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. 2010 Feb 22;588(Pt 8):1361–1368. doi: 10.1113/jphysiol.2009.185694

Localized tyrosine or tetrahydrobiopterin supplementation corrects the age-related decline in cutaneous vasoconstriction

James A Lang 1, Lacy A Holowatz 1, W Larry Kenney 1,2
PMCID: PMC2872739  PMID: 20176627

Abstract

The attenuated reflex vasoconstriction in aged skin may be partly mediated by oxidant-induced reduction in functional substrate and cofactor availability for noradrenaline biosynthesis. We hypothesized that localized supplementation of tyrosine and tetrahydrobiopterin (BH4) in aged human skin could augment reflex- (whole-body cooling) and pharmacologically (tyramine, which displaces noradrenaline from axon terminals) induced vasoconstriction. Four microdialysis fibres were placed in the forearm skin of 10 young and 10 older subjects for infusion of (1) Ringer solution (control), (2) 0.5 mm l-tyrosine, (3) 5 mm BH4, and (4) BH4+l-tyrosine. Cutaneous vascular conductance (CVC) was calculated (laser Doppler flux/mean arterial pressure) and normalized to baseline (%ΔCVCbase). Vasoconstriction was attenuated at the control site in the older subjects during both whole-body cooling (young: −39 ± 3, older: −17 ± 3%ΔCVCbase; P < 0.01) and tyramine infusion (young: −41 ± 3, older: −21 ± 4%ΔCVCbase; P < 0.01). BH4 (cold, young: −37 ± 3, older: −36 ± 3; tyramine, young: −41 ± 2, older: −36 ± 3%ΔCVCbase) and tyrosine (cold, young: −37 ± 4, older: −34 ± 4; tyramine, young: −40 ± 4, older: −45 ± 4%ΔCVCbase) both resolved the age-related decrease in cutaneous vasoconstriction, but BH4+ tyrosine did not further augment vasoconstriction (cold, young: −38 ± 4, older: −31 ± 3; tyramine, young: −36 ± 3, older: −36 ± 5%ΔCVCbase). These data are consistent with the concept that reduced bioavailability of BH4 and/or tyrosine may impair noradrenaline synthesis and contribute to the attenuated vasoconstrictor response in aged skin.

Introduction

Reflex cutaneous vasoconstriction is an early and sustained response to whole-body cold exposure. This thermoregulatory response is attenuated in older adults rendering them more susceptible to greater convective heat loss to the environment (Wagner et al. 1974; Kenney & Armstrong, 1996; Degroot & Kenney, 2007) and possibly hypothermia (CDC, 2002). Even when matched for body composition and fitness, older adults display reduced peripheral vasoconstriction and a relative inability to maintain core temperature during even mild (22°C) cold exposure (Degroot & Kenney, 2007).

Several studies have detailed functional deficits at various locations along the efferent arm of the sympathetic vasoconstrictor reflex in aged skin (Frank et al. 2000; Thompson & Kenney, 2004; Thompson et al. 2005; Lang et al. 2009a,b;). In young skin, ∼40% of the vasoconstriction to whole body cooling is mediated by sympathetic neurotransmitters coreleased with noradrenaline. In aged skin, this cotransmitter component is absent; thus, the vasoconstrictor response depends entirely on a functionally impaired noradrenergic mechanism. Central to this impairment may be elevated oxidative and nitrosative stress that affects key regulatory molecules in the biosynthetic pathway of noradrenaline, putatively tyrosine and tetrahydrobiopterin (BH4) (Lang et al. 2009a). BH4 is an essential cofactor for both nitric oxide synthase (NOS) and tyrosine hydroxylase, the rate-limiting enzyme for catecholamine biosynthesis (Kaufman, 1978; Kumer & Vrana, 1996; Moens & Kass, 2007). BH4 serves as a powerful reducing agent that maintains tyrosine hydroxylase in the ferrous, active form required for catecholamine production (Kaufman, 1978; Dunkley et al. 2004; Urano et al. 2006). Oxidative stress in cultured sympathetic neurons markedly reduces intracellular BH4 concentration (∼90%) resulting in a ∼75% reduction in catecholamine synthesis (Li et al. 2003). In vivo, localized intradermal administration of BH4 offsets the age-related attenuation in cold and tyramine-induced cutaneous vasoconstriction, a result not related to effects of BH4 on NOS (Lang et al. 2009a). These studies suggest that suboptimal tyrosine hydroxylase function contributes to the attenuated reflex vasoconstriction in aged skin.

