Abstract 107: Cytochrome B5 Reductase 3 Sensitizes Soluble Guanylate Cyclase to Nitric Oxide in Vascular Smooth Muscle

The inability nitric oxide (NO) to stimulate soluble guanylate cyclase (sGC) has been linked to numerous cardiovascular diseases (CVD) including hypertension. While several studies have defined the importance of sGC expression in the cardiovascular system, the basic mechanisms that regulate sGC activity remain incompletely understood. Here, we report for the first time that sGC heme iron redox state, which is essential for NO-induced sGC activation, is regulated by cytochrome B5 reductase 3 (CyB5R3). Genetic knockdown and pharmacological inhibition of CyB5R3 in primary rat vascular smooth muscle cells resulted in a 60% loss in cGMP production. Conversely, the sGC activator Bay 58-2667, which activates oxidized or heme free sGC, reversed these effects. Consistent with our cell culture work, purified protein studies demonstrate that CyB5R3 can directly reduce oxidized sGC heme iron and sensitize sGC to NO. To test the functional importance of Cyb5R3 activity, we cultured mouse thoracodorsal arteries with a pharmacological inhibitor of Cyb5R3 (ZINC 747) and performed vascular reactivity studies using pressure myography. Arteries treated with ZINC 747 showed decreased responsiveness the NO donor DETA-NONOate but increase sensitivity to Bay 58-2667. We then treated mice with 10mg/kg/day of ZINC 747 using osmotic mini pumps, which caused an increase in mean arterial blood pressure (107.5±3.4 vs 131±13.16) measured via radio telemetry. Lastly, translational studies reveal that the CyB5R3 T116S polymorphism with allele frequency 0.23 only in African Americans is unable to reduce sGC and correlates with increased blood pressure. Considering the defining role of sGC in NO signaling and the fact that the oxidation state of sGC may predict responses to NO therapies and new classes of sGC activator medications, we anticipate that these studies may significantly impact our understanding of biology, precision therapeutics (right drug for the right patient) and pharmacogenetics (T117S SNP based drug selection).

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Abstract 15696: Cytochrome B5 Reductase 3 Sensitizes Soluble Guanylate Cyclase to Nitric Oxide

Circulation ◽

10.1161/circ.132.suppl_3.15696 ◽

2015 ◽

Vol 132 (suppl_3) ◽

Author(s):

Anh T Nguyen ◽

Mizanur M Rahaman ◽

Stephanie M Mutchler ◽

Megan Miller ◽

Josef T Prchal ◽

...

Keyword(s):

Nitric Oxide ◽

Blood Pressure ◽

Soluble Guanylate Cyclase ◽

Cytochrome B5 ◽

Heme Iron ◽

Proximity Ligation Assay ◽

Cytochrome B5 Reductase ◽

Cgmp Production ◽

Iron Reductase

Impaired soluble guanylyl cyclase (sGC)-dependent nitric oxide (NO) signaling has been linked to numerous cardiovascular diseases (CVD) such as hypertension, myocardial infarction and atherosclerosis. Despite emerging evidence indicating the importance of sGC function within the cardiovascular system, the basic mechanisms that regulate sGC activity remain incompletely understood. Herein, we provide in vitro and in vivo evidence that cytochrome b5 reductase 3 (Cyb5R3) is an sGC heme iron reductase and regulates downstream cGMP signaling. Of major significance, we also demonstrate that a Cyb5R3 T116S polymorphism with allele frequency of 0.23 in African Americans associates with increase blood pressure and is incapable of reducing sGC. Proximity ligation assay (PLA) experiments show that endogenous Cyb5R3 and oxidized sGC associate. Knockdown of Cyb5R3 results in reduced cGMP production and downstream signaling in rat aortic smooth muscle cells (SMC). Overexpression of Cyb5R3 not only rescues cGMP production but also increases baseline cGMP, whereas T116S mutant does not. Finally, inhibition of Cyb5R3 in mice significantly increases systemic blood pressure. Our studies are the first to identify an sGC heme iron reductase, provide evidence for Cyb5R3 as a key biological regulator of sGC activity and vascular tone in SMC, and link a human polymorphism of Cyb5R3 to increased blood pressure; all of which may lead to the development of novel therapeutics targeting Cyb5R3 for the treatment of CVD. Importantly, the co-expression of Cyb5R3 and sGC in multiple cells types suggests that this regulation of sGC activity may have broad applications for multiple physiological and pathophysiological processes. Results: Conclusions:

