Prostaglandins participate in the late phase of the vascular response to acetylcholine iontophoresis in humans

Abstract

The participation of prostaglandins (PGs) in the cutaneous vasodilatation to acetylcholine (ACh) applied via iontophoresis is under debate. Using laser Doppler flowmetry, we studied the long lasting effect (20 min) of iontophoretic application (30 s; 0.1 mA) of ACh on the human forearm. Experiments were repeated (1) using deionized water instead of ACh to test the effect of current application, (2) after scopolamine treatment to inhibit muscarinic cholinergic receptors, and (3) 2 h, 3 days and 10 days following inhibition of PG synthesis with aspirin or a placebo control. Cutaneous vascular conductance (CVC) was calculated at rest (CVC_rest), at peak vasodilatation in the first 5 min following ACh iontophoresis (CVC_peak), and 20 min after iontophoresis (CVC₂₀). The minimal CVC (CVC_min) following iontophoresis was also determined. Cutaneous response to ACh displayed a biphasic pattern with an early and transient peak (CVC_peak: 62 ± 8% of the maximal CVC induced by local heating (MVC)) followed by a long lasting slower vasodilatation (CVC_min: 44 ± 6; CVC₂₀: 56 ± 5%MVC). The current itself had no major effect. Scopolamine almost abolished both phases. The long lasting phase was aspirin sensitive but not the transient phase. At hour 2 post-aspirin, CVC_peak was 61 ± 10, CVC_min 26 ± 6 and CVC₂₀ 29 ± 6%MVC. At day 3, CVC_peak was 53 ± 9, CVC_min 22 ± 3 and CVC₂₀ 25 ± 4%MVC. At day 10, CVC_peak was 67 ± 10, CVC_min 47 ± 7 and CVC₂₀ 50 ± 8%MVC. Placebo had no effect. We conclude that PGs participate in the vasodilator response following ACh iontophoresis. Previous non-steroidal anti-inflammatory drug treatments must be taken into account when studying the effect of ACh iontophoresis.

Among the complications resulting from systemic diseases such as diabetes mellitus or hypertension are cutaneous vascular dysfunctions resulting from altered endothelial vasomotor function. Tests of the vascular effect of acetylcholine (ACh; an endothelium-dependent vasodilator) are often performed to evaluate the endothelium-dependent microvascular responses (Katz et al. 2001; Jagren et al. 2002). Controversies concerning the mechanisms of the ACh-induced cutaneous vasodilatation remain. A variety of vasomotor factors may be released following ACh action on the endothelium including endothelium derived hyperpolarizing factor, nitric oxide (NO) and some prostanoids. However, the exact mechanisms of ACh-induced cutaneous vasodilatation and the influence of these factors remain unresolved. Some investigators have suggested the participation of prostaglandins (PGs) in the ACh-dependent cutaneous vasodilatation (Khan et al. 1997; Noon et al. 1998), whereas others reported no role for PGs (Morris & Shore, 1996). This remains an important question since a role for PGs in the vasodilatation would mean that any use of non-steroidal anti-inflammatory drugs could confound interpretation of this test of endothelial function.

Berghoff et al. (2002) showed recently that the prostanoids did not significantly participate in the vasodilator effect of ACh. However they were involved in the axon-reflex response appearing in the area close to the site of ACh administration. They also proposed that the discrepancy in results found in the literature could be due to an unrecognized axon reflex within the stimulation area.

All of the aforementioned studies used iontophoresis to deliver ACh. However, different aspirin formulations and administration techniques were used to study the participation of prostanoids, making interpretations difficult. Also, analyses of the results were mainly based on peak vascular responses following ACh administration. None of the studies focused on the long-lasting cutaneous vasodilator effects of ACh. We believe that the study of the long-lasting vasodilator effects of ACh could provide essential information about the participation of PGs in this response. We hypothesized that the cutaneous vascular response to ACh applied by iontophoresis results from two parallel mechanisms, one of which would be aspirin sensitive. Using laser Doppler flowmetry (LDF) in humans, we first studied the long lasting effect (20 min) of iontophoretically applied ACh on cutaneous microcirculation and we tested the cholinergic specificity of the responses observed with scopolamine. To study the influence of prostanoids in the vascular response observed and to determine the origin of the prostanoids that may be involved, we also repeated iontophoretic administration of ACh 2 h, 3 days and 10 days following aspirin or placebo administration. These questions have important clinical implications regarding the evaluation of endothelial dysfunction using ACh and could provide important information about the potentially confounding long-lasting effect of aspirin treatment during such procedures.

