Dr Clemence Girardet, a postdoctoral fellow in Dr Andrew Butler’s laboratory, was awarded a scholarship from the Keystone Symposia Future of Science Fund and the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) .
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Department of Pharmacological and Physiology
Room M 362 || 1402 South Grand Blvd
St. Louis, Missouri 63104
Alan H. Stephenson, Ph.D.
Department of Pharmacological and Physiological Science Ph.D., Saint Louis University, 1986
In healthy humans, the erythrocyte, in addition to serving as a vehicle for oxygen (O2) transport, has been shown to participate in the local regulation of vascular resistance in skeletal muscle by its ability to release adenosine triphosphate (ATP) in response to reduced O2 tension. In the vasculature of skeletal muscle, ATP acts on microvascular endothelial purinergic receptors to stimulate local release of endothelium-derived vasodilators as well as inducing retrograde (upstream) vasodilation, resulting in increased perfusion to tissue with reduced O2 tension. This property of the erythrocyte permits it to participate in the matching of O2 delivery with O2 need in skeletal muscle. In humans with type 2 diabetes (DM2), impaired vascular function is a major contributor to their cardiovascular disease. There is convincing evidence that microvascular circulatory control is abnormal in type 2 diabetes. We previously reported that low O2 tension-induced ATP release from erythrocytes requires activation of the heterotrimeric proteins, increases in cAMP levels and normal erythrocyte deformability.
Inhibition of normal erythrocyte deformability with diamide prevents low O2 tension-induced ATP release, suggesting to us a possible mechanistic link between erythrocyte deformation and low O2 tension-induced ATP release. Why is this important? Because humans with type 2 diabetes experience tissue ischemia as a result of inadequate blood flow to skeletal muscle. Identifying a strategy to increase low O2 tension-induced ATP release from type 2 diabetes erythrocytes by targeting deformability may rescue their ability to release ATP in areas of low O2 tension in skeletal muscle and reduce the morbidity due to tissue ischemia.
We have been examining the contribution of RhoA/Rho kinase (ROCK) activation to deformability of erythrocytes and the relationship of ROCK activation to low O2 tension-induced ATP release. A downstream effector of RhoA activation, Rho kinase (ROCK), promotes cytoskeletal stiffening in non-contractile cells and this may also be true in erythrocytes. Y27632, a selective ROCK inhibitor, increases deformability and augments low O2 tension-induced ATP release from erythrocytes of healthy humans.
We have also found that activation of the Gs-coupled prostacyclin receptor (IPR) results in augmented cAMP accumulation and ATP release in DM2 erythrocytes when compared to healthy humans, suggesting a selective defect in the low O2 tension-induced ATP release mechanism in DM2 erythrocytes. Previously, we established that PDE3 hydrolyzes cAMP in the IPR-mediated pathway. We are now examining whether PDE3 activity may be decreased in DM2 erythrocytes, resulting in greater IPR agonist-induced cAMP and ATP release – and/or whether IPR expression or activity may be increased in DM2 erythrocytes.
Honors and Awards:
Fellow of the American Heart Association (FAHA)
Treasurer of the Microcriculatory Society
Sigma Xi – Scientist of the Year
American Heart Association: Lung, Resuscitation and Respiration Study Group Member
NIH – Vascular Cell and Molecular Biology Study Section (VCMB) – Ad hoc Member
NIH – Hypertension & Microcirculation Study Section (HM) – Ad hoc Member
NIH-NHLBI R33 HL089094: “Microvascular O2 Delivery: Impact of Erythrocyte-Released ATP”
2007 – 2012 – Co-investigator
NIH-NHLBI R01HL64180: "Mechanism of Deformation-Induced ATP release from RBCs"
2000 – 2010 Co-investigator
NIH-NHLBI R01 HL52675: "Cytochrome P-450 Eicosanoids and Pulmonary Hemodynamics"
1995 – 2004, Principal Investigator
NIH-NHLBI R01 HL32815: "Lung Perfusion by Positron Emission Tomography"
1985 – 2002, Co-Investigator
NIH-NHLBI SCOR HL30572: "Adult Respiratory Failure",
1988 – 1993, Co-Investigator
NIH-NHLBI SCOR HL30572: "Adult Respiratory Failure", Biochemistry Core",
1988 – 1993, Co-Investigator and Director of Biochemistry Core
American Heart Association Mentor Sponsored Pre-doctoral Award: “Role of CYP2B4 and its Major Metabolite, 5,6-Epoxyeicosatrienoic Acid, in the Pulmonary Vascular Resistance of the Rabbit” 2004 to 2006
American Society for Pharmacology and Experimental Therapeutics
American Physiological Society
American Heart Association, Council on High Blood Pressure Research
The Scientific Research Society of Sigma Xi
The Microcirculatory Society
Editorial Review Boards:
American Journal of Physiology-Regulatory, Integrative and Comparative Physiology
Recent Doctoral Student Graduates:
Josephine Garcia-Ferrer, (2004) “Effects of 5,6-epoxyeicosatrienoic acid on mobilization and metabolism of arachidonic acid”
Jennie (Losapio) Iverson, (2005) “Participation of Rho-kinase in epoxyeicosatrienoic acid-induced increases in pulmonary vascular tone”
Jesse Procknow, (2010) “Effects on Cardiovascular Events by Angiotensin Converting Enzyme and Cyclooxygenase-2 Inhibitors”
Kelly (Thuet) Clapp, (2012) “Effects of increased deformability on ATP release in erythrocytes obtained from healthy humans/rats or humans/rats with type 2 diabetes”
Stephanie Knebel, (2013) “Differential responses to prostacyclin analogs in platelets and erythrocytes from humans with type 2 diabetes”
Current Research Activities
We are currently examining relationships between ROCK activity and low O2 tension-induced ATP release in erythrocytes of humans with type 2 diabetes to learn whether increasing erythrocyte deformability by inhibiting ROCK activity will rescue low O2 tension-induced ATP release from type 2 diabetes erythrocytes.