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Abstract 378: Cytochrome b5 Reductase 3 Regulates Vascular Smooth Muscle Phenotypic Switching

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.378 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Amogha Vijayvargiya ◽

Anh Nguyen ◽

Megan P Miller ◽

Scott Hahn ◽

Adam C Straub

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Cytochrome B5 ◽

Muscle Cells ◽

Phenotypic Switching ◽

Serum Starvation ◽

Contractile State ◽

Cytochrome B5 Reductase ◽

Arterial Blood

Contractile vascular smooth muscle cells (VSMC) play a key role in the regulation of arterial blood vessel tone and cardiovascular health. However, in many vascular diseases, VSMCs undergo a phenotypic switch from a contractile state to a synthetic phenotype, where loss of the contractile markers myosin heavy chain 11 (Myh11), smooth muscle alpha actin (ACTA2) and transgelin (SM22) are observed and proliferation is increased. Recent evidence from our lab demonstrates that cytochrome b5 reductase 3 (Cyb5R3) regulates the redox state of soluble guanylate cyclase to control cGMP levels in VSMCs. Because cGMP modulates protein kinase G activity, a critical kinase that maintains VSMCs in a contractile state, we tested the hypothesis that Cyb5R3 is critical for maintenance of the contractile phenotype. To test this hypothesis, we transduced primary rat aortic smooth muscle cells with non-targeting (NT) or Cyb5R3 shRNA followed by serum starvation for 24, 48, and 72 hours to induce phenotypic switching. After each time point, mRNA measurements of Cyb5R3, Myh11, ACTA2, and SM22 were conducted using RT-PCR. In NT shRNA transduced VSMCs, we observed a significant increase in Cyb5R3, Myh11, ACTA2, and SM22 mRNA, but not in Cyb5R3 knockdown VSMCs. Next, we conducted proliferation studies by serum starving NT shRNA and Cyb5R3 shRNA treated VSMCs for 24 hours followed by stimulation of platelet growth factor BB (PDGF-BB, 40 ng/mL). After 24 hours of PDGF-BB treatment, Cyb5R3 deficient cells showed augmented proliferation compared to control cells measured by 3 H-thymidine incorporation. Together, our data suggest that Cyb5R3 is essential for VSMC phenotypic switching and proliferation, which may unravel a new therapeutic target for treating individuals with cardiovascular disease.

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Interleukin 1 activates soluble guanylate cyclase in human vascular smooth muscle cells through a novel nitric oxide-independent pathway.

Journal of Experimental Medicine ◽

10.1084/jem.179.1.71 ◽

1994 ◽

Vol 179 (1) ◽

pp. 71-80 ◽

Cited By ~ 59

Author(s):

D Beasley ◽

M McGuiggin

Keyword(s):

Nitric Oxide ◽

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Guanylate Cyclase ◽

Soluble Guanylate Cyclase ◽

Interleukin 1 ◽

No Synthase ◽

Guanylate Cyclase Activation

Recent demonstration of cytokine-inducible production of nitric oxide (NO) in vascular smooth muscle cells (VSMC) from rat aorta has implicated VSMC-derived NO as a key mediator of hypotension in septic shock. Our studies to determine whether an inducible NO pathway exists in human VSMC have revealed a novel cytokine-inducible, NO-independent pathway of guanylate cyclase activation in VSMC from human saphenous vein (HSVSMC). Interleukin 1 (IL-1), tumor necrosis factor (TNF), interferon gamma (IFN-gamma) and Escherichia coli lipopolysaccharide (LPS) increased cGMP at 24 h, whereas IL-2 and IL-6 were ineffective. The effect of IL-1 on cyclic guanosine 3',5'-monophosphate (cGMP) was delayed, occurring after 6 h of exposure, and was maximal after 10 h. Methylene blue and LY83583 reversed the IL-1-induced increase in cGMP, suggesting that it was mediated by activation of soluble guanylate cyclase. However, IL-1-induced cGMP in HSVSMC was not inhibited by extracellular hemoglobin. Also, the effect of IL-1 on cGMP was not reversed by nitro- or methyl-substituted L-arginine analogs, aminoguanidine, or diphenyleneiodonium, all of which inhibit IL-1-induced NO synthase in rat aortic VSMC (RAVSMC). IL-1-induced cGMP in HSVSMC was also independent of tetrahydrobiopterin and extracellular L-arginine, as it was not affected by 2,4-diamino-6-hydroxyprytimidine, an inhibitor of tetrahydrobiopterin biosynthesis, and was similar in L-arginine-free and L-arginine-containing media. Analysis of NO synthase mRNA with the use of polymerase chain reaction indicates that levels of mRNA for inducible NO synthase are several orders of magnitude lower in IL-1-treated human HSVSMC than in IL-1-treated RAVSMC. IL-1-induced cGMP was also NO independent in human umbilical artery VSMC, and NO dependent in rat vena cava VSMC. Together these results indicate that IL-1 activates a novel NO-independent pathway of soluble guanylate cyclase activation in human VSMC.