Neuronal activation increases the affinity of tyrosine hydroxylase for its cofactor BH4, thereby making enzyme activity dependent on its saturation with its amino acid substrate l-tyrosine (Weiner, 1978; Fluharty et al. 1985; Kumer & Vrana, 1996). Elevated oxidative and nitrosative stress converts tyrosine to the tyrosyl radical, which is unable to function in catecholamine biosynthesis and can further reduce free tyrosine pools as well as nitrate other proteins thereby compromising their function (Ischiropoulos et al. 1995; Reiter et al. 2000; Kochman et al. 2002). Functionally, tyrosine bioavailability can affect catecholamine production in active nerve terminals (Wurtman et al. 1974; Iuvone et al. 1978; Fernstrom, 1983; Fernstrom et al. 1986). In vivo human studies indicate that tyrosine supplementation may enhance cognitive and psychomotor performance during cold stress (Banderet & Lieberman, 1989; O’Brien et al. 2007). However, no in vivo studies to date have addressed the functional role of tyrosine on thermoregulatory reflex vasoconstriction in aged human skin.

Because the cotransmitter component to reflex vasoconstriction is absent in aged skin, this directed our focus on the remaining noradrenergic mechanisms. Specifically, the purpose of this study was to examine the relative roles of tyrosine and BH4 during cutaneous vasoconstriction. In addition to what we found with BH4, elevated oxidative stress in aged skin may also compromise the available concentration of the tyrosine substrate in the vicinity of tyrosine hydroxylase. Thus, we hypothesized that localized supplementation of tyrosine or BH4 in aged skin would augment reflex and pharmacologically induced vasoconstriction elicited by whole-body cooling (physiological) and tyramine infusion (pharmacological stimulus), respectively. We further hypothesized that these compounds would not affect vasoconstriction to a supraphysiological dose of noradrenaline.

Methods

Subjects

With Pennsylvania State University Institutional Review Board approval and after verbal and written informed consent, 10 young (23 ± 1 years; 5 men, 5 women) and 10 older (73 ± 2 years; 5 men, 5 women) subjects participated in the study. Young women were tested in the early follicular phase (days 1–7) of the menstrual cycle, and older women were post-menopausal and not taking hormone replacement therapy. All subjects were healthy, non-obese, normotensive, normal cholesterolaemic, non-smokers, and not taking any medications or vitamin supplements that would otherwise alter cardiovascular or thermoregulatory function. All procedures conformed to the standards set by the Declaration of Helsinki.

Instrumentation

On the morning of an experiment, between 07.00 and 10.00 h, subjects arrived at the laboratory and were instrumented with four microdialysis (MD) fibres (10 mm, 20 kDa cutoff membrane, MD 2000 Bioanalytical Systems, West Lafayette, IN, USA) placed intradermally in the left ventral forearm using an aseptic technique. MD sites were at least 4.0 cm apart to prevent cross-reactivity of pharmacological agents between sites. Throughout the protocol, subjects were in a semisupine position with the experimental forearm at heart level. Prior to fibre placement, ice packs were applied to MD sites for 5 min to temporarily anaesthetize the skin (Hodges et al. 2009). For each fibre, a 25-gauge needle was inserted horizontally into the dermis such that entry and exit points were ∼2.5 cm apart. After MD fibres were threaded through the needle, the needle was withdrawn leaving the membrane in place. All fibres were taped in place and lactated Ringer solution was initially perfused to test the integrity of the fibre and then during the resolution period following needle insertion trauma. MD sites were then randomly assigned with respect to position on the forearm and each site was perfused with one of the following drugs: (1) lactated Ringer solution serving as control, (2) 0.5 mm l-tyrosine, (3) 5 mm tetrahydrobiopterin (BH4), and (4) 0.5 mm l-tyrosine + 5 mm BH4 (both tyrosine and BH4 were from Sigma-Aldrich, St Louis, MO, USA). Pilot studies were performed to determine the optimal concentration of tyrosine, which was defined as a dose that would maximally affect vasoconstrictor function without altering baseline CVC. Concentrations exceeding 0.5 mm did not further enhance the vasoconstrictor response. The 5 mm dose of BH4 was determined from previous studies in our laboratory (Lang et al. 2009a).

