Open Access
Alexander Holowatz, Lacy Marie
Graduate Program:
Doctor of Philosophy
Document Type:
Date of Defense:
March 09, 2007
Committee Members:
  • William Lawrence Kenney Jr., Committee Chair
  • Kelly J Karpa, Committee Member
  • James Anthony Pawelczyk, Committee Member
  • David Nathan Proctor, Committee Member
  • skin blood flow
  • nitric oxide
  • temperature regulation
  • hypertension
Reflex cutaneous vasodilation is attenuated with primary human aging and hypertensive vascular pathology, rendering these populations more vulnerable to complications from heat-related illness. Full expression of reflex cutaneous vasodilation is dependent on functional nitric oxide (NO), which is reduced with primary human aging and hypertensive vascular pathology. NO bioavailability may be decreased by (1) upregulated arginase (Arg) activity, which reciprocally regulates the NO-synthase (NOS) substrate L-arginine (L-arg) and/or (2) increased oxidant stress. Furthermore, there may be a mechanistic link between upregulated Arg activity and increased oxidant stress through NOS uncoupling. Therefore, the purpose of this series of experiments was to investigate the underlying mechanisms mediating impaired reflex cutaneous vasodilation in aged and hypertensive cutaneous vasculature. In the first study we investigated age-related alterations in exogenous acetylcholine-induced vasodilation. While acetylcholine is capable of modulating reflex-mediated vasodilation, the precise mechanisms through which acetylcholine induces vasodilation and whether those downstream mechanisms change with aging are unclear. We tested the hypotheses that both NO- and prostanoid-mediated pathways contribute to exogenous acetylcholine-induced vasodilation and that both are attenuated with advanced age. Twelve young (Y: 23„b1 yr) and 10 older (O: 69„b1 yr) subjects underwent infusions of 137.5 £gM acetycholine at 4 intradermal microdialysis (MD) sites: control (Co, Ringer¡¦s), NO synthase inhibited (NOS-I, 10 mM L-NAME), cyclooxygenase inhibited (COX-I, 10 mM ketorolac) and NOS-I+COX-I. For all studies red blood cell flux was monitored using laser-Doppler flowmetry and cutaneous vascular conductance (CVC) was calculated (laser-Doppler flux/MAP) and normalized to maximal CVC (%CVCmax) (28mM sodium nitroprusside + local heating to 43„aC). Baseline %CVCmax was increased in the older at COX-I sites (COX-I 16¡Ó1, NOS-I+COX-I 16¡Ó2 vs. Co 10¡Ó1 %CVCmax; p<0.001) but not in the young, suggesting an age-related shift toward COX-derived vasoconstrictors contributing to basal cutaneous vasomotor tone. There was no difference in peak %CVCmax during acetylcholine infusion between age groups and the response was unchanged by NOS-I (O: NOSI 35¡Ó5 vs. Co 38¡Ó5 %CVCmax; p=0.84) (Y: NOSI 41¡Ó4 vs. Co 39¡Ó4 %CVCmax; p=0.67). COX-I and NOS-I+COX-I attenuated the peak CVC response to Ach in both groups (COX-I Y: 22¡Ó2 vs. O: 29¡Ó3 %CVCmax vs. Co; p<0.001 both groups; NOS-I+COX-I Y: 29¡Ó2 vs. O: 32¡Ó3 %CVCmax vs. Co; p<0.001 both groups). We concluded that acetylcholine directly induces cutaneous vasodilation through prostanoid and non-NO-, non-prostanoid-dependent pathways. Further, older subjects have a diminished prostanoid contribution to acetylcholine-induced vasodilation. In the second study we hypothesized that increased Arg activity contributes to attenuated vasodilation in aged skin by limiting L-arg for NOS-mediated NO synthesis. Five MD fibers were placed in forearm skin of 10 young (Y: 23¡Ó1 years) and 9 older (O: 68¡Ó1 years) human subjects serving as Co, NOS-I, Arg-inhibited (Arg-I: 5.0mM (S)-(2-boronoethyl)-L-cysteine + 5.0mM N-hydroxy-nor-L-arginine), L-arg supplemented (L-arg: 10.0mM L-arginine) and combined Arg-inhibited + L-arg sites. For all whole body heating studies reflex vasodilation was induced by using a water-perfused suit to increase oral temperature (Tor) 0.8-1.0¢XC. Cutaneous vasodilation during heating was attenuated in the older subjects (Y: 42¡Ó1 vs. O: 30¡Ó1 %CVCmax, p<0.001) at Co sites. NOS inhibition decreased vasodilation in both age groups compared to Co (Y: 22¡Ó2, O: 18¡Ó2 %CVCmax, p<0.001). Arg inhibition, L-arg supplementation, and Arg inhibition + L-arg supplementation augmented vasodilation in the older subjects (Arg-inhibited: 46¡Ó4, L-arg: 44¡Ó4, Arg-inhibited + L-arg: 46¡Ó5 %CVCmax, p<0.001 vs. Co) but not in young subjects (Arg-inhibited: 46¡Ó4, L-arg: 38¡Ó4, Arg-inhibited + L-arg: 44¡Ó4 %CVCmax, p>0.05 vs. Co). Increasing L-arg for NO synthesis by either Arg inhibition or direct L-arg supplementation abolishes the age-related