Methods

Eight non-smoking healthy volunteers (3 females, 5 males) with no clinical signs of or risk factors for vascular disease (mean ±s.d.; 28 ± 6 years old; height: 171 ± 12 cm; weight: 65 ± 12 kg), participated in this study. Volunteers were not involved in regular exercise training and had not taken any medication during the 3 weeks prior to each experiment. A minimum of 1 week elapsed between each experiment. The study was institutionally approved and performed in accordance with the Declaration of Helsinki. Before their participation, all subjects were thoroughly informed of the methods and procedures and gave their written consent to participate.

Measurements

Experiments were performed in a quiet air-conditioned room (ambient temperature: 23 ± 1°C). Subjects rested supine for 15 min before the experiment was started.

Iontophoresis and measurement of skin blood flow (SkBF) occurred on the volar aspect of the forearm. Iontophoresis is a non-invasive technique that allows transcutaneous delivery of polarized drugs via the application of a continuous low intensity current. We used the PeriIont PF 382, Micropharmacology System from Perimed (Stockholm, Sweden) associated with laser Doppler flowmeters (PeriFlux PF4001 and 5000, Perimed) and temperature regulated heating system (PeriTemp PF4005 and 5000, Perimed) to allow for current application, SkBF recording and local heating, respectively. Technical characteristics of these devices can be found in previous reports (Durand et al. 2002a,b). The ‘active’ electrode of the system was fixed to the skin and connected to the anode of the regulated 9 V intensity regulated current supplier. The sponge of this electrode was wet with 0.2 ml of ACh (2% in deionized water). The cathode was connected to a reference Ag–AgCl electrode placed nearby. In this study, the total charge of anodal current applied was 3 mC (application time: 30 s; intensity: 0.10 mA). Subjects reported no pain during the iontophoresis procedure. Temperature for local heating at the end of the experiments was set to 44°C to allow maximal vasodilatation (Taylor et al. 1984; Johnson et al. 1986; Saumet et al. 1998). Another LDF probe was used as a reference to confirm the absence of response to the current application at an unstimulated site 5 cm adjacent to the ‘active’ electrode. Local cutaneous temperature was measured using a surface thermocouple probe connected to an electronic thermometer (BAT-12, Physitemp Instruments Inc., Clifton, NJ, USA). The thermocouple probe was positioned 5 cm from the LDF probes.

Systemic blood pressure was monitored using a Finapres 2350 (Ohmeda, Englewood, CO, USA) positioned on the 2nd or 3rd finger of the hand contralateral to the sites of SkBF measurements.

Protocols

For all the protocols in this study, recordings consisted of a 2-min rest period followed by the iontophoresis period (30 s). A 20-min recovery period was then followed by local heating for 25 min. As a result, the total duration of each recording was 47.5 min

Protocol 1. Evaluation of non-specific response to anodal current application and effect of ACh iontophoresis on SkBF

The use of continuous monopolar current (i.e. galvanic current) during iontophoresis could lead to a non-specific vasodilatation independent of the effect of the drug diffused (Grossman et al. 1995; Berliner, 1997; Durand et al. 2002a). We assessed the possible role of this non-specific vasodilatation in the response observed following ACh iontophoresis by performing iontophoresis with deionized water alone as a vehicle. Deionized water has been previously used to evaluate the non-specific response to current application (Khan et al. 1997; Durand et al. 2002a; Özbebit et al. 2004). We then used ACh, 2% in deionized water, to assess the cutaneous microvascular response to ACh iontophoresis.

Protocol 2. Sensitivity of ACh-induced vasodilatation to muscarinic receptor inhibition

ACh causes vasodilatation via interaction with muscarinic receptors. To demonstrate the specificity of the response to muscarinic receptors in our model, ACh was applied via iontophoresis at two different sites. One site (site S) was treated with scopolamine in the form of a Scopoderm® patch (Novartis, Rueil-Malmaison, France) for 2 h prior to the start of the experiment to locally inhibit muscarinic receptors while the other site (site C) served as a control. The skin was carefully washed before positioning of the iontophoretic electrodes and laser Doppler probes.

Protocol 3. Sensitivity of ACh-induced vasodilatation to aspirin

Each subject randomly participated in two experiments separated by a minimum of 3 weeks, one under aspirin pretreatment and the other under placebo pretreatment. 1-g aspirin (Aspégic®, SanofiSynthelabo, Paris, France) was dissolved in 125 ml of orange juice in order to disguise the taste and appearance of the aspirin, whereas nothing was added to the orange juice in the placebo experiments. Two hours prior to the beginning of the experiment, subjects drank the orange juice, blinded to the presence or absence of aspirin in the glass. The experiment was repeated 3 and 10 days following the intake of the drink.