To control skin temperature, subjects wore a water-perfused suit that covered the entire body except for the face, feet, hands and forearms. Copper–constantan thermocouples were placed on the surface of the skin at six sites: calf, thigh, abdomen, chest, back and upper arm. The unweighted mean of these sites provided an index of mean skin temperature (Tsk).

To obtain an index of skin blood flow, red blood cell flux was continuously measured with laser Doppler flowmetry (LDF) probes (MoorLAB, Temperature Monitor SH02, Moor Instruments, Devon, UK). LDF probes were placed in the centre of local heaters and positioned directly over each MD fibre site. To specifically isolate reflex mechanisms, local skin temperature was clamped at 34°C throughout the experiment. Arterial blood pressure was measured every 5 min throughout the experiment via brachial auscultation. Mean arterial pressure (MAP) was calculated as the diastolic blood pressure plus one-third the pulse pressure. Cutaneous vascular conductance (CVC) was calculated as the ratio of LDF flux to MAP and expressed as the percentage change from baseline values (%ΔCVCbaseline).

Protocol

After instrumentation with MD fibres, local hyperaemia was allowed to resolve for 60–90 min (depending on how quickly baseline was achieved at all sites) while perfusing sites with their assigned pharamacological agent. The duration and rate of perfusion (2 μl min−1) of each drug were the same across all four MD sites for a given experiment. All drugs were mixed just prior to use, dissolved in lactated Ringer solution, sterilized using syringe microfilters (Acrodisc, Pall, Ann Arbor, MI, USA), and perfused at 2 μl min−1 (Bee Hive controller and Baby Bee microinfusion pumps, Bioanalytical Systems, West Lafayette, IN, USA). Throughout the baseline period, mean Tsk was held constant at 34°C by perfusing thermoneutral water through the suit.

After baseline measurements, cold water was circulated through the suit to induce reflex vasoconstriction. Gradual cooling was performed for 30 min where mean Tsk was uniformly decreased from 34°C to 30.5°C over 30 min. This was followed by an additional 10 min period where Tsk was clamped at 30.5°C; thus, the total duration of cold stress was 40 min. Rewarming for approximately 30 min followed, to return Tsk to 34°C. All sites were then perfused for 20 min with 1 mm tyramine to evoke endogenous noradrenaline release pharmacologically. Exogenous noradrenaline (1 × 10−2m) was then perfused at all sites for 10 min to elicit further vasoconstriction. The CVC established after rewarming was utilized as a baseline to assess tyramine and noradrenaline-mediated vasoconstriction. Full resolution of the robust vasoconstrictor responses to tyramine and noradrenaline prevented the randomization of these steps with whole-body cooling. Lastly, 28 mm sodium nitroprusside was perfused through all sites at a rate of 4 μl min−1 in combination with local heating of the skin to 43°C until a plateau in the vasodilatation response was achieved (∼30 min) at each MD site to ensure that vascular responsivity remained intact post-cooling.

Data acquisition and analysis

Data were collected at 40 Hz, digitized, recorded and stored in a personal computer until data analysis (Windaq, Dataq Instruments, Akron, OH, USA). CVC data were averaged over 3 min intervals during baseline and at each 0.5°C drop in mean Tsk during the cooling period. A three-way mixed model repeated measures analysis of variance was conducted to detect age and treatment differences during whole-body cooling, and tyramine and noradrenaline administration (SAS, v. 9.1.3, Cary, NC, USA). Tukey's post hoc test was performed when appropriate to determine where age and drug treatment differences occurred. Data relating to subject characteristics and absolute baseline CVC values were assessed by Student's t test for paired data. Statistical significance for all analyses was set at α= 0.05. Values are expressed as means ±s.e.m.

