELR.
Animals↗
Nitric oxide limits coronary vasoconstriction by a shear stress-dependent mechanism.
Increases in shear stress promote coronary vasodilation by stimulating the production of nitric oxide (NO). Whether shear stress-induced NO production also limits vasoconstriction in the coronary microcirculation in vivo is unknown. Accordingly, we measured microvascular diameter and flow velocity in the beating heart along with estimated blood viscosity to calculate shear stress during vasoconstriction with endothelin or vasopressin. Measurements were repeated in the presence of NG-monomethyl-L-arginine (L-NMMA) to inhibit NO production and BQ-788 to block NO-linked endothelin type B receptors. BQ-788 did not augment steady-state constriction to endothelin, suggesting that NO production via activation of this receptor is inconsequential. L-NMMA potentiated constriction to both agonists, particularly in small arteries (inner diameter >120 microm). Shear stresses in small arteries were elevated during constriction and further elevated during constriction after L-NMMA. These observations suggest that NO production limits vasoconstriction in the coronary microcirculation and that the principal stimulus for this governance is elevated shear stress. The degree of shear stress moderation of constriction is heterogeneously distributed, with small arteries displaying a higher degree of shear stress regulation than arterioles. These results provide the strongest evidence to date that shear stress-mediated production of NO exerts a "braking" influence on constriction in the coronary microcirculation.
Animals↗
Ischemia-induced coronary collateral growth is dependent on vascular endothelial growth factor and nitric oxide.
BACKGROUND: We hypothesized that ischemia-induced expression of vascular endothelial growth factor (VEGF) and the production of NO stimulate coronary collateral growth. METHODS AND RESULTS: To test this hypothesis, we measured coronary collateral blood flow and VEGF expression in myocardial interstitial fluid in a canine model of repetitive myocardial ischemia under control conditions and during antagonism of NO synthase. Collateralization was induced by multiple (1/h; 8/d), brief (2 minutes) occlusions of the left anterior descending coronary artery for 21 days. In controls, collateral blood flow (microspheres) progressively increased to 89+/-9 mL. min(-1). 100 g(-1) on day 21, which was equivalent to perfusion in the normal zone. Reactive hyperemic responses (a measure of the severity of ischemia) decreased as collateral blood flow increased. In N(G)-nitro-L-arginine methyl ester (L-NAME)- and L-NAME+nifedipine-treated dogs, to block the production of NO and control hypertension, respectively, collateral blood flow did not increase and reactive hyperemia was robust throughout the occlusion protocol (P<0.01 versus control). VEGF expression (Western analyses of VEGF(164) in myocardial interstitial fluid) in controls peaked at day 3 of the repetitive occlusions but waned thereafter. In sham-operated dogs (instrumentation but no occlusions), expression of VEGF was low during the entire protocol. In contrast, VEGF expression was elevated throughout the 21 days of repetitive occlusions after L-NAME. Reverse transcriptase-polymerase chain reaction analyses revealed that the predominant splice variant expressed was VEGF(164). CONCLUSIONS: NO is an important regulator of coronary collateral growth, and the expression of VEGF is induced by ischemia. Furthermore, the induction of coronary collateralization by VEGF appears to require the production of NO.
Animals↗
Effects of transforming growth factor-beta and mechanical strain on osteoblast cell counts: an in vitro model for distraction osteogenesis.
