Shear Stress-induced Nitric Oxide Production Research Articles

Coordinated regulation of endothelial calcium signaling and shear stress-induced nitric oxide production by PKCβ and PKCη

BackgroundProtein Kinase C (PKC) is a promiscuous serine/threonine kinase regulating vasodilatory responses in vascular endothelial cells. Calcium-dependent PKCbeta (PKCβ) and calcium-independent PKCeta (PKCη) have both been implicated in the regulation and dysfunction of endothelial responses to shear stress and agonists. ObjectiveWe hypothesized that PKCβ and PKCη differentially modulate shear stress-induced nitric oxide (NO) production by regulating the transduced calcium signals and the resultant eNOS activation. As such, this study sought to characterize the contribution of PKCη and PKCβ in regulating calcium signaling and endothelial nitric oxide synthase (eNOS) activation after exposure of endothelial cells to ATP or shear stress. MethodsBovine aortic endothelial cells were stimulated in vitro under pharmacological inhibition of PKCβ with LY333531 or PKCη targeting with a pseudosubstrate inhibitor. The participation of PKC isozymes in calcium flux, eNOS phosphorylation and NO production was assessed following stimulation with ATP or shear stress. ResultsPKCη proved to be a robust regulator of agonist- and shear stress-induced eNOS activation, modulating calcium fluxes and tuning eNOS activity by multi-site phosphorylation. PKCβ showed modest influence in this pathway, promoting eNOS activation basally and in response to shear stress. Both PKC isozymes contributed to the constitutive and induced phosphorylation of eNOS. The observed PKC signaling architecture is intricate, recruiting Src to mediate a portion of PKCη's control on calcium entry and eNOS phosphorylation. Elucidation of the importance of PKCη in this pathway was tempered by evidence of a single stimulus producing concurrent phosphorylation at ser1179 and thr497 which are antagonistic to eNOS activity. ConclusionsWe have, for the first time, shown in a single species in vitro that shear stress- and ATP-stimulated NO production are differentially regulated by classical and novel PKCs. This study furthers our understanding of the PKC isozyme interplay that optimizes NO production. These considerations will inform the ongoing design of drugs for the treatment of PKC-sensitive cardiovascular pathologies.

Cholesterol Enrichment Impairs Capacitative Calcium Entry, eNOS Phosphorylation & Shear Stress-Induced NO Production.

Endothelial dysfunction, characterized by decreased production or availability of nitric oxide (NO), is widely believed to be the hallmark of early-stage atherosclerosis. In addition, hypercholesterolemia is considered a major risk factor for development of atherosclerosis and is associated with impaired flow-induced dilation. However, the mechanism by which elevated cholesterol levels leads to decreased production of NO is unclear. NO is released in response to shear stress and agonist-evoked changes in intracellular calcium. Although calcium signaling is complex, we have previously shown that NO production by endothelial nitric oxide synthase (eNOS) is preferentially activated by calcium influx via store-operated channels. We hypothesized that cholesterol enrichment altered this signaling pathway (known as capacitive calcium entry; CCE) ultimately leading to decreased NO. Our results show that cholesterol enrichment abolished ATP-induced eNOS phosphorylation and attenuated the calcium response by the preferential inhibition of CCE. Furthermore, cholesterol enrichment also inhibited shear stress-induced NO production and eNOS phosporylation, consistent with our previous results showing a significant role for ATP autocrine stimulation and subsequent activation of CCE in the endothelial flow response.

Abstract 169: Impaired Shear Stress-induced Endothelial Nitric Oxide Production in High Glucose Condition is Restored by Inhibiton via NADPH Consumption by Polyol Pathway Activation

