Experimental Physiology

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1 674 Exp Physiol 96.7 pp Research Paper Intradermal microdialysis of hypertonic saline attenuates cutaneous vasodilatation in response to local heating Jennifer J. DuPont, William B. Farquhar and David G. Edwards Department of Kinesiology and Applied Physiology, University of Delaware, Newark, DE, USA Experimental Physiology We tested the hypothesis that microdialysis of hypertonic saline would attenuate the skin blood flow response to local heating. Seventeen healthy subjects (23 ± 1 years old) were studied. In one group (n = 9), four microdialysis fibres were placed in the forearm skin and infused with the following: (1) Ringer solution; (2) normal saline (0.9% NaCl); (3) hypertonic saline (3% NaCl); and (4) 10 mm l-name. A second group (n = 8) was infused with the following: (1) normal saline; (2) hypertonic saline; (3) normal saline + l-name; and (4) hypertonic saline + l-name. Red blood cell flux was measured via laser Doppler flowmetry during local heating to 42 C. Sitespecific maximal vasodilatation was determined by infusing 28 mm sodium nitroprusside while the skin was heated to 43 C. Data were expressed as the percentage of maximal cutaneous vascular conductance (%CVC max ). The local heating response at the Ringer solution and normal saline sites did not differ (n = 9; initial peak Ringer solution, 69 ± 6 versus normal saline, 66 ± 2%CVC max ; plateau Ringer solution, 89 ± 4 versus normal saline, 89 ± 5%CVC max ). Hypertonic saline reduced the initial peak (n = 9; normal saline, 66 ± 2 versus hypertonic saline, 54 ± 4%CVC max ; P < 0.05) and plateau (normal saline, 89 ± 5 versus hypertonic saline, 78 ± 2%CVC max ; P < 0.05) compared with normal saline. Plateau %CVC max was attenuated to a similar value at the normal saline + l-name and hypertonic saline + l-name sites (n =8; normal saline + l-name, 39 ± 6 and hypertonic saline + l-name, 39 ± 5%CVC max ). The nitric oxide contribution (plateau %CVC max l-name plateau %CVC max ) was lower at the hypertonic saline site (normal saline, 55 ± 6 versus hypertonic saline, 35 ± 4; P < 0.01). These data suggest an effect of salt on the cutaneous response to local heating, which may be mediated through a decreased production and/or availability of nitric oxide. (Received 11 March 2011; accepted after revision 11 May 2011; first published online 13 May 2011) Corresponding author D. G. Edwards: Department of Kinesiology and Applied Physiology, 541 South College Avenue, 142 HPL, Newark, DE 19716, USA. dge@udel.edu There is evidence for a role of excess dietary salt consumption in the development of hypertension, as well as other cardiovascular diseases (Meneton et al. 2005). An early study in rats found that a high-salt diet results in accelerated arteriosclerosis as well as renal damage and increased mortality (Meneely & Ball, 1958). Considering that modern humans consume many times more salt on a daily basis than our pre-agricultural ancestors (Frassetto et al. 2001), understanding the effects of salt on the cardiovascular system has important clinical and public health implications. In humans, the adverse effects of salt are typically attributed to increased blood pressure; however, at least in rodents, a high-salt diet has been shown to impair endothelial function without altering resting blood pressure (Lenda et al. 2000; Lenda & Boegehold, 2002; Nurkiewicz & Boegehold, 2007; Zhu et al. 2007). These findings in rodents suggest that salt may alter vascular function independent of blood pressure. Flow-mediated dilatation is often used to assess human vascular function in conduit arteries and has been shown to be largely mediated by endothelium-derived nitric oxide (NO; Thijssen et al. 2011). Improved brachial artery flowmediated dilatation has been demonstrated in overweight and obese normotensive humans when they are switched from a diet containing a usual amount of salt to a low-salt diet (Dickinsonet al. 2009). The changes observed in that study were associated with a reduction in blood pressure; DOI: /expphysiol

