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1 J Physiol (2011) pp Effects of cyclooxygenase inhibition on vascular responses evoked in fingers of men and women by iontophoresis of α 1 -andα 2 -adrenoceptor agonists Amar Srinivasa and Janice M. Marshall School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK The Journal of Physiology Non-technical summary Sympathetic nerve fibres in the fingers release noradrenaline, which has the potential to act on α 1 -orα 2 -adrenoreceptors on the blood vessels. We used iontophoresis, which generates a tiny electrical charge, to push selective α 1 -orα 2 -receptor agonists through the skin and recorded changes in finger blood flow before and after aspirin, which inhibits cyclooxygenase (COX), an enzyme which synthesises vasodilator and vasoconstrictor prostaglandins. Our results yielded the novel findings that finger vasoconstriction produced by α 1 -adrenoceptors is blunted by locally synthesised vasodilator COX products in young men and in young women in the high but not the low oestrogen phase of the menstrual cycle. By contrast, finger vasoconstriction evoked by α 2 -adrenoceptors is largely attributable to vasoconstrictor COX products in young men, but overcome by vasodilator COX products in young women. This provides a foundation for testing whether COX products similarly modify vasoconstriction evoked by changes in sympathetic nerve activity. Abstract In 10 men and nine women aged years, we aimed to establish whether endogenous prostanoids synthesised by cyclooxygenase (COX) affect responses evoked in the finger by α 1 -or α 2 -adrenoceptor agonists. Cutaneous red cell flux (crcf) was recorded in dorsal finger during iontophoresis of phenylephrine (PE) or clonidine (0.5 mm, seven 0.1 ma pulses followed by one 0.2 ma pulse: 30 s each at 60 s intervals) before and after the COX inhibitor aspirin (600 mg p.o.). In men, PE evoked a biphasic mean increase/decrease in crcf before but a monophasic mean decrease in crcf of 30 40% after aspirin (P < 0.05). In women in the low oestrogen (E 2 ) phase of the menstrual cycle, PE evoked a decrease in crcf (30 40%; P < 0.05) that was unchanged by aspirin, whereas in the high E 2 phase, PE evoked no change before but a graded decrease in crcf (30 40%;P < 0.05) after aspirin. Clonidine evoked a decrease in crcf ( 30%; P < 0.05) in men before, but not after, aspirin. Clonidine evoked both increases and decreases in crcf before and after aspirin in women in the low and high E 2 phases (P > 0.05). We propose that finger vasoconstriction evoked by extraluminal α 1 -adrenoceptor stimulation is blunted by vasodilator COX products in young men and overcome by their action in women in the high, but not low E 2, phase of the menstrual cycle. By contrast, α 2 -adrenoceptor stimulation evokes finger vasoconstriction that is mediated by vasoconstrictor COX products in young men, but evokes no consistent response in women in the low or high E 2 phases of the menstrual cycle. (Resubmitted 1 July 2011; accepted 25 July 2011; first published online 1 August 2011) Corresponding author J. M. Marshall: School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK. j.m.marshall@bham.ac.uk Abbreviations COX, cyclooxygenase; crcf, cutaneous red cell flux; PE, phenylephrine. DOI: /jphysiol

2 4556 A. Srinivasa and J. M. Marshall J Physiol Introduction There is evidence that the resting level of cutaneous blood flow in the hand is much lower in young women than young men. This has been attributed to greater tonic influence of the sympathetic noradrenergic fibres in young women. (Cooke et al. 1990). It has also been reported that the reflex decrease in finger blood flow evoked by application of a cold stimulus to the neck is of similar magnitude in young women and men (Freedman et al. 1987). However, primary Raynaud s disease, which is characterised by digital vasospasm evoked by cold or emotional stress, is far more common in young women than men and the severity of the disease decreases after the menopause (Cooke & Marshall, 2005). Thus, it seems that under some circumstances, ovarian hormones may promote vasoconstriction in the fingers, even though oestrogen (E 2 ) is considered to play a protective role in vascular function. Post-junctional adrenoceptors on vascular smooth muscle that respond to sympathetically released noradrenaline are predominantly of the α 1 -subtype, while α 2 -adrenoceptors are considered to be pre-junctional and to inhibit transmitter release. However, the preponderance of postsynaptic α 2 -adrenoceptors increases from the radial artery, to the digital arteries (Flavahan et al. 1987). Indeed, vasoconstriction was evoked in the finger both by α 1 -andbyα 