deficit in reflex cutaneous vasodilation. The third study tested the hypothesis that age-related increases in oxidant stress attenuates reflex cutaneous vasodilation, therefore acute antioxidant administration alone and combined with Arg inhibition to increase L-arginine availability would increase reflex cutaneous vasodilation in aged skin. Eleven young (Y, 22¡Ó1 years) and 10 older (O, 68¡Ó1 years) men and women were instrumented with four intradermal MD fibers. MD sites served as Co, NOS-I, L-ascorbate supplemented (Asc: 10mM L-ascorbate), and Asc + Arg-inhibited (AA+Arg-I: 10mM L-ascorbate + 5mM BEC + 5mM nor-NOHA). Cutaneous vasodilation during heating was attenuated in the older subjects (Y: 37¡Ó3 vs. O: 28¡Ó3 %CVCmax, p<0.05). NOS-I significantly decreased vasodilation in both groups compared to the Co site (Y: 20¡Ó4, O: 15¡Ó2 %CVCmax, p<0.05 vs. Co within group). Asc and Asc + Arg-I did increase vasodilation beyond Co in the older subjects (Asc: 35¡Ó4 %CVCmax, Asc + Arg-I: 41¡Ó3 %CVCmax; p<0.001) but did not in the young subjects (Asc: 36¡Ó3 %CVCmax ; Asc + Arg-I: 40¡Ó5 %CVCmax, p>0.05 ). Combined Asc + Arg-I resulted in a greater increase in vasodilation than did Asc alone in the older subjects (p=0.001). Acute Asc supplementation increased reflex cutaneous vasodilation in aged skin. When combined with Arg inhibition to increase L-arg availability Asc supplementation resulted in a further increase in vasodilation above Asc alone, effectively restoring cutaneous vascular conductance to the level of young subjects. In the fourth study we tested the hypothesized that NO-dependent vasodilation would be attenuated in essential hypertensive human skin due to upregulated Arg activity and acute Arg inhibition or L-arg supplementation would augment reflex cutaneous vasodilation. Five MD fibres were placed in skin of 8 unmedicated subjects with essential hypertension (HTN: MAP: 112¡Ó1 mmHg) and 9 age-matched normotensive (AMN) (MAP: 87¡Ó1 mmHg) men and women to serve as: Co, NOS-I, Arg-I, L-arg supplemented, and combined Arg-I + L-arg. The £G%CVCmax between the Co and NOS-I site was calculated as the difference between Co and NOS-I sites. Maximal CVC was attenuated in the HTN subjects by ~25% compared to AMN subjects (p<0.001). With a 1.0¢XC increase in body core temperature (Tor) %CVCmax was not different between the groups (HTN: 43¡Ó3 vs. AMN: 45¡Ó3%CVCmax, p>0.05). NOS-I significantly decreased %CVCmax in both groups but %CVCmax was greater in the HTN group (HTN: 32¡Ó4 vs. AMN: 23¡Ó3%CVCmax, p<0.05). Arg-I alone augmented %CVCmax only in the HTN group (HTN: 65¡Ó5 vs. AMN: 48¡Ó3 %CVCmax, p<0.05). L-arg alone did not effect %CVCmax in either group (HTN: 49¡Ó5 vs. AMN: 49¡Ó3 %CVCmax, p>0.05). Combined Arg-I + L-arg augmented %CVCmax in both subject groups compared to their respective control sites (HTN: 60¡Ó7 vs. AMN: 61¡Ó3 %CVCmax, both p<0.05 vs. respective control sites). The £G %CVCmax between the control and NOS-I sites was attenuated at £GTor>0.5¢XC in the HTN group (p<0.001 vs. AMN). Vasodilation is attenuated with HTN due to decreased NO-dependent vasodilation and can be augmented with Arg inhibition but not L-arg supplementation, suggesting that Arg is upregulated with HTN. In the fifth study we tested the hypothesis that increased oxidant stress contributed to attenuated NO-dependent reflex cutaneous vasodilation in hypertensive human subjects, further antioxidant supplementation, alone and combined with Arg inhibition would augment NO-dependent cutaneous vasodilation. Nine unmedicated subjects with essential hypertension (HTN: MAP=112¡Ó1mmHg) and 9 age-matched normotensive (AMN: MAP=81¡Ó10) men and women were instrumented with 4 intradermal microdialysis (MD) fibers: Co, NOS-I, Asc supplemented, and Asc + Arg-I. Oral temperature (Tor) was increased by 0.8¢XC via a water-perfused suit and L-NAME was then perfused through all MD sites to quantify the change in vasodilation due to NO. Cutaneous vasodilation was attenuated in essential hypertensive skin (HTN: 35¡Ó3 vs. AMN: 42¡Ó4 %CVCmax, p<0.05). Asc and Asc+A-I augmented cutaneous vasodilation in HTN (Asc: 57¡Ó5, Asc + A-I: 53¡Ó6 %CVCmax, p<0.05 vs. Co), but not AMN. %CVCmax after NOS-I in the Asc and Asc + A-I was increased in HTN (Asc: 41¡Ó4, Asc + A-I: 40¡Ó4 vs. Co: 29¡Ó4 %CVCmax; both p<0.05) and was further decreased in HTN (Asc: -19¡Ó4, Asc + A-I: -17¡Ó4 %CVCmax, p<0.05 vs. Co) but not AMN. Antioxidant supplementation alone or combined with Arg inhibition augments attenuated reflex cutaneous vasodilation in HTN skin through NO- and non-NO-dependent mechanisms.