Factors known to regulate bone production during distraction osteogenesis include mechanical strain on bone forming cells and up-regulation of transforming growth factor-beta (TGF-beta) during the distraction, or strain phase of distraction osteogenesis. In the present study, an in vitro model was used to evaluate the functional effect of exogenous TGF-beta1 on mitogenesis in murine-derived MC3T3 osteoblasts during the period of active mechanical strain. The first hypothesis to be tested was that mitogenic suppression of MC3T3 osteoblasts by TGF-beta1 is further enhanced when these cells are also subjected to mechanical strain. To test this hypothesis, MC3T3 osteoblasts were seeded on flexible and rigid membranes. These were subjected to cyclic, vacuum-induced strain, simulating physiologic stress loads. After 24 hours, all cells were transferred to media containing TGF-beta1, and strain was continued for an additional 48 hours. The study was repeated by using two doses of TGF-beta1. This study demonstrated that final cell counts were significantly decreased in the presence of TGF-beta1 in both the nonstrained and strained groups (p < 0.0001). The final cell count in the strained group was significantly less than that in the nonstrained group (p < 0.0001) for both concentrations of TGF-beta1 tested, confirming the initial hypothesis. The second hypothesis to be tested was that alteration in the mitogenic response of MC3T3 osteoblasts after strain is not directly due to autocrine factors produced by the strained osteoblasts. To test this hypothesis, a proliferation assay was performed on nonconfluent MC3T3 osteoblasts by using conditioned media collected from strained and nonstrained osteoblasts. This study demonstrated no significant differences in cell counts after addition of conditioned media collected from strained versus nonstrained cells, confirming the latter hypothesis. The present study demonstrates the functional significance of mechanical strain on osteoblast cell counts. Furthermore, this may help to explain the temporal relationship observed during the early distraction (strain) phase of distraction osteogenesis in rodent models in which peak up-regulation of TGF-beta1 gene expression correlates with peak suppression of osteoblast function as measured by gene expression of extracellular matrix proteins.
Animals↗
Resistance to myocardial ischemia in five rat strains: is there a genetic component of cardioprotection?
There is a need to develop new and more consistent animal models of cardioprotection. Traditionally, outbred dogs, rabbits, and rats have been studied. We determined resistance to ischemia in isolated hearts from inbred strains of rats. Hearts from inbred rats: SS/Mcw (Dahl S, Dahl salt-sensitive), DA/Hsd (Dark Agouti), LEW/Hsd (Lewis), and BN/SsN/Mcw (Brown Norway); and from an outbred rat: Hsd:WIST (Wistar) were subjected to 27 min of global, no-flow ischemia, followed by 3 h of reperfusion. Infarct size in the Brown Norway rat was 2.5 times less than that observed in the Dahl S rat, with the Dark Agouti, Lewis, and Wistar rats intermediate in response. Hearts from Brown Norway rats were also most resistant to ischemia in terms of postischemic enzyme leakage and contractile and vascular function compared with other strains. The average polymorphism rate between strains revealed that such strains were genetically diverse. This study demonstrates strain differences in resistance to myocardial ischemia, suggesting these rats could be used to study a genetic and/or environmental basis for these differences and to provide new animal models for the physiological study of cardioprotection.
Animals↗
Peroxynitrite reversibly inhibits Ca(2+)-activated K(+) channels in rat cerebral artery smooth muscle cells.
Peroxynitrite (ONOO(-)) is a contractile agonist of rat middle cerebral arteries. To determine the mechanism responsible for this component of ONOO(-) bioactivity, the present study examined the effect of ONOO(-) on ionic current and channel activity in rat cerebral arteries. Whole cell recordings of voltage-clamped cells were made under conditions designed to optimize K(+) current. The effects of iberiotoxin, a selective inhibitor of large-conductance Ca(2+)-activated K(+) (BK) channels, and ONOO(-) (10-100 microM) were determined. At a pipette potential of +50 mV, ONOO(-) inhibited 39% of iberiotoxin-sensitive current. ONOO(-) was selective for iberiotoxin-sensitive current, whereas decomposed ONOO(-) had no effect. In excised, inside-out membrane patches, channel activity was recorded using symmetrical K(+) solutions. Unitary currents were sensitive to increases in internal Ca(2+) concentration, consistent with activity due to BK channels. Internal ONOO(-) dose dependently inhibited channel activity by decreasing open probability and mean open times. The inhibitory effect of ONOO(-) could be overcome by reduced glutathione. Glutathione, added after ONOO(-), restored whole cell current amplitude to control levels and reverted single-channel gating to control behavior. The inhibitory effect of ONOO(-) on membrane K(+) current is consistent with its contractile effects in isolated cerebral arteries and single myocytes. Taken together, our data suggest that ONOO(-) has the potential to alter cerebral vascular tone by inhibiting BK channel activity.
Animals↗
Nitric oxide exerts feedback inhibition on EDHF-induced coronary arteriolar dilation in vivo.