2 -adrenoceptor agonists when applied iontophoretically to the skin, or infused intra-arterially (Linblad & Ekenvall, 1986; Coffman & Cohen, 1988). Further, intra-arterial infusion of selective antagonists showed that vasoconstrictor tone in the finger of normal men and women is dependent on both α 1 -adrenoceptor, and α 2 -adrenoceptor stimulation (Cooke et al. 1997). Thus, an obvious question arises as towhethervasoconstrictionevokedinthefingerbyα 1 - and/or α 2 -adrenoceptor stimulation is greater in women than men. In previous studies, intra-arterial infusion of α 1 -or α 2 -agonists into the hand evoked considerably smaller vasoconstrictor responses in the finger of women than men (Freedman et al. 1987). These findings are difficult to interpret in relation to sympathetically mediated vasoconstriction because responses evoked by intraluminally administered α-agonists may be more readily modulated by endothelium-dependent vasodilator and vasoconstrictor substances than noradrenaline released from sympathetic nerves on the extra-luminal surface. Nevertheless, infusion of phenylephrine (PE), a selective α 1 -adrenoceptor agonist, into the hand of women, evoked greater finger vasoconstriction in the luteal phase of the menstrual cycle when oestrogen (E 2 ) levels are relatively high, whereas the α 2 -adrenoceptor agonist clonidine evoked greater vasoconstriction in the follicular phase, when E 2 levels are low. These results suggest that the influences of E 2 may enhance α 1 -butlimitα 2 -evoked finger vasoconstriction (Freedman & Girgis, 2000). Correspondingly, contraction evoked by α 1 -adrenoceptor stimulation in aorta of female rats, was attenuated by ovariectomy, but restored by chronic administration of E 2.Moreover,inaortaofintact female rats, contraction evoked by α 1 -adrenoceptor stimulation was attenuated by endothelium removal, cyclooxygenase (COX) inhibition, or antagonism of the Thromboxane receptor (TP) receptor that binds both PGH 2 and thromboxane (TXA 2 ), which is generated from PGH 2 by TXA 2 synthase. It was therefore proposed that in the presence of E 2, stimulation of α 1 -adrenoceptor on the endothelium causes synthesis and release of PGH 2 /TXA 2, which facilitates contraction evoked directly by α 1 -adrenoceptor stimulation (Fulton & Stallone, 2002). There are also endothelial α 2 -adrenoceptors whose stimulation increases nitric oxide (NO) synthesis and limits vasoconstriction evoked by stimulation of α 2 -adrenoceptors on the vascular smooth muscle (Angus et al. 1986; Bockman et al. 1996). Given the endothelium generates vasodilator, as well as vasoconstrictor COX products and E 2 increases the activity and expression of NO synthase and COX (Chambliss & Shaul, 2002), the cyclical variation in α 2 -adrenoceptor evoked finger vasoconstriction noted by Freedman & Girgis (2000) may reflect greater attenuation by NO and vasodilator COX products when E 2 levels are high. In view of these findings, the present study was performed to elucidate vascular responses evoked in the finger of young men and women by iontophoresis of selective α 1 - and α 2 -adrenoceptor agonists, such that they had access from the extraluminal surface like sympathetically released noradrenaline; we tested responses before and after the COX inhibitor aspirin. We hypothesised that (i) α 1 -mediated vasoconstriction is greater in women than men, particularly in the high E 2 phase of the menstrual cycle, due to α 1 -evoked release of vasoconstrictor COX products and (ii) α 2 -mediated vasoconstrictor responses are smaller in women than men, particularly in the high E 2 phase, reflecting action of vasodilator COX products. Some of these findings have been reported in brief (Srinivasa & Marshall, 2009). Methods We used a modification of the iontophoresis protocol by which we applied noradrenaline, the mixed α 1 -and α 2 -adrenoceptor agonist, to the finger of young men (Hendry & Marshall, 2004). This, in turn, was based on the protocol originally described for acetylcholine by Morris & Shore (1996) and used by us (Hendry & Marshall, 2004; Easter & Marshall, 2005), in which successive pulses of