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RGS5 Attenuates Baseline Activity of ERK1/2 and Promotes Growth Arrest of Vascular Smooth Muscle Cells

Cells ◽

10.3390/cells10071748 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1748

Author(s):

Eda Demirel ◽

Caroline Arnold ◽

Jaspal Garg ◽

Marius Andreas Jäger ◽

Carsten Sticht ◽

...

Keyword(s):

Blood Pressure ◽

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Map Kinase ◽

Vascular Smooth Muscle Cells ◽

Muscle Cells ◽

Genetic Ablation ◽

Variable Expression ◽

Arterial Blood

The regulator of G-protein signaling 5 (RGS5) acts as an inhibitor of Gαq/11 and Gαi/o activity in vascular smooth muscle cells (VSMCs), which regulate arterial tone and blood pressure. While RGS5 has been described as a crucial determinant regulating the VSMC responses during various vascular remodeling processes, its regulatory features in resting VSMCs and its impact on their phenotype are still under debate and were subject of this study. While Rgs5 shows a variable expression in mouse arteries, neither global nor SMC-specific genetic ablation of Rgs5 affected the baseline blood pressure yet elevated the phosphorylation level of the MAP kinase ERK1/2. Comparable results were obtained with 3D cultured resting VSMCs. In contrast, overexpression of RGS5 in 2D-cultured proliferating VSMCs promoted their resting state as evidenced by microarray-based expression profiling and attenuated the activity of Akt- and MAP kinase-related signaling cascades. Moreover, RGS5 overexpression attenuated ERK1/2 phosphorylation, VSMC proliferation, and migration, which was mimicked by selectively inhibiting Gαi/o but not Gαq/11 activity. Collectively, the heterogeneous expression of Rgs5 suggests arterial blood vessel type-specific functions in mouse VSMCs. This comprises inhibition of acute agonist-induced Gαq/11/calcium release as well as the support of a resting VSMC phenotype with low ERK1/2 activity by suppressing the activity of Gαi/o.

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Abstract P726: Cytochrome B5 Reductase 3 Modifies The Brain-Blood Axis Response To Ischemic Stroke In Mice With Sickle Cell Disease

Stroke ◽

10.1161/str.52.suppl_1.p726 ◽

2021 ◽

Vol 52 (Suppl_1) ◽

Author(s):

Katherine C Wood ◽

Heidi M Schmidt ◽

Scott Hahn ◽

Mehdi Nouraie ◽

Mara Carreno ◽

...

Keyword(s):