Results

Subject characteristics are presented in Table 1. Age groups were well matched with regard to height, weight, BMI, MAP, and cholesterol ratio (total cholesterol/HDL cholesterol).

Table 1.

Subject characteristics

Variable Young Older
Sex (M, F) 5, 5 5, 5
Age (years) 23 ± 1 73 ± 2*
Height (cm) 174 ± 3 166 ± 2
Weight (kg) 72 ± 5 68 ± 3
BMI (kg m−2) 24 ± 1 25 ± 1
Resting MAP (mmHg) 85.4 ± 2.4 88.9 ± 1.5
Glucose (mg dl−1) 88 ± 2 90 ± 2
Total cholesterol (mg dl−1) 153 ± 6 190 ± 7*
HDL (mg dl−1) 53 ± 3 68 ± 4*
LDL (mg dl−1) 83 ± 7 106 ± 6*
Cholesterol ratio (total/HDL) 3.0 ± 0.2 2.9 ± 0.2
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Values are means ±s.e.m. for young (n= 10) and older (n= 10) men and women. BMI, body mass index; MAP, mean arterial pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein. *P < 0.05.

The absolute baseline CVC, calculated as laser Doppler flux × mmHg−1, for each MD fibre site is illustrated in Table 2. There were no significant differences in baseline CVC between age groups or between drug treated fibre sites and the control site (P < 0.05).

Table 2.

Baseline absolute cutaneous vascular conductance values

Control BH4 Tyrosine BH4+ tyrosine
Baseline
Y 0.23 ± 0.03 0.30 ± 0.06 0.19 ± 0.03 0.36 ± 0.06
O 0.22 ± 0.03 0.28 ± 0.04 0.25 ± 0.02 0.36 ± 0.07
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Baseline CVC values, calculated as laser Doppler flux × mmHg−1, are means ±s.e.m. for each MD fibre site in young (Y) and older (O) subjects.

The effect of tyrosine and BH4 on adrenergic vasoconstrictor function during whole-body cooling (Tsk= 30.5°C) is illustrated in Fig. 1A. Compared to young subjects, older subjects exhibited a blunted vasoconstrictor response to cooling at control sites (young: −39 ± 3, older: −17 ± 3%ΔCVCbase; P < 0.01). Localized administration of BH4 significantly augmented vasoconstriction in older subjects (−36 ± 3%ΔCVCbase; P < 0.01), but had no effect in young subjects (−37 ± 3%ΔCVCbase; P= 0.82). Localized tyrosine also augmented vasoconstriction in older (−35 ± 3%ΔCVCbase; P < 0.01) but not in young subjects (−38 ± 4%ΔCVCbase; P= 0.87). Similar to the other experimental sites, coadministration of BH4 with tyrosine enhanced vasoconstriction in older (−34 ± 3%ΔCVCbase; P < 0.01) but not young subjects (−39 ± 4%ΔCVCbase; P= 0.96). However, the BH4+ tyrosine site did not further augment the vasoconstrictor response more than either BH4 (young: P= 0.78, older: P= 0.69) or tyrosine (young: P= 0.83, older: P= 0.86) alone.

Figure 1. Average change in maximal cutaneous vasoconstriction in response to whole-body cooling (Tsk= 30.5°C) and following 20 min infusion of 1 mm tyramine at each microdialysis site in young and older subjects.

Figure 1

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The effects of whole-body cooling (A) and tyramine (B) at control, BH4, tyrosine, and BH4+ tyrosine (combo) pretreated sites. n= 20 (10 young, 10 older) subjects. *P < 0.05 versus control; †P < 0.05 versus young.