3 J Physiol Finger vascular responses 4557 Table 1. Baseline values of mean ABP, pulse rate, height, weight, BMI and skin temperature on Day 1 ABP (mmhg) HR (beats min 1 ) Height (cm) Weight (kg) BMI Skin temp. ( C) Males 86.7 ± ± ± ± ± ± 0.4 Females (high E 2 ) 81.5 ± ± ± ± ± ± 0.4 Females (low E 2 ) 82.4 ± ± ± 0.3 Values are means ± SEM. P < 0.05, male vs. female subjects by Student s t test. Comparisons were also made in female subjects between the high and low E 2 phases, but they did not reach statistical significance. the agonists are applied so as to generate a cumulative vascular response that reaches a plateau. In preliminary studies on young men, not reported here, we established the concentration of the α 1 -andα 2 -adrenorecptor agonist, the pulse width and the interval between pulses that allowed a reproducible vascular response to develop such that the crcf value achieved following the penultimate pulse appeared to have reached a maximum response to that agonist; the final pulse was delivered at twice the current with the intention of ensuring a maximum was reached (see Morris & Shore, 1996 and below). The same parameters were used for all studies on young men and women reported below. The studies were carried out on 10 young men (age 21.1 ± 0.13 years; mean ± SEM) and nine young women (age 21.4 ± 0.38 years) all of whom were in good health and had no known cardiovascular or other disorder. A questionnaire was used in order to gain information on medical history along with height and weight, which showed that none of the subjects was obese or underweight (Table 1). None of the volunteers was a smoker, or taking vasoactive medication during the study or in the week preceding it. This includes all drugs that fall into the NSAIDs category, as well as substances such as vitamin C, which might influence oxidant status and thereby vascular responses. None of the women was taking an oral contraceptive pill or using any other medication known to alter levels of reproductive hormones, but they all had regular menstrual cycles, enabling them to predict when menstruation would begin with a confidence of 1 2 days. All subjects abstained from alcohol and strenuous exercise for at least 24 h before the study. They were also asked not to have a heavy meal or consume caffeine within 2 h of any experiment. All subjects gave informed consent. The study was approved by the Local Research Ethics Committee of the South Birmingham Health Authority. All experiments were carried out in a windowless, temperature-controlled room kept at C. Extraneous sound and all distraction within the room was minimised so as to limit the novel or noxious stimuli to which the subjects were exposed. This helped ensure that steady, basal levels of cardiovascular variables were obtained. On arrival, subjects washed their hands in warm water so as to ensure good resting perfusion to the fingers and to facilitate the expected agonist-induced constrictor responses. Each subject was seated throughout the protocol, with both arms and hands supported at heart level by cushions to maximise comfort. Arterial blood pressure (ABP) and heart rate (HR) were recorded from the right hand, while iontophoresis was applied to the left hand (see below), which was placed on an electric heating blanket so as to maintain the hand skin temperature at C. Skin temperature was recorded continuously via a thermistor that was attached to the dorsal surface of the left hand with surgical tape. In our previous study, which was carried out under exactly the same conditions except that we did not use a heating blanket, control crcf was 30 perfusion units (PU) in young male subjects (Hendry & Marshall, 2004) compared with PU in the present study (Table 2). Thus, it is reasonable to assume that we induced tonic vasodilatation. After a 30 min acclimatisation period, when the subjects had adjusted to the temperature of the room and the electric blanket, the cuff of a Finapres device (2300; Ohmeda, Stirling, UK) was placed on the middle finger of the subject s right hand. This allowed continuous recordings of ABP and HR throughout the protocol. The dorsal surface of the subject s left hand was then cleaned with the aid of a sterile alcohol wipe in preparation for iontophoresis to be carried out on the proximal phalynx of the index and middle fingers for phenylephrine (PE) and clonidine, respectively. Iontophoresis was performed and cutaneous red cell flux (crcf) was recorded as described before (Hendry & Marshall, 2004). Briefly, an iontophoresis chamber comprising a Perspex ring containing the anode was attached to the dorsal surface of the finger, while the cathode was fastened onto the dorsal surface of the hand. Both electrodes were connected toabatterypowerediontophoresiscontroller(mic1; Moor Instruments, Axminster, UK). The iontophoresis chamber (8 mm inner diameter) was filled with 0.5 ml of the relevant drug. A laser Doppler probe (DPIT-V2; Moor Instruments) was then placed into the well and connected to a laser Doppler perfusion (DRT 4; Moor Instruments), so as to provide a continuous recording of crcf. The outputs of the iontophoresis controller, crcf, ABP and HR were recorded on a PC (Gateway GP6-400) via MacLab hardware (MacLab/400; ADInstruments, Hastings, UK).