We tested the hypothesis that nitric oxide (NO) inhibits endothelium-derived hyperpolarizing factor (EDHF)-induced vasodilation via a negative feedback pathway in the coronary microcirculation. Coronary microvascular diameters were measured using stroboscopic fluorescence microangiography. Bradykinin (BK)-induced dilation was mediated by EDHF, when NO and prostaglandin syntheses were inhibited, or by NO when EDHF and prostaglandin syntheses were blocked. Specifically, BK (20, 50, and 100 ng. kg(-1). min(-1) ic) caused dose-dependent vasodilation similarly before and after administration of N(G)-monomethyl-L-arginine (L-NMMA) (3 micromol/min ic for 10 min) and indomethacin (Indo, 10 mg/kg iv). The residual dilation to BK with L-NMMA and Indo was completely abolished by suffusion of miconazole or an isosmotic buffer containing high KCl (60 mM), suggesting that this arteriolar vasodilation is mediated by the cytochrome P-450 derivative EDHF. BK-induced dilation was reduced by 39% after inhibition of EDHF and prostaglandin synthesis, and dilation was further inhibited by combined blockade with L-NMMA to a 74% reduction in the response. This suggests an involvement for NO in the vasodilation. After dilation to BK was assessed with L-NMMA and Indo, sodium nitroprusside (SNP, 1-3 microgram. kg(-1). min(-1) ic), an exogenous NO donor, was administered in a dose to increase the diameter to the original control value. Dilation to BK was virtually abolished when administered concomitantly with SNP during L-NMMA and Indo (P < 0.01 vs. before SNP), suggesting that NO inhibits EDHF-induced dilation. SNP did not affect adenosine- or papaverine-induced arteriolar dilation in the presence of L-NMMA and Indo, demonstrating that the effect of SNP was not nonspecific. In conclusion, our data are the first in vivo evidence to suggest that NO inhibits the production and/or action of EDHF in the coronary microcirculation.
Animals↗
Prologue: new insights into the regulation of the coronary microcirculation.
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Animals↗
Adenosine preconditions against endothelin-induced constriction of coronary arterioles.
Myocardial hypoperfusion is accompanied by concomitant increases in adenosine and endothelin-1 (ET-1) production, but the vasodilatory effect of adenosine prevails over that of ET-1. Therefore, we hypothesized that adenosine-induced or ischemic preconditioning reduces the vasoconstrictive effect of ET-1. Coronary arteriolar diameter in vivo was measured using fluorescence microangiography in anesthetized open-thorax dogs. ET-1 (5 ng. kg(-1). min(-1) administered intracoronary, n = 10) induced progressive constriction over 45 min [25 +/- 6% (SE)]. The constriction was blocked by preconditioning with adenosine (25 microgram. kg(-1). min(-1) administered intracoronary) for 20 min and 10 min of washout (n = 10) or attenuated by ischemic preconditioning (four 5-min periods of ischemia, 9 +/- 5% at 45 min). To investigate the receptor involved in this process, coronary arterioles (50-150 micrometer) were isolated and pressurized at 60 mmHg in vitro. The ET-1 dose-response curve (1 pM-5 nM) was rightward shifted after preconditioning with adenosine (1 microM) for 20 min and 10 min of washout (n = 11). Blockade of A(2) receptors [8-(3-chlorostyryl)caffeine, 1 microM, n = 9] but not A(1) receptors (8-cyclopentyl-1,3-dipropylxanthine, 100 nM, n = 7) prevented this shift. These results suggest that adenosine confers a vascular preconditioning effect, mediated via the A(2) receptor, against endothelin-induced constriction. This effect may offer a new protective function of adenosine in preventing excessive coronary constriction.
Adenosine↗
Brief commentary on coronary wave-intensity analysis.
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Animals↗
Regulation of shear stress in the canine coronary microcirculation.