2 Exp Physiol 96.7 pp Hypertonic saline and local heating of the skin 675 hence, in that study, the beneficial effects of reducing salt intake on endothelial function cannot be separated from the effects of blood pressure, because blood pressure also declined. Likewise, a low-salt diet was shown to be associated with a higher flow-mediated dilatation in older adults with elevated systolic blood pressure (Jablonski et al. 2009). Thus, the effect of salt on the vasculature in normotensive humans independent of blood pressure is not fully understood. Alterations in microvascular function may occur early in the progression of cardiovascular disease (Levy et al. 2001; Cohuet & Struijker-Boudier, 2006; Holowatz et al. 2008). Therefore, understanding the effect of salt on the microvasculature may provide insight into early vascular pathological consequences. The cutaneous microcirculation has been proposed as an accessible vascular bed for the assessment of in vivo human microvascular function and may provide insight into systemic vascular function (Abularrage et al. 2005; Rossi et al. 2006; Holowatz et al. 2008). The purpose of the present study was to determine the acute effects of salt on the cutaneous microvasculature in normotensive humans when blood pressure is unaltered. We used intradermal microdialysis of hypertonic saline (3% NaCl) coupled with laser Doppler flowmetry to assess cutaneous vasodilatation in response to local heating. Local heating of the skin results in an initial peak in cutaneous blood flow largely due to an axon reflex (Minson et al. 2001) through the activation of transient receptor potential vanilloid type-1 (TRPV-1) channels (Wong & Fieger, 2010), followed by a secondary plateau that is largely mediated by NO (Kellogg et al. 1999; Minson et al. 2001), which is synthesized by endothelial nitric oxide synthase (enos; Kellogg et al. 2008, 2009). Our experiments allowed for examination of the acute effect of salt on local microvascular function without potentially confounding systemic effects. We hypothesized that microdialysis of hypertonic saline would attenuate the secondary plateau in cutaneous vasodilatation elicited by local heating. Furthermore, we hypothesized that any observed attenuation in the plateau would be the result of a reduction in the NO contribution to dilatation. Methods Subjects Seventeen healthy subjects (23± 1 years old; 12 male and five female) were studied. All subjects were healthy, non-obese, normotensive, non-smokers, and not taking any medications that may alter cardiovascular function. Females were studied during the early follicular phase of the menstrual cycle to control for any effects of the menstrual cycle on the skin blood flow response to local heating. The study was approved by the University of Delaware Institutional Review Board and conformed to the guidelines set out by the Declaration of Helsinki. Written and verbal consent was obtained from each subject. Instrumentation Subjects arrived at the laboratory and were instrumented with four microdialysis (MD) fibres (10 mm, 30 kda cutoff membrane, MD 2000; Bioanalytical Systems, West Lafayette, IN, USA) in the ventral side of the nondominant forearm using sterile technique. Ice was applied to the forearm for 10 min prior to fibre insertion to provide temporary anaesthesia to the area (Hodges et al. 2009). A 25 gauge needle was then inserted into the skin, with entry and exit points 2.5 cm apart. Microdialysis fibres were threaded through the lumen of the needle, which was removed once the semi-permeable membrane of the fibre was in place. The fibres were secured with tape, and lactated Ringer solution was infused at 2 μlmin 1 (Bee Hive controller, Baby Bee microinfusion pumps; Bioanalytical Systems) for min, allowing for local hyperaemia from needle insertion trauma to subside. Cutaneous red blood cell (RBC) flux, an index of skinbloodflow,wasmeasuredfrom1.5mm 2 of skin with a multifibre laser Doppler probe placed in a local heater (MoorLAB, Temperature Monitor SH02; Moor Instruments, Axminster, UK) on the skin directly above each MD membrane. A blood pressure cuff was placed on the contralateral arm for measurement of blood pressure via an automated oscillometric sphygmomanometer (Dinamap Dash 2000; GE Medical Systems, Waukesha, WI, USA). Experimental protocol We used a standard non-painful heating protocol (Kellogg et al. 1999; Minson et al. 2001) to test the hypothesis that hypertonic saline attenuates cutaneous vasodilatation. Upon resolution of hyperaemia in the subject s arm, we set the local heaters to 33 C. The first group of subjects (n =9, five male and four