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

Weanling Arteriolar Responsiveness to ACh is Normal and Sensitive to NOS Inhibition Dilation (% of Maximum Response) Current Dose of ACh (nAmp) * * * * * * * * * * P<0.05 vs Normal * +L-NMMA (10 -4 M) Juvenile Weanling

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 *

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  

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

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)

Picospritzer Ejection Pressure (PSI) Dilation (% of Maximum Response) * * * * * * Weanling Arteriolar Responsiveness to A23187 is Normal and Sensitive to NOS Inhibition P<0.05 vs Normal * +L-NMMA (10 -4 M) Juvenile Weanling

Arteriolar VEGF (5  g/ml) Infusion (Ca 2+ -Independent) P<0.05 vs Weanling * P<0.05 vs Vehicle † Juvenile Weanling Vehicle-juvenile Diameter (  m) Picospritzer Ejection Pressure (PSI) * † * * * * † Passive

Picospritzer Ejection Pressure (PSI) Dilation (% of Maximum Response) * * * * * Juvenile Arteriolar Responsiveness to VEGF is Sensitive to NOS Inhibition P<0.05 vs Normal * +L-NMMA (10 -4 M) Juvenile Weanling

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  

Diameter (  m) Picospritzer Ejection Pressure (PSI) * * Passive Arteriolar Simvastatin (1 mM) Infusion (Ca 2+ -Independent) Juvenile Weanling Vehicle-juvenile P<0.05 vs Weanling *

Dilation (% of Maximum Response) Picospritzer Ejection Pressure (PSI) * * Juvenile Arteriolar Responsiveness to Simvastatin is Sensitive to NOS Inhibition P<0.05 vs Normal * +L-NMMA (10 -4 M) Juvenile

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

Dilation(% of Maximum Response) Current Dose of ACh (nAmp) Juvenile Weanling Arteriolar Responsiveness to ACh is Identical (Ca 2+ -Dependent)

Arteriolar Responsiveness to A23187 is Identical (Ca 2+ -Dependent) Dilation (% of Maximum Response) Picospritzer Ejection Pressure (PSI) Juvenile Weanling

Dilation (% of Maximum Response) Picospritzer Ejection Pressure (PSI) * * * * Juvenile Weanling P<0.05 vs Weanling * Weanling Arteriolar Responsiveness to VEGF is Attenuated (Ca 2+ -Independent)

Dilation (% of Maximum Response) Picospritzer Ejection Pressure (PSI) * * Juvenile Weanling P<0.05 vs Weanling * Weanling Arteriolar Responsiveness to Simvastatin is Attenuated (Ca 2+ -Independent)