Endothelial dysfunction leading to cardiovascular disease risk involves a decrease in nitric oxide (NO) production. In physiological conditions shear stress is a potent stimulation of endothelium-derived NO production and flow mediated NO production is regulated by the activation of endothelial NO synthase (eNOS). In endothelial cells, eNOS, aldose reductase (AR), a rate limiting enzyme of polyol pathway, and glutathione reductase (GR) share a NADPH as an obligate cofactor. In diabetec condition intracellular polyol pathway is activated and this may decrease shear stress-induced endothelial NO production and increase intracellular oxidative stress via inhibition of eNOS and GR by NADPH consumption. Therefore we investigated whethter AR inhibitor epalrestat improved endothelial NO production under high glucose condition to elucidate the mechanism of endothelial dysfunction in diabetes. We incubated human umbilical vein endothelial cells (HUVECs) in normal (5mM) and high (30mM) glucose condition for 72 hours, with or without epralrestat, or 100U/ml superoxide dismutase (SOD), respectively. After exchange of medium for Krebs’ buffer, HUVECs were exposed to 12dyne/cm2 steady laminar fluid shear stress for 5 minutes. NO release from HUVECs was measured as NO2 using a NOx analyzing HPLC system by Griess reaction. Next we harvested the cells in lysis buffer and analyzed phosphorylation of Akt (shear induced intracellular signal transduction) and eNOS by western blotting, and measured intracellular 8-OHdG and ratio of NADPH/NADP. In high glucose condition NO2 was decreased and 8-OHdG increased compared to low glucose. NO2 was restored and 8-OHdG was reduced by epalrestat significantly (p<0.01, p<0.05, respectively, vs. high glucose condition). In SOD-treated HUVECs, NO2 was not restored (n.s. vs. high glucose condition) despite of complete reduction of 8-OHdG (p<0.01). Both Akt and eNOS phosphorylation by shear stress was affected neither by high glucose, epalrestat nor SOD. Intracellular NADPH/NADP ratio was decreased in high glucose condition, but this reduction was restored by epalrestat. These results showed that polyol pathway activation plays a key role in endothelial NO production under high glucose condition via a cofactor NADPH.

NOS1-dependent negative feedback regulation of the epithelial sodium channel in the collecting duct.

With an increase in urine flow there is a significant increase in shear stress against the renal epithelium including the inner medullary collecting duct, resulting in an increase in nitric oxide (NO) production. The mechanisms of the shear stress-mediated increases in NO are undetermined. Previous studies found that shear stress increases epithelial sodium channel (ENaC) open probability and endothelin (ET)-1 production in an ENaC-dependent mechanism in the collecting duct (CD). Given that ET-1 stimulates NO production in the CD, we hypothesized that shear stress-induced NO production is downstream of shear stress-induced ENaC activation and ET-1 production in a negative feedback loop. We determined that nitric oxide synthase 1 (NOS1) and NOS3 contribute to shear stress-mediated NO production in the CD, that is attenuated by low doses of the ENaC inhibitors amiloride and benzamil. Moreover, ETB receptor blockade significantly blunted the shear stress-mediated NO production. We further elucidated whether mice lacking NOS1 in the collecting duct (CDNOS1KO) have an impaired renal ET-1 system in the CD. Although urinary ET-1 production and inner medullary ET receptor expression were similar between flox control and CDNOS1KO mice, acute ET-1 treatment significantly reduced ENaC open probability in CDs from flox mice but not CDNOS1KO mice compared with basal. Basal ENaC activity in CDs was similar between the genotypes. We conclude that during acute shear stress across the CD, ENaC acts in a negative feedback loop to stimulate NO production in an ETB/NOS1-dependent manner resulting in a decrease in ENaC open probability and promoting natriuresis.

Shear Stress-Induced NO Production is Dependent on ATP Autocrine Signaling and Capacitative Calcium Entry

Flow-induced production of nitric oxide (NO) by endothelial cells plays a fundamental role in vascular homeostasis. However, the mechanisms by which shear stress activates NO production remain unclear due in part to limitations in measuring NO, especially under flow conditions. Shear stress elicits the release of ATP, but the relative contribution of autocrine stimulation by ATP to flow-induced NO production has not been established. Furthermore, the importance of calcium in shear stress-induced NO production remains controversial, and in particular the role of capacitive calcium entry (CCE) has yet to be determined. We have utilized our unique NO measurement device to investigate the role of ATP autocrine signaling and CCE in shear stress-induced NO production. We found that endogenously released ATP and downstream activation of purinergic receptors and CCE plays a significant role in shear stress-induced NO production. ATP-induced eNOS phophorylation under static conditions is also dependent on CCE. Inhibition of protein kinase C significantly inhibited eNOS phosphorylation and the calcium response. To our knowledge, we are the first to report on the role of CCE in the mechanism of acute shear stress-induced NO response. In addition, our work highlights the importance of ATP autocrine signaling in shear stress-induced NO production.