female) were instrumented with four MD fibres, and each MD site was randomly assigned to receive one of the following: (1) lactated Ringer solution; (2) normal saline solution (0.9% NaCl); (3) hypertonic saline solution (3% NaCl); or (4) 10 mml-name (Sigma). These experiments allowed us to determine the effect of hypertonic saline on the cutaneous response to local heating as well as to compare two control substances (Ringer solution and normal saline) typically used in local heating experiments. A second group of subjects (n = 8, seven male and one female) were instrumented with four MD fibres, and each MD site was randomly assigned to receive one of the following: (1) normal saline;

3 676 J. J. DuPont and others Exp Physiol 96.7 pp Table 1. Parameter Subject characteristics Value Sex 12 male, 5 female Age (years) 23 ± 1 Body mass index (kg m 2 ) 25.9 ± 1 Systolic blood pressure (mmhg) 115 ± 2 Diastolic blood pressure (mmhg) 67 ± 2 Mean arterial pressure (mmhg) 84 ± 2 Table 2. Blood pressure during local heating experiments Blood pressure (mmhg) Baseline Initial peak Nadir Plateau Systolic 116 ± ± ± ± 3 Diastolic 67 ± 2 65 ± 2 68 ± 2 68 ± 2 Mean arterial 83 ± 2 82 ± 2 84 ± 2 83 ± 2 (2) hypertonic saline; (3) normal saline + L-NAME; or (4) hypertonic saline + L-NAME.Testingthesecond group of subjects by co-infusing L-NAME with hypertonic saline or normal saline allowed us to determine the nitric oxide contribution at the normal saline and hypertonic saline sites. Microdialysis sites were infused at 2 μlmin 1 for at least a 30 min baseline period. Blood pressure was measured every 10 min throughout. Following baseline measurements, the temperature of the local heating units was increased by 0.5 Cevery5stoatemperatureof 42 C, where they were held constant throughout the local heating protocol. The initial peak in cutaneous blood flow occurs in the first 10 min and is largely due to an axon reflex (Minson et al. 2001) via activation of TRPV1 channels on sensory fibres (Wong & Fieger, 2010). Following the nadir, a secondary plateau in cutaneous blood flow occurs after approximately 30 min more of heating and is largely mediated by NO (Kellogg et al. 1999; Minson et al. 2001). Once RBC flux reached a stable plateau (approximately min), we set the local heaters to 43 C and infused 28 mm sodium nitroprusside (SNP; Nitropress; Hospira, Inc., Lake Forest, IL, USA) in all four sites at a rate of 2 μlmin 1 to determine maximal cutaneous vasodilatation. A dose of 10 mm L-NAME was chosen because this dose has been shown to sufficiently inhibit nitric oxide synthase in the skin (Minson et al. 2001). A dose of 28 mm SNP was chosen because this dose has been shown to elicit maximal vasodilatation in the skin (Minson et al. 2001). The L-NAME and SNP were mixed just prior to use. All pharmacological substances were sterilized using syringe microfilters (Whatman Puradisc 13 mm Syringe Filters, Florham Park, NJ, USA). Data analysis Data were collected at 40 Hz using a National Instruments PCI-6221 DAQ board and LabView software (National Instruments Corp., Austin, TX, USA). Blood pressure was used to calculate cutaneous vascular conductance (CVC) as RBC flux/mean arterial blood pressure. Baseline and plateau CVC were obtained over a stable 10 min period. Initial peak and nadir CVC were calculated by averaging the highest and lowest values, respectively, over a stable 60 s period. All data are expressed as a percentage of maximal CVC obtained during SNP infusion (%CVC max ). We calculated the NO contribution by subtracting the normal saline + L-NAME plateau from the normal saline plateau %CVC max and the hypertonic saline + L-NAME plateau from the hypertonic saline plateau %CVC max.we performed a one-way ANOVA to test for differences in %CVC max across treatment sites and used Tukey s post hoc test to determine differences between treatment sites when appropriate. We also used Student s paired t test to compare the percentage NO contribution to the plateau between normal and hypertonic saline. The value of α was set at 0.05, and values are expressed as means ± SEM. Figure 1. Baseline, initial peak, nadir and plateau percentage of maximal cutaneous vascular conductance at Ringer solution and normal saline sites in response to local heating (n = 9) Values are means ± SEM. No differences were observed between Ringer solution and