BACKGROUND: Physical forces, such as pressure and flow, are well known to affect vascular function in the coronary circulation. Increases in shear stress produce vasodilation in coronary arterioles in vitro, and constant-flow preparations suggest a role for shear stress-induced vasodilation during adjustments to metabolic demand in vivo. Hypothetically, the regulation of shear stress can be viewed as a negative feedback control scheme (increased velocity --> increased shear --> vasodilation --> decreased velocity --> shear normalized). Therefore, we hypothesized that shear stress would be at least partially regulated during conditions of elevated flow. METHODS AND RESULTS: We used fluorescence microangiography to measure microvascular diameters and velocities in the coronary circulation in vivo and used these variables to calculate shear stress. Measurements were obtained under basal conditions, during maximal coronary blood flow, and after inhibition of NO synthase. Basal shear stress in the coronary circulation averaged 10 dyn/cm2 in small arteries and 19 dyn/cm2 in arterioles. Regulation of shear stress was observed in small arteries during adenosine-induced increases in coronary blood flow, but arterioles showed minimal regulation. NO synthase blockade had no effect on basal shear stress but completely abolished its regulation in small arteries during vasodilation. CONCLUSIONS: Our data provide the first quantitative estimates of microvascular shear stress in the coronary circulation. Moreover, our results suggest that shear stress in small coronary arteries is regulated by NO release from the endothelium.
Adenosine↗
Functional characteristics of the coronary microcirculation.
For over 50 years, it has been recognized that coronary blood flow is precisely matched to cardiac metabolism. The interactions which govern this matching remain unknown. In the current review, 3 specific aspects of coronary flow regulation will be discussed: Specialization of function in different microvascular domains, influence of cardiac region on microvascular function and the interactions of vasoactive agents in control of coronary blood flow. Each level of the coronary microcirculation is affected by different physical and chemical forces within the heart. These forces place special demands on these vessels and are in turn met by specialized vasodilator responses, including metabolic and flow-mediated vasodilation. Perfusion of the heart is also profoundly affected by the region perfused. The endocardium is affected by forces, notably cardiac contraction, in a different manner than the epicardium. Thus, the microcirculation has specialized to meet these demands. Finally, the factors determining microvascular tone appear to be coordinated such that the loss of any individual dilator, such as nitric oxide, can be compensated for by the increased contribution of another, such as adenosine. This interplay may serve to protect the heart from ischemia during the early phases of coronary vascular disease when individual dilators may be impaired.
Animals↗
Multifactorial basis for coronary collateralization: a complex adaptive response to ischemia.
Angiogenesis and vasculogenesis are adaptive responses of the coronary collateral circulation to myocardial ischemia. This review focuses on the concerted action of growth factors, growth factor receptors, extracellular matrix, and inflammatory cellular responses to regulate angiogenesis and vasculogenesis in response to myocardial ischemia and alterations in shear stress. Therapeutic angiogenesis represents a novel approach to increase myocardial perfusion in patients with coronary artery disease and provides an opportunity to further clarify the mechanisms that regulate collateral development. Impairment of angiogenic adaptive responses to ischemia during disease states is an important subject for future investigation.
Animals↗
Endothelin antagonists block alpha1-adrenergic constriction of coronary arterioles.
We have previously observed that intracoronary administration of the alpha1-adrenergic agonist phenylephrine (PE) over a period of minutes induced both an immediate and long-lasting (2 h) vasoconstriction of epicardial coronary arterioles. Because it is unlikely that alpha1-adrenergic constriction would persist for hours after removal of the agonist, this observation supports the view that another constrictor(s) is released during alpha1-adrenergic activation and induces the prolonged vasoconstriction. Therefore, we hypothesized that the prolonged microvascular constriction after PE is due to the production of endothelin (ET). We focused on ET not only because this peptide produces potent vasoconstriction but also because its vasoconstrictor action is characterized by a long duration. To test this hypothesis, the diameters of coronary arterioles (<222 micrometers) in the beating heart of pentobarbital-anesthetized dogs with stroboscopic intravital microscopy were measured during a 15-min intracoronary infusion of PE (1 microgram. kg-1 . min-1) and at 15-min intervals for a total of 120 min. All experiments were performed in the presence of beta-adrenergic blockade with propranolol. At 120 min, arterioles in the PE group were constricted (-23 +/- 9% change in diameter vs. baseline). Pretreatment with the ET-converting enzyme inhibitor phosphoramidon or the ETA-receptor antagonist FR-139317 prevented the PE-induced constriction at 120 min (-1 +/- 3 and -6 +/- 3%, respectively, P < 0.01 vs. PE). Pretreatment with the selective alpha1-adrenergic antagonist prazosin (Prz) also prevented the sustained constriction (0 +/- 2%, P < 0.01 vs. PE) but Prz given 60 min after PE infusion did not (-13 +/- 3%). In the aggregate, these results show that vasoconstriction of epicardial coronary arterioles via alpha1-adrenergic activation is blocked by an ET antagonist and an inhibitor of its production. From these data, we conclude that alpha1-adrenergic activation promotes the production and/or release of ET, which produces or facilitates microvascular constriction of epicardial canine coronary arterioles.