Similar to whole-body cooling, older subjects exhibited an attenuated vasoconstrictor response to local tyramine infusion at control sites (young: −41 ± 4, older: −21 ± 4%ΔCVCbase; P < 0.01) (Fig. 1B). Localized BH4 supplementation enhanced vasoconstriction in older subjects (−35 ± 3%ΔCVCbase; P < 0.02) but not in young subjects (−41 ± 2%ΔCVCbase; P= 0.94). Localized tyrosine also augmented vasoconstriction in older (−44 ± 4%ΔCVCbase; P < 0.01) but not young subjects (−40 ± 4%ΔCVCbase; P= 0.95). The increase in vasoconstriction with tyrosine was not greater than the increase in vasoconstriction observed with BH4 in older subjects (P= 0.13). Compared to the control site, coinfusion of BH4 with tyrosine enhanced the vasoconstrictor response in older (−37 ± 6%ΔCVCbase; P < 0.01) but not young subjects (−37 ± 3%ΔCVCbase; P= 0.57). However, the BH4+ tyrosine site did not further augment vasoconstriction more than BH4 (Y: P= 0.52, O: P= 0.75) or tyrosine (Y: P= 0.62, O: P= 0.23) alone.

In Fig. 2, the CVC response at every 0.5°C decrease in mean Tsk during whole-body cooling is illustrated. Throughout cooling, the drug treated sites did not differ from the control site in young subjects (Fig. 2A). Compared to young subjects, older subjects exhibited a blunted vasoconstrictor response in the control site (mean Tsk < 32.5°C; P < 0.05). In older subjects (Fig. 2B), the vasoconstrictor response was augmented at the drug treated sites, which was significant at mean Tsk≤ 32.0°C (BH4, BH4+ tyrosine) and mean Tsk≤ 33.0°C (tyrosine; P < 0.05). No differences were observed between the drug-treated sites in older subjects.

Figure 2. CVC responses to 0.5°C incremental decreases in skin temperature during whole-body cooling in young and older subjects.

Figure 2

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A, young subjects (n= 10); B, older subjects (n= 10). Vasoconstriction was augmented at the drug treated sites in older individuals (panel B), which was significant at mean Tsk≤ 32.0°C (BH4, BH4+ tyrosine) and mean Tsk≤ 33.0°C (tyrosine). *P < 0.05 versus control; †P < 0.05 versus young.

During infusion of a supraphysiological concentration (1 × 10−2m) of noradrenaline (Fig. 3), there were no differences in noradrenaline-mediated vasoconstriction between control (Y: −70 ± 5, O: −67 ± 4%ΔCVCbase; P= 0.52), BH4 (Y: −74 ± 3, O: −66 ± 5%ΔCVCbase; P= 0.14), tyrosine (Y: −64 ± 5, O: −73 ± 4%ΔCVCbase; P= 0.12), and BH4+ tyrosine (Y: −71 ± 6, O: −74 ± 4%ΔCVCbase; P= 0.57) sites.

Figure 3. Maximal vasoconstriction induced following a supraphysiological dose (1 × 10−2m) of noradrenaline at each microdialysis site in young and older subjects.

Figure 3

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The effects of noradrenaline at control, BH4, tyrosine, and BH4+ tyrosine (combo) pretreated sites. n= 20 (10 young, 10 older) subjects. *P < 0.05 versus control; †P < 0.05 versus young.

Discussion

The primary finding from this study was that both local tyrosine and BH4 administration augmented the vasoconstrictor response induced by either whole-body cooling or tyramine infusion in aged, but not in young, skin. Similar to BH4, localized tyrosine supplementation offset the age-associated decrement in cutaneous vasoconstriction. Furthermore, perfusing tyrosine and BH4 concomitantly did not have an additive effect on the cutaneous vasoconstrictor response in older subjects. Lastly, neither tyrosine nor BH4 affected the vasoconstrictor response to an exogenous supraphysiological dose of noradrenaline, which suggests that the putative effects of these compounds were localized to peripheral nerve terminals. In summary, these data suggest that the synthesis of noradrenaline in response to a physiological or pharmacological stimulus is compromised in older subjects and this may be occurring at the tyrosine hydroxylase, or rate-limiting, step in the biosynthetic pathway. Consequently, blunted noradrenaline synthesis and axonal release from sympathetic nerves are likely to contribute to the attenuated cutaneous vasoconstriction observed in older subjects.