normal saline. Results Subject characteristics are presented in Table 1. Subjects were young, healthy, normotensive and non-obese. Blood pressure was unaltered during the local heating experiments, as shown in Table 2. Skin blood flow responses to local heating, expressed as %CVC max,were not different between Ringer solution and normal saline in the group of subjects receiving both control substances (baseline Ringer solution, 11 ± 6; baseline normal saline, 7 ± 4; initial peak Ringer solution, 69 ± 6; initial peak normal saline, 66 ± 2; nadir Ringer solution, 61 ± 8; nadir normal saline, 56 ± 5; plateau Ringer solution, 89 ± 4; and plateau normal saline, 89 ± 5; Fig. 1).

4 Exp Physiol 96.7 pp Hypertonic saline and local heating of the skin 677 Inthefirstgroupofsubjects(n = 9), baseline %CVC max did not differ across the three MD sites (normal saline, 7 ± 4; hypertonic saline, 7 ± 1; and L-NAME, 6 ± 1). Initial peak %CVC max was significantly lower at the hypertonic saline site compared with the normal saline site (normal saline, 66 ± 2; hypertonic saline, 54 ± 4; P < 0.05; Fig. 2B). We also infused L-NAME at one site. Initial peak %CVC max did not differ between hypertonic saline and L-NAME (54 ± 5%; P > 0.05 versus hypertonic saline); however, L-NAME attenuated the initial peak %CVC max compared with normal saline (P < 0.01 versus normal saline). The plateau %CVC max was significantly attenuated at the hypertonic saline site compared with the normal saline site (normal saline, 89 ± 5; hypertonic saline, 78 ± 2; P < 0.05; Fig. 2B). The L-NAME significantly reduced plateau %CVC max compared with both normal saline and hypertonic saline sites (normal saline + L- NAME, 42 ± 5; P < 0.001), confirming that a significant part of the plateau was NO mediated. In the second group of eight subjects, the initial peak %CVC max was attenuated to a similar level in the normal saline + L-NAME and hypertonic saline + L- NAME sites (normal saline + L-NAME, 48 ± 7; hypertonic saline + L-NAME, 48 ± 4; P < 0.01 versus normal saline, 71 ± 4). There were no significant differences in the initial peak %CVC max between the two L-NAME sites and the hypertonic saline site (hypertonic saline, 56 ± 3; P = 0.50). The plateau %CVC max was significantly attenuated at the hypertonic saline site compared with the normal saline site, similar to our findings in the first group of subjects (normal saline, 87 ± 2; hypertonic saline, 73 ± 5; P < 0.05; Fig. 3). The %CVC max was attenuated to a similar level in both L-NAME sites (normal saline + L- NAME, 39 ± 6; hypertonic saline + L-NAME, 39 ± 5; both P < 0.01 versus normal saline and hypertonic saline sites, respectively; P > 0.05 versus each other). The NO contribution to cutaneous vasodilatation was significantly lower at the hypertonic saline site (normal saline, 55 ± 6 versus hypertonic saline, 35 ± 4; P < 0.05; Fig. 3). Absolute maximal CVC was not different between the normal saline and hypertonic saline sites across all 17 subjects (normal saline, 2.22 ± 0.22; hypertonic saline, 2.22 ± 0.22 a.u.), indicating that the maximal dilatory capacity of the skin vasculature was not affected by hypertonic saline. Discussion The purpose of this study was to determine the effects of intradermal microdialysis of hypertonic saline on cutaneous vasodilatation in response to local heating in normotensive adults. Hypertonic saline attenuated both the initial peak and the plateau phase in response to local heating. When L-NAME was co-infused during local heating, it reduced the plateau to a similar level in the normal saline- and hypertonic saline-infused sites, Figure 2 Local heating response during infusion of normal and hypertonic saline A, representative response to local heating at normal saline and hypertonic saline sites. B, baseline, initial peak, nadir and plateau percentage of maximal cutaneous vascular conductance at the normal saline and hypertonic saline sites in response to local heating (n = 9). Values are means ± SEM. P < 0.05 versus normal saline. Figure 3. Plateau percentage of maximal cutaneous vascular conductance (%CVC max ) in the normal saline and hypertonic saline sites, plateau %CVC max in the normal saline + L-NAME and hypertonic saline + L-NAME sites, and NO contribution at the normal saline and hypertonic saline sites (n = 8) Values are means ± SEM. P < 0.01 versus normal saline.