Nitric Oxide ◽

Ischemic Stroke ◽

Sickle Cell Disease ◽

Sickle Cell ◽

African Ancestry ◽

Cytochrome B5 ◽

Heme Iron ◽

Wild Type ◽

Cell Disease ◽

Cytochrome B5 Reductase

Introduction: Stroke and silent infarcts are serious complications of sickle cell disease (SCD), occurring frequently in children. Decreased nitric oxide bioavailability and responsiveness contribute to neurovascular disease. Cytochrome b5 reductase 3 (Cyb5R3) is a heme iron reductase that reduces oxidized soluble guanylate cyclase heme iron (Fe 3+ --> Fe 2+ ) to preserve nitric oxide signaling. A loss-of-function Cyb5R3 missense variant (T117S) occurs with high frequency (0.23 minor allele) in persons of African ancestry. Hypothesis: We hypothesized that impaired reductase function of T117S Cyb5R3 exacerbates brain damage after ischemic stroke in SCD. Methods: Bone marrow transplant was used to create male SCD mice with wild type (SS/WT) or T117S (SS/T117S) Cyb5R3. Blood was sampled before and after middle cerebral artery occlusion (55 minutes occlusion, 48 hours reperfusion). Infarct volume (IV) was determined by 2,3,5-triphenyltetrazolium chloride. Intravascular hemolysis and correlation (Pearson’s R) of hematology changes with IV were determined. Baseline Walk-PHaSST (NCT00492531) data were analyzed for stroke occurrence. Results: Brain IV (63 vs 27 cm 3 , P=0.003) and mortality (3/6 vs 0/8) were greater in SS/T117S vs SS/WT. Red blood cells, hemoglobin and hematocrit declined as IV increased. Plasma oxyhemoglobin increased in parallel with IV (r = 0.74, P=0.09). There were different signatures to hematologic changes that occurred with IV in SCD. Relative to wild type, T117S contracted the erythroid compartment (red blood cell: -13% vs 13%, P=0.003; hematocrit: -20% vs 1%, P=0.008; hemoglobin: -18% vs 2%, P=0.007). Mean platelet volume correlated with IV in SS/T117S (r = 0.87, P=0.06), while the inverse occurred in SS/WT (r = -0.63, P=0.09) Monocytes increased in parallel with IV in SS/T117S (r = 0.73, P=0.16), but followed the opposite trajectory in SS/WT (r = -0.77, P=0.04). WalkPHaSST participants with T117S Cyb5R3 self-reported more ischemic stroke (7.4% vs 5.1%) relative to wild type. Conclusion: Cyb5R3 is an important modifier of the evolution and outcome of ischemic brain injury in SCD and its hematologic consequences. Our findings indicate a bidirectional relationship between stroke and anemia in SCD that may axially turn on Cyb5R3 activity.

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Cullin-3 Regulates Vascular Smooth Muscle Function and Arterial Blood Pressure via PPARγ and RhoA/Rho-Kinase

Cell Metabolism ◽

10.1016/j.cmet.2012.08.011 ◽

2012 ◽

Vol 16 (4) ◽

pp. 462-472 ◽

Cited By ~ 66

Author(s):

Christopher J. Pelham ◽

Pimonrat Ketsawatsomkron ◽

Séverine Groh ◽

Justin L. Grobe ◽

Willem J. de Lange ◽

...

Keyword(s):

Blood Pressure ◽

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Arterial Blood Pressure ◽

Muscle Function ◽

Rho Kinase ◽

Arterial Blood ◽

Smooth Muscle Function ◽

Cullin 3

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Pulmonary soluble guanylate cyclase, a nitric oxide receptor, is increased during the perinatal period

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1997.272.3.l400 ◽

1997 ◽

Vol 272 (3) ◽

pp. L400-L406 ◽

Cited By ~ 26

Author(s):

K. D. Bloch ◽

G. Filippov ◽

L. S. Sanchez ◽

M. Nakane ◽

S. M. de la Monte

Keyword(s):

Nitric Oxide ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

Guanylate Cyclase ◽

Soluble Guanylate Cyclase ◽

Muscle Cells ◽

Perinatal Period ◽

Mrna Levels ◽

Old Rats ◽

Beta1 Subunit

Nitric oxide (NO) has an important role in the pulmonary vasodilatation associated with the transition from fetal to neonatal life. NO activates pulmonary soluble guanylate cyclase (sGC), an obligate heterodimer composed of alpha1- and beta1-subunits, increasing synthesis of guanosine 3',5'-cyclic monophosphate (cGMP) and leading to vasodilation. In this study, regulation of sGC subunit expression during pulmonary development was examined. RNA blot hybridization revealed abundant alpha1- and beta1-subunit mRNA in lungs of late-gestation fetal and neonatal Sprague-Dawley rats, with markedly reduced levels detected in adult lungs. Pulmonary sGC enzyme activity in the presence of 1 mM sodium nitroprusside, a NO-donor compound, was approximately sevenfold greater in 1- and 8-day-old rats than in adult rats (P < 0.03). With the use of immunoblot techniques, pulmonary alpha1-subunit concentrations closely correlated with mRNA levels. With in situ hybridization, alpha1- and beta1-subunit mRNAs were readily detected in pulmonary vascular and bronchial smooth muscle cells as well as alveolar and serosal epithelial cells in lungs of 1-day-old rats. In adult lungs, sGC subunit mRNAs were present at low levels and were found nearly exclusively in bronchial and vascular smooth muscle cells. These results demonstrate that abundant pulmonary sGC is available to respond to the increased NO produced during the perinatal period. High-level expression of sGC subunit genes outside the vasculature of lungs of 1-day-old rats suggests an important role for NO-cGMP signal transduction in the perinatal regulation of pulmonary epithelial function and bronchial tone.