4 4558 A. Srinivasa and J. M. Marshall J Physiol Table 2. Baseline values of mean RCF recorded in male and female subjects at start of each iontophoresis protocol Control (PU) 30 min post-aspirin (PU) 180 min post-aspirin (PU) 24 h post-aspirin (PU) Males Phenylephrine 66.9 ± ± ± ± 9.1 Clonidine 54.4 ± ± ± ± 7.8 Females Low E 2 Phenylephrine 73.8 ± ± ± ± 15.1 Clonidine 47.1 ± ± ± ± 6.9 High E 2 Phenylephrine 41.8 ± ± ± ± 12.4 Clonidine 43.2 ± ± ± ± 11.9 Values are means ± SEM. PU: perfusion units. Comparisons were also made between females during the high and low E 2 phases: they did not reach statistical significance. Protocols Day 1. When a steady baseline crcf had been maintained for at least 5 min, the iontophoresis protocol for either 0.5 mm PE or 0.5 mm clonidine was begun: seven pulses of 0.1 ma for 30 s, followed by a final pulse of 0.2 ma for 30 s with 60 s intervals between pulses. After an interval of 5 min, this protocol was repeated with the other drug. The order of applying PE and clonidine was randomised. Day 2. On the following day the subject was given 600 mg of soluble aspirin dissolved in water to take orally. The equipment was connected as described above and the iontophoresis protocol just described was repeated at 30 min post-aspirin and at 3 4 h post-aspirin for PE and for clonidine, each being applied at the same site as on Day 1. This dose given orally can be expected to provide 86% inhibition of prostacyclin (PGI 2 ) synthesis attributable to endothelial COX at 30 min, 70% inhibition at 90 min and full recovery at 6 h, whereas 99% inhibition of TXA 2 synthesis by platelets is achieved from 30 min to 6 h due to their inability to synthesise new COX (Heavey et al. 1985). Day 3. Approximately 24 h post-aspirin, a final repeat of the iontophoresis protocol was performed for both PE and clonidine each at the same site as on the previous days. Exactly the same protocol was used on the male and female subjects. However, female subjects attended for two complete runs of the protocol, the first in the low E 2 phase of the menstrual cycle (days 0 5) and the second in the high E 2 phase (days 9 12). Water control. The iontophoresis protocol described above was performed on five male subjects, with sterile water in the chamber. Drugs Phenylephrine hydrochloride and clonidine hydrochloride (Sigma, Poole, Dorset) were dissolved in sterile water for injection (Steri-Amp Water for injection BP; Norton, Runcorn, Cheshire, UK) to create stock solutions of 1 mm of PE and clonidine which were kept refrigerated at 3 C. These stock solutions were used to create the 0.5mM solutions for iontophoresis on the day of the experiment by dilution with sterile water. New stock solutionsweremadeupweekly.allsolutionsofpewere wrapped in aluminium foil due its photosensitivity and both test solutions were kept on ice until use during the experiment. Introduction of the ice-cold solution of PE or clonidine into the iontopheresis chamber did not result in any significant change in crcf until the iontophoresis currents were applied. Analysis of results All results are expressed as means ± SEM. In each iontophoresis protocol, the responses evoked by successive pulses were calculated as the mean crcf over the last 20 s of the interval between successive current pulses, so that the change in RCF in response to each pulse could be calculated (Hendry & Marshall, 2004). As can be seen from Table 2, there was variation between subjects within each group in the baseline level of crcf. Therefore, in order to normalise the crcf data, the values recorded during iontophoresis were expressed as the percentage change from the baseline recorded before iontophoresis. Changes in crcf within each iontophoresis protocol for PE and for clonidine in the absence of and at different time intervals after aspirin were analysed by using ANOVA for repeated measures with Scheffé s post hoc test to detect the time within the protocol at which a change reached significance. Student s paired and unpaired t test was used to compare baseline values without and after aspirin within and between groups. Statistical significance was taken as P < 0.05.