5 678 J. J. DuPont and others Exp Physiol 96.7 pp indicating that hypertonic saline resulted in a decreased contribution of NO to cutaneous vasodilatation. As these effects occurred without any alteration in systemic blood pressure (due to the use of local infusion), these findings suggest that an increase in interstitial salt concentration may have an effect on microvascular function independent of blood pressure, which may be mediated through a decreased production and/or availability of NO. The plateau phase of dilatation in response to local heating is largely mediated by NO (Kellogg et al. 1999; Minson et al. 2001) and has been shown to be mediated by enos (Kellogg et al. 2008), while others have reported a role for neuronal NOS in postural tachycardia syndrome (Stewart et al. 2007). We found that hypertonic saline reduced the NO contribution to the plateau phase of dilatation in response to local heating. We did not hypothesize that hypertonic saline would reduce the initial peak in %CVC max. The initial increase in skin blood flow in response to local heating is largely due to an axon reflex (Minson et al. 2001, 2002) via activation of TRPV1 channels (Wong & Fieger, 2010); however, a portion can be attenuated by NOS antagonism (Minson et al. 2001, 2002). Interestingly, we found that co-infusion of L-NAME resulted in similar initial peak responses in the presence of hypertonic saline and normal saline, indicating that hypertonic saline probably reduced the initial peak by reducing the NO contribution to this response as well. Alternatively, hypertonic saline may have impaired sympathetic vasoconstrictor nerve function, which is required for full expression of the skin blood flow response to local heating, in particular the axon reflex (Houghton et al. 2006; Hodges et al. 2008). The local infusion of hypertonic saline in the present study is likely to have resulted in a large increase in local sodium concentration, but may nevertheless have relevance to the deleterious effects of dietary salt. For example, impaired arteriolar responses to acetylcholine, an endothelium-dependent dilator, have been reported in mice fed a high-salt diet compared with mice fed a lowsalt diet (Nurkiewicz & Boegehold, 2007). Additionally, an improvement in brachial artery flow-mediated dilatation wasobservedinanoverweightandobesenormotensive population when switched from a diet containing a usual amount of salt to a low-salt diet (Dickinson et al. 2009). The acute effects of local hypertonic saline infusion on cutaneous vasodilatation in the present study are consistent with these studies. Dietary sodium reduction appears to reduce the occurrence of cardiovascular diseases in humans (Cook et al. 2007). The long-term follow up of two intervention trials designed to reduce dietary sodium found a 25% reduction in cardiovascular events despite very small changes in systolic and diastolic blood pressure (less than 2 mmhg; Cook et al. 2007). Importantly, the reduction in NO-mediated cutaneous vasodilatation in the present study occurred in the absence of systemic blood pressure changes due to the local infusion of hypertonic saline, suggesting that there may be an independent effect of salt on the vasculature. An excess intake of dietary salt can potentially increase plasma sodium levels from 2 to 4 mmol L 1 in both normotensive and hypertensive humans (de Wardener et al. 2004). The local infusion of hypertonic saline is likely to increase interstitial sodium concentration, but the mechanism by which it may be altering NO bioavailability is unknown. An increase in sodium concentration has been shown to alter the stiffness and deformability of endothelial cells when cultured with physiological levels of aldosterone (Oberleithner et al. 2007). In these conditions, a reduction in NO release was observed in the high-sodium-treated endothelial cells. This effect was dependent