Data analysis

Analog output from all measured data was recorded on a computer via a 16-bit analog to digital converter (Biopac Systems, Inc., Goleta, CA, USA) with a sample rate of 3 Hz. The LDF signal was expressed in arbitrary units (AU). Due to instantaneous variability of the LDF signal resulting from vasomotion, individual results were averaged over 5-s intervals throughout each experiment. To index active changes in the cutaneous vasculature, vasodilatation was assessed as cutaneous vascular conductance (CVC) and calculated as the ratio of LDF to mean arterial pressure for each 5-s interval. Maximal CVC response to local heating (MVC) was calculated from each experiment from the average of CVC values observed over the last minute of the heating period. CVC values were normalized and presented as a percentage of the MVC (%MVC).

For data analysis and interpretation we defined the following points: CVC_rest was the last 5-s interval recorded before the current application; CVC_peak was the maximal value recorded during, or in the 5 min following, current application; CVC_min was the minimal CVC values observed following current application and CVC₂₀ was the value recorded 20 min after the current application, just prior to local heating. An example of the determination of these key points is presented in Fig. 1. All time references reported in the Results are from the start of the current application and were estimated for a protocol from the mean result, not from the mean of the individual times.

Figure 1. Illustration of the different points analysed on the laser Doppler flowmetry signals.

Data presented are the mean response ±s.e.m. (n = 8) observed before, during and after an ACh iontophoretic administration (30 s; 0.1 mA) at the forearm skin level. Results are expressed as a percentage of the maximal cutaneous vascular conductance (%MVC) observed after a 25-min local heating period (44°C). The white arrow indicates the period of ACh iontophoresis. Note the biphasic response to ACh iontophoresis with an early peak in CVC followed by a slow and long-lasting vasodilatation.

Student's t test was used to compare CVC data within a protocol. The influence of aspirin and placebo treatment on CVC (protocol 3) was assessed by two-way repeated measures ANOVA (Prism 2.01, GraphPad Software Inc., San Diego, CA, USA). Data are expressed as means ±s.e.m.P < 0.05 was considered significant in all statistical analyses. N.S. is reported in the text for statistically non-significant results.

Results

In all experiments, compared with starting values, no significant changes were observed in mean blood pressure, local skin temperature or skin blood flow at the control probe.

Protocol 1. Evaluation of the non-specific response to anodal current application and effect of ACh iontophoresis on SkBF

Effect of anodal current application on SkBF

CVC_rest was 11 ± 3%MVC. Iontophoresis of deionized water did not result in any immediate vasodilatation (Table 1). CVC_min was 9 ± 2%MVC (NS versus CVC_rest) at min 1.0 and CVC_peak was 14 ± 3%MVC at min 5.1 (NS versus CVC_rest). However, a slow drift in CVC could be observed during the experiment with CVC₂₀ reaching 18 ± 4%MVC (P < 0.05versus CVC_rest and versus CVC_min).

Table 1.

Effect of anodal iontophoresis of acetylcholine, 2% in deionized water, and deionized water alone (DW) on cutaneous microcirculation measured by laser Doppler flowmetry

	ACh	DW
	CVCrest	11 ± 2	11 ± 3
	CVCpeak	62 ± 8†	14 ± 3
	CVCmin	44 ± 6†*	9 ± 2
	CVC20	56 ± 5†‡	18 ± 4†‡

Anodal current application: 30 s; 0.1 mA. Results are expressed as a percentage of the maximal cutaneous vascular conductance observed after a 25-min local heating period (44°C).

^†P < 0.05 compared to CVC_rest.

^*P < 0.05versus CVC_peak.

^‡P < 0.05versus CVC_min.