Mathematical Modeling of Shear Stress-induced Nitric Oxide Production in Endothelial Cell

Mathematical Modeling of Shear Stress-induced Nitric Oxide Production in Endothelial Cell

Mechanisms of shear stress mediated nitric oxide production by inner medullary collecting duct cells

We previously reported that increased shear stress (SS) stimulates nitric oxide (NO) production in mouse inner medullary collecting duct‐3 (mIMCD3) cells. Recent work in mpk‐cortical collecting duct cells determined that SS‐induced endothelin‐1 (ET1) expression was mediated via activation of the epithelial sodium channel (ENaC). We have shown that ET1 via ETB receptor stimulation of NOS1 mediates NO production in mIMCD3 cells. Thus, we hypothesized that SS‐induced NO production in mIMCD3 cells is dependent on an ENaC‐ET1‐ETB‐NOS1 mechanism. mIMCD3 cells were grown to confluency on glass slides, subjected to SS (30 dynes/cm2 for 1 h), and nitrite production measured by HPLC and calculated as fold change from static conditions. Cells were pretreated with ENaC inhibitors, 1 μM amiloride or 0.2 μM benzamil, ETB antagonist 1000 nM BQ‐788, or 100 nM 1400W (NOS2‐selective inhibitor) or 1 μM VNIO (NOS1‐selective inhibitor), and nitrite production determined. Both ENaC inhibitors significantly blunted the SS‐induced increase in nitrite (control = 1.8 ± 0.3 vs 1.3 ± 0.2 and 0.9 ± 0.3 respectively N = 4, P < 0.05). BQ‐788 attenuated the SS mediated increase in NO (control = 3.9 ± 1.0 vs 1.7 ±0.1, N =5, P < 0.05). NOS2 inhibition had no significant effect on SS‐induced nitrite production (p>;0.05), while NOS1 inhibition lead to an ~50% reduction in SS‐mediated nitrite (control = 2.8 ± 0.5 vs 1.5 ± 0.4 N= 5–10, P < 0.05). In conclusion, mIMCD3 SS induced NO production is mediated via activation of ENaC, ETB receptor, and NOS1. Future studies will determine whether ENaC via ET1/ETB/NOS1 directly regulates mIMCD3 NO production. Funding support from AHA to KAH and NIH to JSP.

Endothelial nitric oxide gene haplotype reduces the effect of a single bout of exercise on the vascular reactivity in healthy subjects

Polymorphisms in the endothelial nitric oxide synthase (eNOS) gene reduce shear stress-induced nitric oxide production. Thus, we investigated the individual and combined impact of 3 variants in the eNOS gene (-786T>C, intron 4b4a, and 894G>T) on vascular reactivity before and after exercise. Sedentary, healthy subjects were studied (105 women/26 men, age 32 ± 1 years [mean ± standard error of the mean]). Genotypes were determined by polymerase chain reaction restriction fragment length polymorphism, and haplotypes were determined by a Bayesian-based algorithm. Vascular reactivity was evaluated by the percentage of change in forearm vascular conductance provoked by 5 minutes of circulatory occlusion before (baseline) and 10, 60, and 120 minutes after a maximal cardiopulmonary exercise test. Vascular reactivity increased 10 minutes after exercise in the entire sample (baseline: 218 ± 11% vs 10 minutes: 284 ± 15%, P < 0.001), remained increased at 60 minutes (239 ± 12%, P = 0.02 vs baseline), and returned to baseline at 120 minutes (210 ± 10%, P = 0.83 vs baseline). Genotype analysis showed that subjects with the 894G>T polymorphism had lower vascular reactivity than wild counterparts (group effect, P = 0.05). Furthermore, subjects with haplotype 2 (H2), containing the -786T>C and 894G>T polymorphisms, had lower vascular reactivity than wild counterparts (haplotype 1 [H1]) (group effect, P = 0.05), whereas subjects with haplotype 4 (H4), containing only the 894G>T polymorphism, had vascular reactivity similar to that of wild counterparts (H1) (group effect, P = 0.35). Altogether, these results indicate that the 894G>T polymorphism reduced exercise-mediated increase in vascular reactivity, particularly when it occurred concomitantly with the -786T>C polymorphism.