We are also examining potential mechanisms by which erythrocytes of humans with type 2 diabetes accumulate greater amounts of cAMP and release greater amounts of ATP than erythrocytes of healthy humans when stimulated with the prostacyclin receptor agonists, iloprost and UT-15C. We are examining how these effects differ from those of the platelet.
Recent Peer Reviewed Publications (older to newest – of 93):
1. Losapio, J.L., Lonigro, A.J., Sprague, R.S., and Stephenson, A.H. 5,6-Epoxyeicosatrienoic acid contracts rabbit pulmonary artery via Rho-kinase activation. Appl Physiol: 99: 1391 – 1396, 2005.
2. Moreland, T., Procknow, J.D., Sprague, R.S., Iverson, J.L. Lonigro, A.J. and Stephenson, A.H. Selective Inhibition of Cyclooxygenase-1 Inhibits 5,6-EET-Induced Contraction of Rabbit Intralobar Pulmonary Arteries. J. Pharmacol Exp Ther. 321: 446 – 454, 2007.
3. Sridharan M, Sprague RS, Adderley SP, Bowles EA, Ellsworth ML and Stephenson AH: Diamide decreases deformability of rabbit erythrocytes and attenuates low oxygen tension-induced ATP release. Exp Biol & Med 235: 1142–1148, 2010 (PMC3138790).
4. Adderley, SP, Sridharan, M, Bowles, EA, Stephenson, AH, Sprague, RS and Ellsworth, ML. Inhibition of ATP release from Erythrocytes: A role for EPACs and PKC. Microcirculation, 18(2): 128-35. Feb 2011. (PMC3034780)
5. Adderley, SP, Thuet, KM, Sridharan, M, Bowles, EA, Stephenson, AH, Ellsworth, ML, and Sprague, RS. Identification of cytosolic phosphodiesterases in the erythrocyte: A possible role for PDE5. Med Sci Monit, 17(5): CR241-247, May 2011. (PMC3366467).
6. Thuet, KM, Bowles, EA, Ellsworth, ML, Sprague, RS, Stephenson, AH. The Rho kinase inhibitor, Y27632 increases erythrocyte deformability and low oxygen tension-induced ATP release. Am J Physiol Heart Circ Physiol. Nov 2011; 301: H1891 - H1896. (PMC3213963).
7. Sprague, RS, Bowles, EA, Achilleus, D, Stephenson, AH, Ellis, CG, and Ellsworth, ML. A selective phosphodiesterase 3 inhibitor rescues low PO2-induced ATP release from erythrocytes of humans with type 2 diabetes: Implication for vascular control. Am J Physiol Heart Circ Physiol, Dec 2011; 301: H2466 - H2472. (PMC3233804).
8. Bowden JA, Shao F, Albert CJ, Lally JW, Brown RJ, Procknow JD, Stephenson, AH, Ford DA. Electrospray Ionization Tandem Mass Spectrometry of Sodiated Adducts of Cholesteryl Esters. Lipids. Dec 2011; 46(12): 1169-79 (PMC3215890)
9. Sridharan, M., Bowles, EA., Richards, JP, Krantic, M., Davis, KL., Dietrich, KA., Stephenson, AH., Ellsworth, ML., and Sprague, RS. Prostacyclin Receptor-Mediated ATP Release from Erythrocytes Requires the Voltage-Dependent Anion Channel (VDAC). Am J Physiol Heart Circ Physiol, Feb 2012; 302: H553 - H559. (PMC3353798).
10. Goldman, D, Fraser, GM, Ellis, CG, Sprague, RS, Ellsworth, ML and Stephenson, AH. Towards a multiscale description of microvascular flow regulation: O2-dependent release of ATP from human erythrocytes and the distribution of ATP in capillary networks. Front. Physio. 2012;3:246. doi: 10.3389/fphys.2012.00246. Epub 2012 Jul 16.
11. Clapp, KM, Ellsworth, ML, Sprague, RS, and Stephenson, AH. Simvastatin and GGTI-2133, a geranylgeranyl transferase inhibitor, increase erythrocyte deformability, but reduce low oxygen tension-induced ATP release. Am J Physiol Heart Circ Physiol, Mar 2013; 304: H660 - H666.
12. Knebel, SM, Elrick, MM, Bowles, EA, Zdanovec, AK, Stephenson, AH, Ellsworth, ML and Sprague, RS. Synergistic effects of prostacyclin analogs and phosphodiesterase inhibitors on cAMP accumulation and ATP release from human erythrocytes. Experimental Biology and Medicine, Jun 2013, accepted for publication.