on physiological levels of aldosterone, which activates epithelial sodium channels on endothelial cells. Oberleithner et al. (2007) proposed that an acute rise in sodium concentration leads to an increase in endothelial stiffness and a loss of the deformability potential of the cell, which leads to reduced NO release. Dietary salt loading has also been shown to downregulate renal and vascular NOS expression (Ni & Vaziri, 2001), as well as to decrease the conversion of L-arginine to NO (Higashi et al. 1996) and upregulate the endogenous NOS inhibitor, asymmetrical dimethyl-l-arginine (Fujiwara et al. 2000). One potential mechanism for the acute decrease in NO-mediated dilatation observed in the present study is increased reactive oxygen species. Nitric oxide and superoxide react in a diffusion-limited reaction to form peroxynitrite (OONO ), which is a strong oxidant (Munzel et al. 1997). Increased superoxide and peroxynitrite can also lead to oxidation of tetrahydrobiopterin (BH 4 ), a critical cofactor for NO synthesis (Milstien & Katusic, 1999; Landmesser et al. 2003). Oxidation of BH 4 leads to an uncoupling of enos, which results in production of superoxide by enos instead of NO (Vasquez-Vivar et al. 2002; Landmesser et al. 2003). As previously stated, several studies in rodents have demonstrated that endothelial function is impaired by a high-salt diet without a change in blood pressure (Lenda et al. 2000; Lenda & Boegehold, 2002; Nurkiewicz & Boegehold, 2007; Zhu et al. 2007). The mechanism for impaired NO release and endothelialdependent dilatation has been shown via an increase in reactive oxygen species (Lenda et al. 2000), specifically superoxide (Nurkiewicz & Boegehold, 2007; Zhu et al. 2007). The source of superoxide production appears to be NAD(P)H oxidase (Lenda et al. 2000; Zhu et al. 2007), xanthine oxidase (Lenda et al. 2000; Zhu et al. 2007) and enos (Nurkiewicz & Boegehold, 2007). Future studies are warranted to determine whether the reduced NO contribution to cutaneous vasodilatation as a result of hypertonic saline observed in the present study is mediated by reactive oxygen species.

6 Exp Physiol 96.7 pp Hypertonic saline and local heating of the skin 679 As hypertonic saline was delivered via intradermal microdialysis into the interstitial space, an alternative mechanism by which cutaneous vasodilatation is impaired by hypertonic saline may be mediated by a shift in fluid from the intravascular space to the interstitial space. Sodium and water can diffuse rapidly between the interstitial space and the intravascular space, so sodium concentrations should eventually reach equilibrium. In spite of this, the continuous infusion (2 μlmin 1 )of hypertonic saline could have caused an acute shift in fluid from the intravascular space to the interstitial space that may have reduced local blood flow and contributed to the observed reduction in vasodilatation. However, we found that maximal dilatation (raw CVC max ) was similar at normal saline and hypertonic saline sites, indicating that the capacity of the vessels to vasodilate was not affected by the infusion of hypertonic saline; however, future studies could be designed to determine the effects of acutely changing interstitial tonicity alone on the cutaneous response to local heating. A limitation of the present study is that the local infusion of hypertonic saline is likely to have resulted in a greater local sodium concentration compared with a chronic high-salt diet; therefore, we do not know whether a high-salt diet would have similar effects. Furthermore, the local nature of our experiments controlled for systemic neuroendocrine effects of sodium; however, these effects may be important in vascular control. Therefore, future studies using dietary salt manipulation or intravenous infusion of hypertonic saline are warranted. 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