Shear Stress Generated by Different Fluid Compositions Induces Differential Endothelial Signaling in Intact Venules

Disturbed shear stress promotes atherosclerosis, thrombosis, and inflammation of the vessel wall. Most shear stress studies have been conducted in large arterial segments or cultured endothelial cells (ECs). Shear stress induced EC signaling and its role in the regulation of microvessel function have not been well explored in intact venules. The objective of this study was to investigate the EC [Ca2+]i and nitric oxide (NO) responses at the onset of shear stress generated by different fluids. Shear stress was quantified by measuring the flow velocity and fluid viscosity using a high speed camera and cone‐plate viscometer. The changes of EC [Ca2+]i and NO were measured in Fura‐2 AM and DAF‐2 DA loaded individually perfused rat mesenteric venules. Transient increases in EC [Ca2+]i occurred only with whole blood perfusion. The peak EC [Ca2+]i of 203 ± 17 and 288 ± 18 nM were observed with shear stress at 11 and 32 dyn/cm2(n=5), respectively. Shear stress generated by the perfusion of albumin–Ringer, plasma, and Dextran 70‐albumin‐Ringer solution caused no changes in EC [Ca2+]i. However, both plasma and whole blood perfusion induced shear magnitude‐dependent NO production. These results indicated that in intact venules shear stress‐induced increases in EC [Ca2+]i require the presences of erythrocytes, while shear stress‐induced NO production can be independent of Ca2+ signaling. Supported by HL56237 and HL084338.

Simulation of NO and O2 transport facilitated by polymerized hemoglobin solutions in an arteriole that takes into account wall shear stress-induced NO production

A mathematical model was developed to study nitric oxide (NO) and oxygen (O2) transport in an arteriole and surrounding tissues exposed to a mixture of red blood cells (RBCs) and hemoglobin (Hb)-based O2 carriers (HBOCs). A unique feature of this model is the inclusion of blood vessel wall shear stress-induced production of endothelial-derived NO, which is very sensitive to the viscosity of the RBC and HBOC mixture traversing the blood vessel lumen. Therefore in this study, a series of polymerized bovine Hb (PolyHb) solutions with high viscosity, varying O2 affinities, NO dioxygenation rate constants and O2 dissociation rate constants that were previously synthesized and characterized by our group was evaluated via mathematical modeling, in order to investigate the effect of these biophysical properties on the transport of NO and O2 in an arteriole and its surrounding tissues subjected to anemia with the commercial HBOC Oxyglobin® and cell-free bovine Hb (bHb) serving as appropriate controls. The computer simulation results indicated that transfusion of high viscosity PolyHb solutions promoted blood vessel wall shear stress dependent generation of the vasodilator NO, especially in the blood vessel wall and should transport enough NO inside the smooth muscle layer to activate vasodilation compared to the commercial HBOC Oxyglobin® and cell-free bHb. However, NO scavenging in the arteriole lumen was unavoidable due to the intrinsic high NO dioxygenation rate constant of the HBOCs being studied. This study also observed that all PolyHbs could potentially improve tissue oxygenation under hypoxic conditions, while low O2 affinity PolyHbs were more effective in oxygenating tissues under normoxic conditions compared with high O2 affinity PolyHbs. In addition, all ultrahigh molecular weight PolyHbs displayed higher O2 transfer rates than the commercial HBOC Oxyglobin® and cell-free bHb. Therefore, these results suggest that ultrahigh molecular weight PolyHb solutions could be used as safe and efficacious O2 carriers for use in transfusion medicine. It also suggests that future generations of PolyHb solutions should possess lower NO dioxygenation reaction rate constants in order to reduce NO scavenging, while maintaining high solution viscosity to take advantage of wall shear stress-induced NO production. Taken together, we suggest that this mathematical model can be used to predict the vasoactivity of HBOCs and help guide the design and optimization of the next generation of HBOCs for use in transfusion medicine.