Adrenergic alpha-Agonists↗
Changes in coronary endothelial cell Ca2+ concentration during shear stress- and agonist-induced vasodilation.
Increases in intraluminal shear stress are thought to cause vasodilation of coronary arterioles by activation of Ca2+-dependent endothelial nitric oxide synthase followed by release of nitric oxide. We tested the hypothesis that endothelium-dependent vasodilation of isolated coronary arterioles to shear stress and agonists is necessarily preceded by an increase in endothelial cell Ca2+ concentration ([Ca2+]i). After selective loading of endothelium in isolated rabbit coronary arterioles with fura 2, simultaneous changes in diameter and [Ca2+]i were recorded. Vasodilations recorded in response to ACh, substance P, or shear stress were accompanied by significant increases in endothelial cell [Ca2+]i. Vasodilations to shear stress were accompanied by smaller changes in endothelial cell [Ca2+]i than equivalent dilations evoked by substance P or ACh. To test the role for Ca2+ as an activator of endothelial nitric oxide synthase, the endothelium was treated with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid. 1,2-Bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid eliminated significant changes in endothelial cell [Ca2+]i and inhibited dilations to ACh and substance P but did not significantly affect shear stress-induced vasodilation. The data indicate that endothelium-dependent vasodilation of coronary arterioles in response to agonists and shear stress is mediated in part through a rise in endothelial cell [Ca2+]i but that a substantial component of the shear stress-induced response occurs through a Ca2+-insensitive pathway.
Acetylcholine↗
In vivo location and mechanism of EDHF-mediated vasodilation in canine coronary microcirculation.
Responses of epicardial coronary arterioles to ACh were measured using stroboscopic fluorescence microangiography in dogs (n = 38). ACh (0.1 and 0.5 microg. kg(-1). min(-1) ic) dilated small (<100 micron, 11 +/- 2 and 19 +/- 2%, respectively) and large (>100 micron, , 6 +/- 3 and 13 +/- 3%, respectively) arterioles at baseline. Combined administration of N(omega)-monomethyl-L-arginine (L-NMMA; 1. 0 micromol/min ic) and indomethacin (10 mg/kg iv) eliminated ACh-induced dilation in large coronary arterioles but only partially attenuated that in small arterioles. Suffusion of a buffer containing 60 mM KCl (high KCl) completely abolished cromakalim-induced dilation in arterioles and in combination with L-NMMA plus indomethacin completely blocked ACh-induced dilation in small arterioles. This indicated that the vasodilation to ACh that persists in small arterioles after administration of L-NMMA and indomethacin is mediated via a hyperpolarizing factor. The ACh-induced vasodilation remaining after L-NMMA and indomethacin was completely blocked by the large-conductance potassium-channel antagonist iberiotoxin or by epicardial suffusion of miconazole or metyrapone, inhibitors of cytochrome P-450 enzymes. These observations are consistent with the view that endothelium-derived hyperpolarizing factor (EDHF) is a product of cytochrome P-450 enzymes and produces vasodilation by the opening of large-conductance potassium channels. We conclude that ACh-induced dilation in large coronary arterioles is mediated mainly by nitric oxide (NO), whereas, in small arterioles both NO and EDHF mediate dilation to ACh. These data provide the first direct evidence for an in vivo role of EDHF in small coronary arterioles.
Acetylcholine↗
Prologue: ischemic preconditioning in cardiac vascular muscle.
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Adenosine Triphosphate↗