SYMPATHETIC NORADRENERGIC MECHANISMS OF THERMOREGULATORY VASOCONSTRICTION IN AGED HUMAN SKIN

Open Access
Author:
Lang, James Andrew
Graduate Program:
Kinesiology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
February 19, 2010
Committee Members:
  • William Lawrence Kenney Jr., Dissertation Advisor
  • William Lawrence Kenney Jr., Committee Chair
  • James Anthony Pawelczyk, Committee Member
  • Lacy Marie Alexander, Committee Member
  • Kelly J Karpa, Committee Member
Keywords:
  • aging
  • adrenergic
  • skin blood flow
  • tetrahydrobiopterin
Abstract:
Reflex vasoconstriction (VC) is attenuated in older human skin, resulting in greater blood flow and convective heat loss during cold exposure, which may in turn increase their risk of developing hypothermia. Central to this impairment may be the effects of elevated oxidative and nitrosative stress on key enzymes that modulate noradrenergic VC such as tyrosine hydroxylase (TH) and rho-kinase (ROCK). The purpose of this series of four studies was to investigate the neural and vascular mechanisms underlying the attenuated VC response in aged skin, and specifically, the role of these enzymes in contributing to this deficit. The first study tested the hypothesis that compromised noradrenergic VC in aged skin is due to the depletion of an essential cofactor, tetrahydrobiopterin (BH4), needed in the rate-limited step of norepinephrine (NE) biosynthesis. Functional changes in forearm skin blood flow were assessed with Laser Doppler flowmetry in response to gradual whole-body cooling and tyramine infusion, which evokes NE release from storage vesicles in sympathetic nerve terminals. Four microdialysis (MD) fibers were placed in the forearm skin of eleven young (Y) and eleven older (O) human subjects for infusion of 1) Ringers solution (control), 2) 5 mM BH4, 3) BH4 + 10mM ascorbate, and 4) BH4 + adrenoreceptor blockade (5mM yohimbine + 1mM propranolol). The VC response was lower at the control site in O during both cooling (Y: -34 ± 2, O: -17 ± 2 %&#916;CVCbase; P<0.001) and tyramine infusion (Y: -33 ± 4, O: -15 ± 3 %&#916;CVCbase; P<0.001). BH4 infusion normalized O to Y values during both cooling (Y: -34 ± 4, O: -34 ± 2 %&#916;CVCbase; P<0.001) and tyramine infusion (Y: -38 ± 4, O: -35 ± 3 %&#916;CVCbase; P<0.001). The addition of adrenoreceptor blockade abolished VC in aged skin indicating that BH4 acts through noradrenergic, not cotransmitter, mechanisms. Local BH4 supplementation augments reflex and tyramine-induced VC in aged skin, suggesting that reduced BH4 bioavailability may contribute to the attenuated VC during whole-body cooling. The second study tested the hypothesis that attenuated reflex VC in aged skin may be partly mediated by oxidant-induced reduction in functional substrate and/or cofactor availability required by TH for NE biosynthesis; i.e., tyrosine and BH4, respectively. Functional changes in skin blood flow were assessed with Laser Doppler flowmetry in response to gradual whole-body cooling and tyramine infusion. Four microdialysis fibers were placed in the forearm skin of 10 Y and 10 O subjects for infusion of 1) Ringer’s, 2) 0.5 mM L-tyrosine, 3) 5 mM BH4, and 4) BH4 + L-tyrosine. VC was attenuated at the control site in O during whole-body cooling (Y: -39 ± 3, O: -17 ± 3 %&#916;CVCbase; P<0.01) and tyramine infusion (Y: -41 ± 3, O: -21 ± 4 %&#916;CVCbase; P<0.01). Similar to BH4 (cold, Y: -37 ± 3, O: -36 ± 3; tyramine, Y: 41 ± 2, O: -36 ± 3 %&#916;CVCbase), tyrosine also augmented the VC in O during cooling (Y: -37 ± 4, O: -34 ± 4 %&#916;CVCbase) and tyramine infusion (Y: -40 ± 4, O: -45 ± 4 %&#916;CVCbase), but BH4 + tyrosine did not further augment VC during cooling (Y: -38 ± 4, O: -31 ± 3 %&#916;CVCbase) or tyramine infusion (Y: -36 ± 3, O: -36 ± 5 %&#916;CVCbase). Individually, both tyrosine and BH4 infusion augmented VC in aged skin, suggesting that their reduced bioavailability may impair NE synthesis and contribute to the attenuated VC response. The third study tested the hypothesis that the contribution of ROCK to reflex VC is greater in aged skin. NOS inhibited sites would be used to control for the putative effects Cutaneous VC was assessed with Laser Doppler flowmetry and was elicited by 1) whole-body cooling (Tsk = 30.5oC) and 2) infusion of a local physiological concentration of NE (1x10-6M). Four microdialysis fibers were placed in the forearm skin of 8 Y and 8 O subjects for infusion of 1) Ringers solution, 2) 3 mM fasudil (ROCK inhibition), 3) 20 mM NG-L-arginine methyl ester (L-NAME) (NOS inhibition), and 4) both ROCK + NOS inhibitors. VC was reduced at the control site in O during both cooling (Y: -34 ± 3, O: -18 ± 3 %&#916;CVCbaseline; P<0.001) and NE infusion (Y: -53 ± 4, O: -41 ± 9 %&#916;CVCbaseline; P=0.006). Fasudil attenuated VC in both age groups during mild cooling; however, this reduction remained only in O but not Y skin during moderate cooling (Y: -30 ± 5, O: -7 ± 1 %&#916;CVCbaseline; P=0.016), and was not altered by NOS inhibition. Fasudil blunted NE-mediated VC in both age groups (Y: -23 ± 4, O: -7 ± 3 %&#916;CVCbaseline; P<0.01). Cumulatively, these data suggest that although ROCK modestly contributes to the VC response in young, it is upregulated in aged skin during moderate cooling and mediates approximately half of the total reflex VC response in aged skin. The fourth study tested the hypothesis that localized BH4 supplementation would not affect end-organ VC responsiveness to exogenous NE after localized sympathetic nerve blockade. Two microdialysis fibers were placed in bretylium tosylate-pretreated sites (presynaptically blocks neurotransmitters release from sympathetic adrenergic nerve terminals; iontophoresis, 200 &#956;A for 20 min) on the forearm skin of 10 Y and 10 older O subjects for infusion of 1) Ringer’s (control) and 2) 5 mM BH4. While local skin temperature was clamped at 34oC, 6 concentrations of NE (10-12, 10-10, 10-8, 10-6, 10-4, 10-2 M) were randomly infused (except the supraphysiological doses) at each laser Doppler site. Despite prejunctional adrenergic blockade, NE-mediated VC was blunted in aged skin at each NE dose (10-12: -12 ± 2 vs. -21 ± 2, 10-10: -15 ± 2 vs. -27 ± 1, 10-8: -22 ± 2 vs. -32 ± 2, 10-6: -27 ± 2 vs. -38 ± 1, 10-4: -52 ± 3 vs. -66 ± 5, 10-2: -62 ± 3 vs. -75 ± 4 %&#916;CVCbase; P<0.01), and this response was not affected by pretreatment with BH4 (P>0.05). Localized BH4 did not affect end-organ responsiveness to exogenous NE suggesting that the effects of BH4 on cutaneous VC are primarily limited to the NE biosynthetic pathway in prejunctional adrenergic nerve terminals. The collective results of this series of studies suggest that compromised noradrenergic function in aged skin has both a pre- and postjunctional component. The ability for TH to upregulate during cold exposure is suboptimal in aged skin, which is likely due to reduced tyrosine and BH4 bioavailability. Furthermore, ~50% of the extant reflex VC response in aged skin relies on ROCK; an enzyme that is also upregulated with various age-related vascular diseases.