Pro-survival Effects of 17β-Estradiol on Osteocytes Are Mediated by Nitric Oxide/cGMP via Differential Actions of cGMP-dependent Protein Kinases I and II

Estrogens promote bone health in part by increasing osteocyte survival, an effect that requires activation of the protein kinases Akt and ERK1/2, but the molecular mechanisms involved are only partly understood. Because estrogens increase nitric oxide (NO) synthesis and NO can have anti-apoptotic effects, we examined the role of NO/cGMP signaling in estrogen regulation of osteocyte survival. Etoposide-induced death of MLO-Y4 osteocyte-like cells, assessed by trypan blue staining, caspase-3 cleavage, and TUNEL assays, was completely prevented when cells were pre-treated with 17β-estradiol. This protective effect was mimicked when cells were pre-treated with a membrane-permeable cGMP analog and blocked by pharmacological inhibitors of NO synthase, soluble guanylate cyclase, or cGMP-dependent protein kinases (PKGs), supporting a requirement for NO/cGMP/PKG signaling downstream of 17β-estradiol. siRNA-mediated knockdown and viral reconstitution of individual PKG isoforms demonstrated that the anti-apoptotic effects of estradiol and cGMP were mediated by PKG Iα and PKG II. Akt and ERK1/2 activation by 17β-estradiol required PKG II, and cGMP mimicked the effects of estradiol on Akt and ERK, including induction of ERK nuclear translocation. cGMP induced BAD phosphorylation on several sites, and experiments with phosphorylation-deficient BAD mutants demonstrated that the anti-apoptotic effects of cGMP and 17β-estradiol required BAD phosphorylation on Ser(136) and Ser(155); these sites were targeted by Akt and PKG I, respectively, and regulate BAD interaction with Bcl-2. In conclusion, 17β-estradiol protects osteocytes against apoptosis by activating the NO/cGMP/PKG cascade; PKG II is required for estradiol-induced activation of ERK and Akt, and PKG Iα contributes to pro-survival signaling by directly phosphorylating BAD.

Cytokeratin-1 Regulates Shear Stress-Induced Nitric Oxide Production in Endothelial Cells

Cytokeratin-1 Regulates Shear Stress-Induced Nitric Oxide Production in Endothelial Cells

Abstract 1232: Regulation of Mitochondria membrane Potential by Shear Stress: Implication of NO Signaling

Introduction : Mitochondria are the most important source of intracellular reactive oxygen species (ROS) in vascular endothelial cells (EC). Mitochondrial membrane potential (ΔΨm) is an indicator of mitochondrial energy state and mtROS production. Shear stress is known to induce eNOS and produce nitric oxide (NO). Hypothesis: We assessed whether NO is implicated in shear stress-induced ΔΨm and mtROS generation. Methods : Confluent HAEC were subjected to three conditions: static control (τ ave = 0 dyn.cm −2 at ∂τ/∂t = 0); pulsatile shear stress (PSS) at a mean shear stress (τ ave ) of 23 dyn.cm −2 with a temporal variation (∂τ/∂t) at 71 dyn.cm −2 .s −1 ; and oscillatory shear stress (OSS) at τ ave = 0.02 with ∂τ/∂t at ±3 dyn.cm − 2.s −1 . Tetramethyl rhodamine methyl ester (TMRM) and Mitosox red fluorescent dyes were used to assess mitochondrial ΔΨm and O 2 ·− production respectively. Intracellular H 2 O 2 production was assessed by carboxy-H 2 DCFDA. HAEC were treated with DETA-NO (NO donor) and L-NAME (eNOS inhibitor) to assess how shear stress-induced NO production regulates ΔΨm. Results: Delta;Ψm was increased by 12±5.3 mV in response to OSS, and by 33.5±8.9 mV in response to PSS (n=3, P &lt;0.05) relative to static control. DETA-NO at 0.21mM and 0.94mM was used to simulate PSS-induced NO production during 1 hour and 4 hours of flow and it increased Delta;Ψm by 9.6±4.9 mV and 18.9 ±3.2 mV, respectively (n=3, P&lt;0.05). L-NAME reduced Delta;Ψm (n=3, P&lt;0.05) suggesting that NO is involved in Delta;Ψm regulation. PSS-induced Delta;Ψm was accompanied by a 1.4 fold increase in mtROS production (n=3, P&lt;0.05) and a concomitant 10 fold upregulation in mitochondrial manganese superoxide dismutase (MnSOD) mRNA expression (n=6, P&lt;0.05). Conclusion: Nitric oxide modulates Delta;Ψm and mtROS production. PSS, which upregulates eNOS, is more potent than OSS at increasing Delta;Ψm and mtROS, implicating NO as a signaling molecule in mitochondrial and endothelial function.