These data corroborate previous findings in our laboratory indicating that BH4 augments reflex- and tyramine-induced vasoconstriction in aged skin even after controlling for the putative effects of BH4 on nitric oxide synthase (Lang et al. 2009a). Additionally, BH4 did not affect cotransmitter- or noradrenaline (1 × 10−2m)-mediated vasoconstriction suggesting that its effects were adrenergically mediated and localized to peripheral nerve terminals. Using a similar experimental design, the current study demonstrates that tyrosine also selectively augments cutaneous vasoconstriction in aged skin. Reduced noradrenaline biosynthesis due to diminished tyrosine and BH4 bioavailability is likely to contribute to the attenuated vasoconstrictor response in aged skin.

Catecholamine synthesis and storage requires functionally active tyrosine hydroxylase. To this end, both BH4 and tyrosine must be present in sufficient concentrations to maintain optimal tyrosine hydroxylase function (Kumer & Vrana, 1996; Dunkley et al. 2004). BH4 is a critical cofactor in this process because it catalytically activates the enzyme and enables hydroxylation of the amino acid substrate tyrosine (Kumer & Vrana, 1996; Dunkley et al. 2004; Thony et al. 2008). In response to cold exposure, the affinity of tyrosine hydroxylase for BH4 is considerably enhanced, thereby augmenting noradrenaline biosynthesis (Weiner, 1978; Fluharty et al. 1985; Kumer & Vrana, 1996). However, in aged sympathetic ganglia, markedly reduced noradrenaline fluorescence has been reported (Hervonen et al. 1978; Santer, 1979). Furthermore, the number of transporters for noradrenaline in synaptomsomes declines with age (Snyder et al. 1998). Thus, the present data support the hypothesis that the apparent age-associated deficits in noradrenaline synthesis and storage may be secondary to reduced precursor substrates and cofactors. Alternatively, it is possible that a chronic and compensatory increase in tyrosine hydroxylase expression occurs in aged skin and thus, adding BH4 or tyrosine may in fact augment the noradrenergic component above that observed in young skin.

It is plausible that reduced BH4 and tyrosine bioavailability may be secondary to the globalized elevation in oxidative and nitrosative stress associated with ageing (Finkel & Holbrook, 2000). Although BH4 has not been directly measured in human skin, its concentration is diminished in other aged tissues and this has been linked to oxidative stress at various points along the BH4 biosynthetic and salvage pathways (Williams et al. 1980; Moens & Kass, 2007; Delp et al. 2008). Tyrosine bioavailability may be decreased by its oxidation to tyrosyl radical. Peroxynitrite or its highly reactive degradation products may convert free tyrosine to an inactive noradrenaline substrate (Ischiropoulos et al. 1995; Reiter et al. 2000; Kochman et al. 2002). For example, in human skin 3-nitrotyrosine is elevated in photoaged skin and can increase acutely in response to oxidative stress generated from ultraviolet light exposure (Nishigori et al. 2003). Thus, elevated peroxynitrite in aged skin may reduce the tyrosine pool available for catecholamine production.

In addition to oxidative stress, tyrosine bioavailability may be diminished due to increased tonic noradrenaline release in aged skin (Seals & Dinenno, 2004). At rest, tyrosine is not thought to be limiting because its concentration is well above the substrate Km of tyrosine hydroxylase (Fernstrom, 1983). Moreover, plasma tyrosine concentrations appear to be unaltered with age (Caballero et al. 1991). However, in animal studies tyrosine supplementation augments the concentration of the noradrenaline precursor, l-DOPA, in activated neurons (Wurtman et al. 1974; Iuvone et al. 1978; Fernstrom, 1983; Fernstrom et al. 1986). This suggests that upon neuronal activation tyrosine concentration in the vicinity of tyrosine hydroxylase may be well below saturation, and in the case of aged skin, this may be compounded by elevated oxidative stress. The cumulative effects of oxidative stress and elevated tonic noradrenaline synthesis may deplete tyrosine in aged skin, thereby limiting noradrenaline biosynthesis and consequently the ability to sustain a significant vasoconstrictor response during cold exposure.