5 J Physiol Finger vascular responses 4559 Results The height and weight of the subjects and the mean values of ABP and HR in males and in females in the low and high E 2 phases of the menstrual cycle are shown in Table 1. The male subjects weighed more than the females and had higher skin temperature than the females in the high E 2 phase. There were no significant differences between groups or between experimental days within groups (data not shown). Within each group, ABP did not change significantly during the iontophoresis protocol under any condition. Therefore the changes in crcf that occurred during iontophoresis can be taken as an index of the vascular response in the cutaneous circulation of the finger. Vascular reponses evoked in male subjects Baseline RCF values in the absence of and at different times after aspirin were not significantly different for the PE or clonidine protocol (see Table 2). Phenylephrine. In males, the control response to successive pulses of PE comprised a biphasic change in mean crcf: crcf increased in 5 of the 10 subjects in response to pulses 1 4 and decreased thereafter, whereas in the remaining subjects, crcf decreased throughout (see Fig. 1A). The response over the entire iontophoresis protocol was statistically significant by ANOVA, but changes evoked by individual pulses did not reach significance by post hoc test.however,at30andat180min post-aspirin on Day 2, PE evoked constriction in all subjects (Fig. 1B and C), the changes from baseline reaching significance by post hoc test at the sixth to eighth pulses at 180 min after aspirin, (Fig. 1C). At 24 h post-aspirin on Day 3, the mean response evoked by PE was again biphasic and similar to the control response on Day 1 with 5/10 subjects showing dilatation to pulses 1 4 (Fig. 1D), Clonidine. The control response to successive pulses of clonidinewasadecreaseincrcf(seefig.2a). By contrast, at 30 and 180 min post-aspirin, clonidine evoked no significant change in crcf (Fig. 2B and C). However, at 24 h post-aspirin the response evoked by clonidine was again vasoconstriction comparable to the control response (Fig. 2D). Vascular reponses evoked in female subjects Baseline crcf values were not significantly different in the absence of or at different times after aspirin for the PE or clonidine protocols in the low or high E 2 phases of the menstrual cycle (see Table 2). Phenylephrine. In the low E 2 phase of the menstrual cycle, the control response to PE was a decrease in crcf (see Fig. 3A, upper row). This response was not obviously altered by aspirin (Fig. 3B and C upper row), although the response evoked at 24 h post-aspirin did not reach statistical significance (P = 0.07), reflecting dilator responses to the second to fifth pulses of PE in three subjects. By contrast, in the high E 2 phase of the cycle, the control response to PE was very variable between individuals in the direction and magnitude of the changes in crcf (Fig. 3A, lower row). However, at 30 and 180 min post-aspirin, PE evoked significant changes in crcf, vasoconstriction occurring by the sixth to eighth pulses (Fig. 3B and C lower row). At 24 h post-aspirin, PE again evoked no significant change in crcf (Fig. 3D). Figure 1. Cutaneous vascular responses evoked in finger of young men by iontophoresis of phenylephrine before and at different times after aspirin In each graph, RCF is shown as percentage change from baseline (mean ± SEM) following each iontophoretic current pulse, as shown on the abscissa. A, responses evoked by PE before aspirin (control response). B D, responses evoked 30 min, 180 min, 24 h post-aspirin, respectively. n = 10.,, : P < 0.05, 0.01, respectively by ANOVA. P < 0.05 vs. baseline by post hoc Scheffé s test.