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Constitutive LH receptor activity impairs NO-mediated penile smooth muscle relaxation

Reproduction ◽

10.1530/rep-20-0447 ◽

2021 ◽

Vol 161 (1) ◽

pp. 31-41

Author(s):

Deepak S Hiremath ◽

Fernanda B M Priviero ◽

R Clinton Webb ◽

CheMyong Ko ◽

Prema Narayan

Keyword(s):

Nitric Oxide ◽

Smooth Muscle ◽

Erectile Dysfunction ◽

Sodium Nitroprusside ◽

Signaling Pathway ◽

Guanylate Cyclase ◽

Soluble Guanylate Cyclase ◽

Muscle Relaxation ◽

Smooth Muscle Relaxation ◽

Activating Mutations

Timely activation of the luteinizing hormone receptor (LHCGR) is critical for fertility. Activating mutations in LHCGR cause familial male-limited precocious puberty (FMPP) due to premature synthesis of testosterone. A mouse model of FMPP (KiLHRD582G), expressing a constitutively activating mutation in LHCGR, was previously developed in our laboratory. KiLHRD582G mice became progressively infertile due to sexual dysfunction and exhibited smooth muscle loss and chondrocyte accumulation in the penis. In this study, we tested the hypothesis that KiLHRD582G mice had erectile dysfunction due to impaired smooth muscle function. Apomorphine-induced erection studies determined that KiLHRD582G mice had erectile dysfunction. Penile smooth muscle and endothelial function were assessed using penile cavernosal strips. Penile endothelial cell content was not changed in KiLHRD582G mice. The maximal relaxation response to acetylcholine and the nitric oxide donor, sodium nitroprusside, was significantly reduced in KiLHRD582G mice indicating an impairment in the nitric oxide (NO)-mediated signaling. Cyclic GMP (cGMP) levels were significantly reduced in KiLHRD582G mice in response to acetylcholine, sodium nitroprusside and the soluble guanylate cyclase stimulator, BAY 41-2272. Expression of NOS1, NOS3 and PKRG1 were unchanged. The Rho-kinase signaling pathway for smooth muscle contraction was not altered. Together, these data indicate that KiLHRD582G mice have erectile dysfunction due to impaired NO-mediated activation of soluble guanylate cyclase resulting in decreased levels of cGMP and penile smooth muscle relaxation. These studies in the KiLHRD582G mice demonstrate that activating mutations in the mouse LHCGR cause erectile dysfunction due to impairment of the NO-mediated signaling pathway in the penile smooth muscle.

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BDNF augments rat internal anal sphincter smooth muscle tone via RhoA/ROCK signaling and nonadrenergic noncholinergic relaxation via increased NO release

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00247.2019 ◽

2020 ◽

Vol 318 (1) ◽

pp. G23-G33

Author(s):

Arjun Singh ◽

Ipsita Mohanty ◽

Jagmohan Singh ◽

Satish Rattan

Keyword(s):