In addition to prejunctional noradrenergic mechanisms, attenuated cutaneous vasoconstrictor function in aged skin may be explained by multiple deficits along the efferent arm of the sympathetic reflex including, (1) absent functional contribution of coreleased sympathetic neurotransmitters, putatively ATP and NPY (Thompson & Kenney, 2004), (2) reduced adrenoreceptor responsivity (Thompson et al. 2005), and (3) altered downstream vascular signalling mechanisms (Lang et al. 2009b). Furthermore, although the apparent effects of BH4 and tyrosine are likely to be isolated to tyrosine hydroxylase in sympathetic nerve terminals, additional effects on end-organ responsiveness cannot be ruled out. In the present study, vasoconstriction induced by a supraphysiological dose of noradrenaline was not different between control and drug treatment sites suggesting that neither BH4 nor tyrosine significantly influenced end-organ responsivity; however, further investigation is required to assess whether this is consistent with more physiological concentrations of noradrenaline.

We also investigated the combined role of BH4 and tyrosine on vasoconstrictor function. Contrary to our hypothesis, BH4 did not additionally enhance vasoconstrictor function when administered with tyrosine. Thus, administering either BH4 or tyrosine appears sufficient to augment vasoconstrictor function in older adults during sympathetic activation. This presumably occurs through two different mechanisms acting on tyrosine hydroxylase, (1) greater cofactor increases the amount of catalytically active enzyme and (2) greater substrate increases the saturation of enzyme that is already active. Alternatively, it is possible that cooling of greater duration or intensity is required to observe additive effects of BH4 and tyrosine. Moreover, a basement effect may have occurred where the signal gain is minimized to a point where no detectable changes in the combination site could be observed. However, further reduction of the LDF signal occurred with exogenous noradrenaline infusion.

Limitations

Because tyramine was perfused after whole-body cooling, it is possible that an order effect may influence the interpretation of the tyramine data. However, the large dose of tyramine that was administered would be likely to reduce this effect and preclude the discrimination between releasing noradrenaline as opposed to coreleased neuropeptides. Additionally, the functional response to tyrosine and BH4 were assessed in this study; not noradrenaline release per se. Thus, the existence of BH4- or tyrosine-mediated postjunctional or vascular effects cannot be discounted. However, to our knowledge, there is no known postjunctional mechanism for tyrosine. In contrast, BH4 functions as a cofactor for NOS and may have an antioxidant effect, both of which would putatively diminish the vasoconstrictor response. However, this was not apparent in the present study.

In summary, local supplementation of either tyrosine or BH4 resolved the compromised adrenergic vasoconstrictor response to both cooling and local tyramine infusion in aged skin. However, combined tyrosine and BH4 infusion did not further enhance cutaneous vasoconstriction. Additionally, vasoconstriction to a supraphysiological dose of noradrenaline was not affected by either tyrosine or BH4 indicating that adrenoreceptor sensitivity was unaltered by these compounds. These results suggest that optimal tyrosine hydroxylase function is required to fully express the cutaneous vasoconstrictor response, and that reduced functional substrate and cofactor for tyrosine hydroxylase contribute to the attenuated vasoconstriction in aged skin.

Acknowledgments

The authors gratefully acknowledge the subjects who participated in this study. We also specially thank Jane Pierza, Rebecca Bruning, Anna Stanhewicz and John Jennings for their technical and data collection assistance and the General Clincial Research Center (GCRC) for medical assistance. This research was supported by the National Institutes of Health grants RO1-AG-07004-19 and GCRC M01RR10732.

Glossary

Abbreviations

BH4

tetrahydrobiopterin

CVC

cutaneous vascular conductance

LDF

laser Doppler flowmetry

MAP

mean arterial pressure

MD

microdialysis

NOS

nitric oxide synthase

NPY

neuropeptide Y

O

older

Y

young

Author contributions

L.A.H. and W.L.K. both contributed to the design of experiments, the collection and interpretation of results and the drafting and revising of the manuscript. This study was completed at Penn State University and this manuscript, in its final form, has been approved by all authors.

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