6 4560 A. Srinivasa and J. M. Marshall J Physiol Clonidine. In the low and high E 2 phases of the cycle, responses evoked by clonidine were very variable both with and between individuals such that there were no significantchangesincrcfeitherbeforeoratdifferent times post-aspirin (Fig. 4). Visual inspection of the site of iontophoresis when the chamber had been removed generally showed a circular halo of pale skin around patchy flushing which seemed to be located around hair follicles. Thus, it appeared that clonidine evoked competing vasoconstrictor and dilator responses. This contrasted with the general situation in men and women in that the skin usually appeared uniformly pale when PE or clonidine evoked a net decrease in crcf and flushed when there was a net increase in crcf. Water. Iontophoresis of water with the same protocol as used for PE and clonidine evoked no significant changes in crcf (see Fig. 5). Discussion In the present study, iontophoresis of successive pulses of the α 1 -adrenoceptor agonist PE evoked biphasic dilatation/constriction in the finger of young men, but vasoconstriction after COX inhibition with aspirin. By contrast, in women in the low E 2 phase of the menstrual cycle, PE evoked finger vasoconstriction that was not altered by aspirin, whereas in the high E 2 phase, PE evoked no response until aspirin was given; then it evoked finger vasoconstriction. Further, the α 2 -adrenoceptor agonist clonidine evoked vasoconstriction in the finger of men that was unchanged by aspirin, but no net response in the finger of women in the low or high E 2 phases, whether or not aspirin had been taken. These findings contrast in several respects with our working hypotheses (see Introduction), but before interpreting them we need to consider the conditions under which our study was done and its limitations. In previous studies, resting blood flow in the hand was lowerinyoungmenthanwomen(cookeet al. 1990). By contrast, in the present study, baseline levels of crcf in the finger were similar in men and women, presumably because we warmed the hand to facilitate vasoconstrictor responses to α-agonists. This would have achieved a relatively low level of sympathetic vasoconstrictor tone and possibly active vasodilatation mediated by dilator nerve fibres and local heating, both of which involve NO (Kellogg, 2006). Our results must therefore be considered in this context (see below). In order to compare responses evoked in the low and high E 2 phases of the menstrual cycle, we performed experiments on days 0 5 and on days 9 12, respectively. The women who volunteered had regular cycles, but we acknowledge uncertainty, particularly in estimating the high E 2 phase. This may explain some of the variability in the evoked changes in crcf in the high E 2 phase (see below). We used aspirin at 600 mg p.o. because this dose achieves a maximum inhibitory effect on COX activity in nucleated cells at 30 min, with recovery by 6 h (Heavey et al. 1985). In men and in women in the low and high E 2 phase of the menstrual cycle, aspirin had no effect on baseline levels of crcf suggesting that tonically synthesised vasoconstrictor or dilator COX products do not contribute to finger vascular tone. This accords with previous findings when COX inhibitors were given systemically (e.g. Morris & Shore, 1996; Khan et al. 1997; Hendry & Marshall, 2004; Easter & Marshall, 2005). Further, when the control response evoked by α 1 -orα 2 -adrenoceptor stimulation was changed at 30 and 180 min post-aspirin, the response Figure 2. Cutaneous vascular responses evoked in finger of young men by iontophoresis of clonidine before and at different times after aspirin In each graph, RCF is shown as percentage change from baseline (mean ± SEM) following each iontophoretic current pulse, as shown on the abscissa: A, responses evoked by clonidine before aspirin (control response). B D, responses evoked 30 min, 180 min, 24 h post-aspirin respectively. n = 10., : P < 0.05, 0.01 respectively by ANOVA.

7 J Physiol Finger vascular responses 4561 evoked at 24 h was generally comparable to the control response. Thus, we can reasonably attribute such changes to inhibition of COX, rather than to time, or inherent variability within or between subjects. Given iontophoresis of water evoked no change in crcf, we can attribute the substantial changes in crcf evoked by PE and clonidine to α 1 -orα 2 -adrenoceptor stimulation rather than to the iontophoresis current per se. Responses evoked by α 1 -adrenoceptor stimulation On the basis of previous studies, we hypothesised that iontophoresis of α 1 -agonist would evoke greater finger vasoconstriction in women than men, particularly in the high E 2 phase of the menstrual cycle due to α 1 -evoked release of vasoconstrictor COX products. In fact, iontophoresis of PE evoked pure finger vasoconstriction only in women in the low E 2 phase. The increases in crcf evoked by the early pulses of PE in young men were converted to decreases after aspirin such that pure vasoconstriction occurred, crcf decreasing by 30 40%. Assuming the concentration of PE in superficial skin gradually increases during successive pulses and that PE gradually diffuses more deeply, it is likely the initial dilator responses reflected α 1 -stimulated release of a dilator COX product from vascular smooth muscle or other cells close to the extraluminal surface of blood vessels, although we cannot exclude the possibility of diffusion to endothelium. Vascular smooth muscle cells and endothelial cells express COX-1 and COX-2 in aortae of male and female rats and both cell types synthesise dilator and constrictor COX products (Shepherd & Katusic, 1991; Li et al. 2008). Thus, irrespective of the cells involved, we can propose that in young men, COX-dependent vasodilator products may blunt finger vasoconstriction evoked by α 1 -adrenoceptors. Figure 3. Cutaneous vascular responses evoked in finger of young women by iontophoresis of phenylephrine in the low (above) and high (below) E 2 phases of the menstrual cycle, before and at different times after aspirin In each graph, RCF is shown as percentage change from baseline (mean ± SEM) following each iontophoretic current pulse, as shown on the abscissa: A, responses evoked by PE before aspirin (control response). B D, responses evoked 30 min, 180 min, 24 h post-aspirin respectively. n = 9. P < 0.05, P < 0.01, P < by ANOVA. P < 0.05 vs. baseline by post hoc Scheffé s test.