Chronic high blood flow potentiates shear stress-induced release of NO in arteries of aged rats

Aging impairs shear-stress-dependent dilation of arteries via increased superoxide production, decreased SOD activity, and decreased activation of endothelial nitric oxide (NO) synthase (eNOS). In the present study, we investigated whether chronic increases in shear stress, elicited by increases in blood flow, would improve vascular endothelial function of aged rats. To this end, second-order mesenteric arteries of young (6 mo) and aged (24 mo) male Fischer-344 rats were selectively ligated for 3 wk to elevate blood flow in a first-order artery [high blood flow (HF)]. An in vitro study was then conducted on first-order arteries with HF and normal blood flow (NF) to assess shear stress (1, 10, and 20 dyn/cm(2))-induced release of NO into the perfusate. In HF arteries of both age groups, shear stress-induced NO production increased significantly. In 24-mo-old rats, the reduced shear stress-induced NO production in NF arteries was normalized by HF to a level similar to that in NF arteries of 6-mo-old rats. The increased NO production in HF arteries of 24-mo-old rats was associated with increased shear stress-induced dilation, expression of eNOS protein, and shear stress-induced eNOS phosphorylation. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, reduced shear stress-induced eNOS phosphorylation and vasodilation. Superoxide production decreased significantly in HF compared with NF arteries in 24-mo-old rats. The decreased superoxide production was associated with significant increases in CuZn-SOD and extracellular SOD protein expressions and total SOD activity. These results suggest that stimulation with chronic HF restores shear-stress-induced activation of eNOS and antioxidant ability in aged arteries.

Impaired shear stress-induced nitric oxide production through decreased NOS phosphorylation contributes to age-related vascular stiffness

Endothelial dysfunction and increased arterial stiffness contribute to multiple vascular diseases and are hallmarks of cardiovascular aging. To investigate the effects of aging on shear stress-induced endothelial nitric oxide (NO) signaling and aortic stiffness, we studied young (3-4 mo) and old (22-24 mo) rats in vivo and in vitro. Old rat aorta demonstrated impaired vasorelaxation to acetylcholine and sphingosine 1-phosphate, while responses to sodium nitroprusside were similar to those in young aorta. In a customized flow chamber, aortic sections preincubated with the NO-sensitive dye, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, were subjected to steady-state flow with shear stress increase from 0.4 to 6.4 dyn/cm(2). In young aorta, this shear step amplified 4-amino-5-methylamino-2',7'-difluorofluorescein fluorescence rate by 70.6 +/- 13.9%, while the old aorta response was significantly attenuated (23.6 +/- 11.3%, P < 0.05). Endothelial NO synthase (eNOS) inhibition, by N(G)-monomethyl-l-arginine, abolished any fluorescence rate increase. Furthermore, impaired NO production was associated with a significant reduction of the phosphorylated-Akt-to-total-Akt ratio in aged aorta (P < 0.05). Correspondingly, the phosphorylated-to-total-eNOS ratio in aged aortic endothelium was markedly lower than in young endothelium (P < 0.001). Lastly, pulse wave velocity, an in vivo measure of vascular stiffness, in old rats (5.99 +/- 0.191 m/s) and in N(omega)-nitro-l-arginine methyl ester-treated rats (4.96 +/- 0.118 m/s) was significantly greater than that in young rats (3.64 +/- 0.068 m/s, P < 0.001). Similarly, eNOS-knockout mice demonstrated higher pulse wave velocity than wild-type mice (P < 0.001). Thus impaired Akt-dependent NO synthase activation is a potential mechanism for decreased NO bioavailability and endothelial dysfunction, which likely contributes to age-associated vascular stiffness.

