1. Absence of Calcium-Independent Release of Endothelial Nitric Oxide (NO) in Young Rats Timothy R. Nurkiewicz & Matthew A. Boegehold West Virginia University School of Medicine Department of Physiology & Pharmacology Morgantown, West Virginia

2. Weanling Arteriolar Responsiveness to ACh is Normal and Sensitive to NOS Inhibition Dilation (% of Maximum Response) Current Dose of ACh (nAmp) 0 20 40 60 80 100 51020 40 80 0 20 40 60 80 100 * * * * * * * * * * P<0.05 vs Normal * +L-NMMA (10 -4 M) Juvenile Weanling

3. 6008001000120014001600 20 30 40 50 60 Diameter (  m) Wall Shear Rate (s -1 ) Control Occlusion * Weanling Arteriolar Responsiveness to Shear Stress is Absent (Ca 2+ -Independent) Juvenile Weanling P<0.05 vs Control *

4. Endothelium-derived NO Ca 2+ -independent and Ca 2+ –dependent transduction pathways Tyrosine Kinase PI-3 Kinase Akt / PKB eNOS ERK ½ MAPK Tyr-P Ser-P + + + + ACh [Ca 2+ ] A23187 Ca 2+ VEGF EGF  

5. Methods Weanling rats: 4 wks Juvenile Rats: 8 wks Intravital microscopy of the spinotrapezius muscle Arcade bridge arterioles Local, luminal perfusion: 1.A23187 2.VEGF 3.Simvastatin

6. 0102030 40 60 80 100 120 Diameter (  m) Picospritzer Ejection Pressure (PSI) * † † † Passive Juvenile Weanling Vehicle-juvenile P<0.05 vs Weanling * P<0.05 vs Vehicle † Arteriolar A23187 (10 -7 M) Infusion (Ca 2+ -Dependent)

7. Picospritzer Ejection Pressure (PSI) Dilation (% of Maximum Response) 0 20 40 60 80 100 0 20 40 60 80 100 5102040 * * * * * * Weanling Arteriolar Responsiveness to A23187 is Normal and Sensitive to NOS Inhibition P<0.05 vs Normal * +L-NMMA (10 -4 M) Juvenile Weanling

8. Arteriolar VEGF (5  g/ml) Infusion (Ca 2+ -Independent) P<0.05 vs Weanling * P<0.05 vs Vehicle † Juvenile Weanling Vehicle-juvenile 40 60 80 100 120 0 10 20 30 40 Diameter (  m) Picospritzer Ejection Pressure (PSI) * † * * * * † Passive

9. Picospritzer Ejection Pressure (PSI) Dilation (% of Maximum Response) 5102040 0 20 40 60 80 100 0 20 40 60 80 100 * * * * * Juvenile Arteriolar Responsiveness to VEGF is Sensitive to NOS Inhibition P<0.05 vs Normal * +L-NMMA (10 -4 M) Juvenile Weanling

10. Endothelium-derived NO Ca 2+ -independent and Ca 2+ –dependent transduction pathways Tyrosine Kinase PI-3 Kinase Akt / PKB eNOS ERK ½ MAPK Tyr-P Ser-P + + + + ACh [Ca 2+ ] A23187 Ca 2+ VEGF EGF  

11. Diameter (  m) Picospritzer Ejection Pressure (PSI) 40 60 80 100 120 0 10 20 30 40 * * Passive Arteriolar Simvastatin (1 mM) Infusion (Ca 2+ -Independent) Juvenile Weanling Vehicle-juvenile P<0.05 vs Weanling *

12. Dilation (% of Maximum Response) Picospritzer Ejection Pressure (PSI) 2040 0 20 40 60 80 100 * * Juvenile Arteriolar Responsiveness to Simvastatin is Sensitive to NOS Inhibition P<0.05 vs Normal * +L-NMMA (10 -4 M) Juvenile

13. Conclusions 2.Endothelial NO production in response to Ca 2+ -dependent stimuli (ACh & A23187) is present in weanling arterioles. 3.Endothelial NO production in response to Ca 2+ -independent stimuli (shear stress, VEGF, Simvastatin) is absent or not fully functional in weanling arterioles. 1.Endothelium-dependent arteriolar dilation in response to Ca 2+ - dependent, and -independent stimuli is present in juvenile rats, and is sensitive to NOS inhibition

15. Dilation(% of Maximum Response) Current Dose of ACh (nAmp) 5102040 80 0 20 40 60 80 100 Juvenile Weanling Arteriolar Responsiveness to ACh is Identical (Ca 2+ -Dependent)

16. Arteriolar Responsiveness to A23187 is Identical (Ca 2+ -Dependent) Dilation (% of Maximum Response) Picospritzer Ejection Pressure (PSI) 0 20 40 60 80 100 5102040 Juvenile Weanling

17. Dilation (% of Maximum Response) Picospritzer Ejection Pressure (PSI) 5102040 0 20 40 60 80 100 * * * * Juvenile Weanling P<0.05 vs Weanling * Weanling Arteriolar Responsiveness to VEGF is Attenuated (Ca 2+ -Independent)

18. Dilation (% of Maximum Response) Picospritzer Ejection Pressure (PSI) 20 40 0 20 40 60 80 100 * * Juvenile Weanling P<0.05 vs Weanling * Weanling Arteriolar Responsiveness to Simvastatin is Attenuated (Ca 2+ -Independent)