8 4562 A. Srinivasa and J. M. Marshall J Physiol Finger vasoconstriction evoked by PE in women in the low E 2 phase was not affected by aspirin. Thus, there is no reason to propose it was partly mediated by vasoconstrictor COX products as we hypothesised from in vitro studies (Fulton & Stallone, 2002). That the evoked change in crcf did not reach statistical significance at 24 h post-aspirin in the low E 2 phase may be explained if E 2 level was rising in some women by this time (see above). Indeed, our finding that in the high E 2 phase, PE had no significant effect on crcf in the absence of aspirin, but evoked finger vasoconstriction at 30 and 180 min post-aspirin suggests that α 1 -stimulation released vasodilator COX products that overwhelmed the α 1 -evoked constriction. This proposal accords with evidence that E 2 increases the activity and expression of COX (Orshal & Khalil, 2003). The finger vasoconstriction evoked by PE when COX was blocked, was comparable in the high E 2 and low E 2 phases, crcf decreasing by 30 40%. Thus, even though E 2 increases endothelial NO synthase (enos) activity and expression (Chambliss & Shaul, 2002) and NO blunts α 1 -evoked vasoconstriction (Zhang & Davidge, 1999; Tuttle & Fallone, 2001), there seems no reason to suppose that NO has a menstrual phase-related blunting effect on finger vasoconstrictor responses to α 1 -stimulation. Responses evoked by α 2 -adrenoceptor stimulation There are three subtypes of α 2 -adrenoceptor: α 2A -, α 2B - and α 2C -receptors (Bylund et al. 1994). The α 2C -adrenoceptor has been specifically implicated in the vasoconstrictor response to cold in extremities such as fingers and mouse-tail. In contrast to vasoconstriction evoked by α 1 -, and other subtypes of α 2 -receptors, that evoked by α 2C -receptors is augmented by cooling because these receptors are translocated to the plasma membrane by cooling (Chotani et al. 2000). Also, E 2 increases the Figure 4 Cutaneous vascular responses evoked in finger of young women by iontophoresis of clonidine in the low (above) and high (below) E 2 phases of the menstrual cycle, before and at different times after aspirin In each graph, RCF is shown as percentage change from baseline (mean ± SEM) following each iontophoretic current pulse, as shown on the abscissa. A, responses evoked by clonidine before aspirin (control response). B D, responses evoked 30 min, 180 min, 24 h post-aspirin respectively. n = 9. None of these responses was statistically significant at P < 0.05.