Laminar Shear Stress Up-Regulates Inducible Nitric Oxide Synthase in the Endothelium

Shear stress caused by blood flow is a potent physiological stimulus for the generation of nitric oxide (NO) in endothelial cells, which is believed to derive from the up-regulation and post-transcriptional activation of endothelial constitutive NO synthase (ecNOS). However, it has yet to be demonstrated that inducible NO synthase (iNOS) plays a significant role in shear stress-induced NO production from endothelial cells. We used parallel plate-type flow chambers that detect fluid shear stress to determine that shear stress, as quantified by a real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR), increased iNOS gene transcripts in cultured endothelial cells, which resulted in increased NO production. Shear stress-induced iNOS expression was inhibited by pyrrolidine dithiocarbamate (PDTC), an antioxidant and nuclear factor kappaB (NF-kappaB) blocker, and by MG132, an aldehyde peptide proteasome inhibitor that antagonizes I kappaB-kinase. Laminar shear stress increased the transcriptional activity of NF-kappaB, whereas over-expression of an I kappaB-alpha mutant that inhibits the activation of NF-KB in a dominant-negative fashion was found to attenuate the induction of endothelial iNOS by shear stress. The present results demonstrate that shear stress induces iNOS in the endothelium, mainly via the activation of NF-kappaB.

Table of Contents—A new look for the New Year

a major strength of the American Journal of Physiology-Cell Physiology is the diversity of cellular events covered by the Journal and the large array of cell types examined in the course of these studies. Unfortunately, this may also lead to some uncertainty regarding the type of manuscript that we

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.

Control of the pulmonary circulation in the fetus and during the transitional period to air breathing

During fetal life and the transition to extra-uterine air breathing, pulmonary vascular tone is regulated by a complex, interactive group of mechanisms. Arachidonic acid metabolites play an important role in this regulation. Although prostaglandins may not be central to regulation of the resting fetal pulmonary circulation, PGI 2 acts to modulate tone and thereby maintain pulmonary vascular resistance relatively constant. PGI 2 also may play an important role as one of the components involved in the major changes that occur with the onset of air breathing. Leukotrienes, also metabolites of arachidonic acid and potent smooth muscle constrictors, may play an active role in maintaining the normally high fetal pulmonary vascular resistance, because leukotriene receptor blockade or synthesis inhibition increases pulmonary blood flow about eight-fold; the presence of leukotrienes in fetal tracheal fluid further supports this. In addition to PGI 2, vascular endothelial cells produce other vasoactive factors. These include potent vasodilators, such as endothelium-derived relaxing factor (EDRF). EDRF, known to be nitric oxide (NO) and often called endothelium-derived nitric oxide (EDNO), is produced by endothelial cells in response to varied stimuli, generally involving specific receptors and the activation of endothelial NO synthetase (eNOS); subsequent smooth muscle relaxation is produced by a NO/guanylyl cyclase/cGMP–mediated mechanism. NO clearly is involved in regulation of vascular tone in the fetal pulmonary circulation, although it plays a far more important role in the postnatal transition to air breathing. Superfused fetal sheep pulmonary arteries release NO when stimulated with bradykinin. In fetal lambs the vasodilating effects of bradykinin are attenuated by methylene blue and resting tone falls with N ω-nitro- l-arginine, an inhibitor of NO synthesis, suggesting that a NO/cGMP-dependent mechanism continuously modulates or offsets the increased tone of the resting fetal pulmonary circulation. Inhibition of NO synthesis blocks the pulmonary vasodilation with oxygenation of fetal lungs in utero. Shear stress-induced NO production as well as the relationship of oxygenation to NO production further support the important function of NO in the transition. Although endothelin-1 (ET-1) has potent vasoactivity as well as ontogenetic differences in effect on pulmonary vascular resistance, its exact physiological role has not been defined. Adrenomedullin and calcitonin gene-related peptide (CGRP), two additional vasoactive substances, have profound, and prolonged, vasodilating effects in the fetal pulmonary circulation. Their physiological roles have not yet been established.