Nitric Oxide ◽

Smooth Muscle ◽

Anal Sphincter ◽

Guanylate Cyclase ◽

Internal Anal Sphincter ◽

Soluble Guanylate Cyclase ◽

Muscle Tone ◽

Tyrosine Kinase Receptor ◽

Therapeutic Targeting ◽

Nanc Relaxation

Presently, there are no studies examining the neuromodulatory effects of brain-derived neurotropic factor (BDNF) on the basal internal anal sphincter (IAS) tone and nonadrenergic noncholinergic (NANC) relaxation. To examine this, we determined the neuromuscular effects of BDNF on basal IAS smooth muscle tone and the smooth muscle cells (SMCs) and the effects of NANC nerve stimulation before and after high-affinity receptor tyrosine kinase receptor B (TrkB) antagonist K252a. We also investigated the mechanisms underlying BDNF-augmented increase in the IAS tone and NANC relaxation. We found that BDNF-increased IAS tone and SMC contractility were TTX resistant and attenuated by K252a. TrkB-specific agonist 7,8-dihydroxyflavone, similar to BDNF, also produced a concentration-dependent increase in the basal tone, whereas TrkB inhibitors K252a and ANA-12 produced a decrease in the tone. In addition, BDNF produced leftward shifts in the concentration-response curves with U46619 and ANG II (but not with bethanechol and K+ depolarization), and these shifts were reversed by K252a. Effects of Y27632 and Western blot data indicated that the BDNF-induced increase in IAS tone was mediated via RhoA/ROCK. BDNF-augmented NANC relaxation by electrical field stimulation was found to be mediated via the nitric oxide (NO)/soluble guanylate cyclase (sGC) pathway rather than via increased sensitivity to NO. In conclusion, the net effect of BDNF was that it caused an increase in the basal IAS tone via RhoA/ROCK signaling. BDNF also augmented NANC relaxation via NO/sGC. These findings may have relevance to the role of BDNF in the pathophysiology and therapeutic targeting of the IAS-associated rectoanal motility disorders. NEW & NOTEWORTHY These studies for the first time to our knowledge demonstrate that increased levels of brain-derived neurotrophic factor (BDNF; conceivably released from smooth muscle cells and/or the enteric neurons) has two major effects. First, BDNF augments the internal anal sphincter (IAS) tone via tyrosine kinase receptor B/thromboxane A2-receptor, angiotensin II receptor type 1/RhoA/ROCK signaling; and second, it increases nonadrenergic noncholinergic relaxation via nitric oxide/soluble guanylate cyclase. These studies may have relevance in therapeutic targeting in the anorectal motility disorders associated with the IAS.

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Abstract 524: Extracellular Guanosine Regulates Extracellular Adenosine Levels

Hypertension ◽

10.1161/hyp.60.suppl_1.a524 ◽

2012 ◽

Vol 60 (suppl_1) ◽

Author(s):

Edwin K Jackson ◽

Delbert G Gillespie

Keyword(s):

Blood Pressure ◽

Endothelial Cells ◽

Smooth Muscle ◽

Coronary Artery ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Muscle Cells ◽

Extracellular Adenosine ◽

Arterial Blood

Extracellular adenosine modulates cardiovascular and renal function. While measuring extracellular purines in biological samples, we observed a correlation between levels of adenosine and guanosine. This observation led us to test the hypothesis that extracellular guanosine regulates extracellular adenosine levels in the cardiovascular and renal systems. Rat preglomerular vascular smooth muscle cells in culture were incubated with adenosine and/or guanosine. In the absence of added adenosine, exogenous guanosine (30 μmol/L) had little effect on extracellular adenosine levels, indicating that extracellular guanosine does not trigger the release or production of adenosine. Without added guanosine and 1 hour after adding 3 μmol/L of exogenous adenosine, extracellular adenosine levels were only 0.125 ± 0.020 μmol/L, indicating rapid disposition of extracellular adenosine by a monolayer of cells. In contrast, extracellular adenosine levels 1 hour after adding 3 μmol/L of adenosine plus guanosine (30 μmol/L) were 1.173 ± 0.061 μmol/L (9-fold higher; p<0.0001), indicating slow disposition of extracellular adenosine in the presence of extracellular guanosine. Extracellular guanosine impeded the disposition of extracellular adenosine not only in preglomerular vascular smooth muscle cells, but also in rat preglomerular vascular endothelial cells, mesangial cells, cardiac fibroblasts and kidney epithelial cells, as well as in human aortic vascular smooth muscle cells, coronary artery vascular smooth muscle cells and coronary artery endothelial cells. In rats, infusions of guanosine per se had little effect on cardiovascular/renal variables, yet markedly enhanced the effects of co-infusions of adenosine. For example, in control rats, adenosine (0.3 μmol/kg/min) only modestly decreased mean arterial blood pressure (from 114 ± 4 to 100 ± 4 mm Hg). In contrast, in guanosine-treated rats (10 μmol/kg/min), adenosine profoundly decreased blood pressure (from 109 ± 4 to 79 ± 3 mm Hg; p<0.0001 vs non-guanosine treated group). Conclusion: Extracellular guanosine powerfully regulates extracellular adenosine levels by altering adenosine disposition and this occurs in many, perhaps most, cell types in the cardiovascular system and kidneys.

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