9 J Physiol Finger vascular responses 4563 expression of α 2C -receptors in vascular smooth muscle (Eid et al. 2007). However, we maintained the hand at Cwhenα 2C -receptors are silent (Chotani et al. 2000). Thus, iontophoretically applied clonidine probably had access only to α 2A -andα 2B -receptors. We hypothesised that α 2 -mediated finger vasoconstriction evoked by iontophoresis of clonidine would be smaller in women than men, particularly in the high E 2 phase, reflecting the action of vasodilator COX products. However, our finding that iontophoresis of α 2 -agonist failed to evoke an overriding decrease in crcf in women in the low or high E 2 phases of the menstrual cycle irrespective of whether COX was inhibited is consistent with an overriding tonic or α 2 -stimulated influence of NO and does not implicate COX products at all. That α 2 -stimulation did evoke an underlying vasoconstriction is supported by the visual observation of skin blanching (see Results). It may be that the patchy flushing around hair follicles reflects their ability to give easier access of the agonist to endothelial α 2 -adrenoceptors. By contrast, in men, α 2 -stimulation evoked graded vasoconstriction that was attenuated by aspirin indicating it was largely attributable to vasoconstrictor COX products. To our knowledge, this is a novel finding. However, α 2 -stimulation evoked contraction in endothelium-intact rat mesenteric arteries that was partly attributable to a COX-dependent vasoconstrictor product and attenuated by castration (Tejera et al. 1999). Further, in pig coronary artery, testosterone facilitated release of an endothelium-derived vasoconstrictor that was probably acoxproduct(farhatet al. 1995), while testosterone increased TP receptor expression in vascular smooth muscle (Masuda et al. 1991). As indicated above COX-1 and COX-2 are expressed in vascular smooth muscle and endothelium (Li et al. 2008). Thus, it may be that diffusion of α 2 -agonist from the extraluminal surface evokes finger vasoconstriction in young men mediated by COX products that are released from vascular smooth muscle or endothelium, whose action on TP receptors is facilitated by testosterone. This proposal is consistent with our finding that vasoconstrictor COX products limit vasodilatation evoked by iontophoresis of ACh in the finger of young men, but not young women (Hendry & Marshall, 2004; Easter & Marshall, 2005). Indeed, our findings directly contrast with the proposal that E 2 potentiates vasoconstriction evoked by a range of agonists even in normal females, by releasing vasoconstrictor COX products that act on TP receptors (see Sellars & Stallone, 2007). Rather, our results indicate that in the finger, release of such a substance is a characteristic of healthy young men, but not healthy young women, even in the high E 2 phase of the menstrual cycle. That a vasoconstrictor COX product limits ACh-evoked finger vasodilatation in healthy, post-menopausal women and in pre- and post-menopausal women with primary Figure 5. Cutaneous vascular responses evoked in finger of young men by iontophoresis of water RCF is shown as percentage change from baseline (mean ± SEM) following each iontophoretic current pulse, as shown on the abscissa. Raynaud s disease (Easter & Marshall, 2005), suggests that such substances become important in women in the absence of E 2, or pathologically. In summary, our results allow the novel proposal that under conditions of low sympathetic vasoconstrictor tone, iontophoresis of α 1 -adrenoceptor agonist into the finger, giving access from the extraluminal surface like sympathetically released noradrenaline, evokes finger vasoconstriction that is of similar magnitude in young men and women in the low and high E 2 phases of the menstrual cycle, decreasing crcf by 30 40%. However, vasodilator COX products overcome the vasoconstriction evoked by low concentrations of α 1 agonist in men, and completely attenuate finger vasoconstriction in women in the high E 2 phase of the menstrual cycle, whilst having no modulatory effect in the low E 2 phase. They also allow the novel proposal that iontophoresis of α 2 agonist evokes finger vasoconstriction in young men that is largely mediated by vasoconstrictor COX products. By contrast, in young women, in both the low and high E 2 phases of the menstrual cycle, any finger vasoconstrictor response to α 2 -stimulation is overcome by vasodilator influences that probably include NO, but not COX products. It remains to be determined whether vasodilator and vasoconstrictor COX products similarly modulate α 1 and α 2 components of vasoconstriction evoked in fingers by sympathetic nerve activity. References Angus JA, Cocks TM & Satoh K (1986). The α adrenoceptors on endolethelial cells. Fed Proc 45,

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Clin Sci 97, Tuttle JL & Fallone JC (2001). Nitric oxide release during α 1 -adrenoceptor-mediated contraction of arterioles. Am J Physiol Heart Circ Physiol 281, H873 H881. Zhang Y & Davidge ST (1999). Effect of estrogen replacement on vasoconstrictor responses in rat mesenteric arteries. Hypertension 34, Author contributions J.M.M. conceived the study and planned it. A.S. performed the experiments and analysed the data under the guidance of J.M.M. A.S. produced a first draft of the paper. J.M.M. set the study in context and played the major role in completing the paper. Both authors have approved the final version. Acknowledgements This study was generously supported by the Raynaud s and Scleroderma Association.