Effect of ACh iontophoresis on SkBF

A typical recording of SkBF following ACh iontophoresis in one subject is presented in Fig. 2. The mean response to ACh iontophoresis is shown in Fig. 1. CVC_rest was 11 ± 2%MVC. Iontophoresis of ACh induced a rapid and large vasodilatation. This cutaneous vasodilatation displays a biphasic pattern with an early peak (phase 1 of the vasodilatation) and a long lasting slower vasodilatation (phase 2 of the vasodilatation). At the end of the current application (i.e. end of ACh administration), CVC reached 23 ± 4%MVC (P < 0.01versus CVC_rest). It was 38 ± 6 and 59 ± 7%MVC 15 s and 1 min later, respectively. CVC_peak (62 ± 8%MVC, P < 0.001versus CVC_rest) was attained at 2.2 min following the end of iontophoresis. CVC then transiently decreased (end of phase 1). CVC_min reached 44 ± 6%MVC (P < 0.001versus CVC_rest and P < 0.05versus CVC_peak) at min 9.0. A progressive and slow vasodilatation followed (phase 2) and CVC₂₀ was 56 ± 5%MVC (NS versus CVC_peak; P < 0.05versus CVC_min and CVC_rest).

Figure 2. Typical cutaneous vascular response to the iontophoretic application of ACh (30 s; 0.1 mA) in forearm skin.

From top to bottom, recordings are: SkBF under the ‘active’ electrode where the ACh iontophoresis was performed (Laser v1); systemic blood pressure monitored via Finapres (Pressure); local cutaneous temperature in the area close to the iontophoresis site (Skin Temperature); and SkBF at an unstimulated control site (Laser control).

Protocol 2. Sensitivity of ACh-induced vasodilatation to muscarinic receptor inhibition

No differences in CVC_rest were observed between site S and site C (12 ± 2 versus 12 ± 3%MVC, respectively). Scopolamine profoundly blunted the ACh-dependent vasodilatation. There was a significant difference in CVC_peak between site S and site C. CVC_peak at site C reached 57 ± 14%MVC but was only 20 ± 6%MVC at site S (NS versus CVC_rest and P < 0.05versus CVC_peak at site C). CVC remained stable at site S in the 20 min following the end of the iontophoresis, whereas the late vasodilatation was still observed at site C. At min 20, CVC₂₀ was 40 ± 12%MVC at site C (P < 0.05versus CVC_rest and CVC_peak) and 18 ± 5%MVC at site S (NS versus CVC_rest and CVC_peak; Fig. 3).

Figure 3. Effect of muscarinic receptor inhibition by scopolamine on the cutaneous vasodilatation induced by iontophoretic administration of ACh.

Results are expressed as a percentage of the maximal cutaneous vascular conductance (%MVC) observed after a 25-min local heating period (44°C).

Protocol 3. Sensitivity of ACh-induced vasodilatation to aspirin

The effects of aspirin and placebo pretreatments on the vascular response to ACh iontophoresis are presented in Fig. 4 and Table 2.

Figure 4. Mean cutaneous vascular conductance (CVC) observed before, during and 20 min following the ACh iontophoresis (30 s; 0.1 mA), at different intervals from aspirin (bottom graphs) or placebo (top graphs) treatment: hour 2 (H2), day 3 (D3) and day 10 (D10).

Results are expressed as a percentage of the maximal cutaneous vascular conductance (%MVC) observed after a 25-min local heating period (44°C). The white arrow indicates the period of ACh iontophoresis. The heating period is not presented, to simplify the graphs.

Table 2.

Mean CVC_rest, CVC_peak, CVC_min and CVC₂₀ observed following ACh iontophoresis 2 h (H2), 3 days (D3) or 10 days (D10) after aspirin or placebo treatment

	Aspirin treatment			Placebo treatment
	H2	D3	D10	H2	D3	D10
CVCrest	12 ± 1	12 ± 2	16 ± 2	12 ± 2	10 ± 2	15 ± 2
CVCpeak	61 ± 10†	53 ± 9†	67 ± 10†	61 ± 6†	56 ± 6†	64 ± 10†
CVCmin	26 ± 6†*	22 ± 3†*	47 ± 7†*	39 ± 7†*	31 ± 7†*	38 ± 7†*
CVC20	29 ± 6†*	25 ± 4†*	50 ± 8†	47 ± 6†‡	†‡53 ± 10	49 ± 8†‡

Results are expressed as a percentage of the maximal cutaneous vascular conductance observed after a 25-min local heating period (44°C).

^†P < 0.05 versus CVC_rest.

^*P < 0.05 versus CVC_peak.

^‡P < 0.05 versus CVC_min. Note that at D10 after aspirin treatment, a response almost similar to the one observed during the placebo experiments can be observed.

Placebo treatment did not affect the response to iontophoretic ACh administration at all time points. CVC_peak was significantly higher than CVC_rest. Then CVC transiently decreased with CVC_min significantly different from CVC_peak. CVC₂₀ was not different from CVC_peak but different from CVC_min.

Aspirin pretreatment did not modify the first phase of the vasodilatation induced by ACh iontophoresis. No differences were observed in CVC_peak under aspirin or placebo treatment at any of the time points we studied (hour 2, day 3 or day 10). The second phase of the vasodilator response was clearly affected suggesting PG involvement. Two hours after aspirin intake, this phase 2 vasodilatation was abolished. A plateau was observed at a level higher than CVC_rest but lower than CVC_peak. CVC_min was not different from CVC₂₀. In contrast to protocol 1, CVC₂₀ was lower than CVC_peak and it was also lower than CVC₂₀ at 2 h following placebo treatment. A progressive recovery of the slow vasodilatation occurred in the 10 days following aspirin treatment. On day 3, following the early peak vasodilatation, the plateau was changed to a progressive fall in CVC throughout the recovery period. CVC_min was not different from CVC₂₀ and CVC₂₀ was lower than CVC_peak. Compared to placebo treatment, CVC₂₀ was diminished after aspirin (P < 0.05 placebo versus aspirin). At day 10, the phase 2 vasodilatation returned, such that CVC₂₀ was not different from CVC_peak. However, despite the similarities with the responses observed in protocol 1 and under placebo treatment, the time course of this slow and long lasting vasodilatation was slightly different from what was observed in the previous protocols, resulting in CVC_min being lower than CVC_peak but not different from CVC₂₀ (P = 0.2).

Discussion

The long lasting effect of iontophoretic administration of ACh in the skin has not been well described. We report in the present study that the cutaneous vasodilator response to iontophoretic administration of ACh at the forearm displays a biphasic pattern and that PGs are involved in the second phase of this cutaneous microvascular response.

In the present study, the vascular response to 30-s iontophoretic administration of ACh (total electrical charge = 3 mC) at the cutaneous level included an early (within 5 min following the start of current application), short lasting, peak vasodilatation followed by a late, slower prolonged vasodilatation. This vasodilatation was still present after 20 min and resulted in CVC levels in the same range of the peak vasodilatation observed in the early vasodilator response. Iontophoresis has been previously reported to induce a non-specific long-lived cutaneous vasodilatation due to the use of the current itself (Grossmann et al. 1995; Durand et al. 2002a,b). ACh-induced vasodilatation is based on the effect of ACh on cutaneous M2 muscarinic receptors. Involvement of these receptors in the mechanism of the non-specific vasodilatation is not known. However after local cutaneous inhibition of the muscarinic receptors, no significant vasodilatation was observed following ACh iontophoresis, suggesting that both phases of the cutaneous vasodilatation were specifically related to ACh. The slow drift of SkBF accompanying the iontophoresis of deionized water led to a very limited vasodilatation (from 13 ± 3 to 18 ± 4%MVC). This also suggests that there was no major influence of the technique itself.

Aspirin is known to inhibit the synthesis of prostanoids through an irreversible blockade of cyclooxygenase (Vane, 1971). The early phase of the ACh-induced vasodilatation was insensitive to aspirin treatment. No change in the peak vasodilatation was observed. This observation is consistent with the results of Berghoff et al. (2002) and Morris & Shore (1996) but differs with the previous reports of Noon et al. (1998) and Khan et al. (1997). These latter authors observed a significant decrease in the vascular response to ACh applied via iontophoresis following aspirin treatment. All of the aforementioned (Morris & Shore, 1996; Khan et al. 1997; Noon et al. 1998; Berghoff et al. 2002) performed dose–response studies to investigate the participation of PG in the response to ACh iontophoresis, focusing only on the peak response observed shortly after ACh iontophoresis. Morris & Shore (1996), using a laser imager, recorded the responses at 0, 60 and 120 s after the iontophoresis; Khan et al. (1997) recorded changes in flux for 4 min between each dose applied; Berghoff et al. (2002) applied the current every 3 min while Noon et al. (1998) analysed the response observed within the 2 min maximum following iontophoresis. Reasons for discrepancies in their conclusions are unknown but could be attributed to differences in the iontophoretic protocol, the maximal current charge used (from 4 to 56 mC), and the method of aspirin administration.

Vallance et al. (1989a,b) concluded that NO participates in the ACh-mediated vasodilatation in human arteries and veins in vivo. The early peak vasodilator response could reflect the release of NO due to the action of ACh on the endothelial M2 muscarinic receptors. Those receptors can activate constitutive endothelial NO synthases via a G protein mechanism leading to the production of NO. NO has a short half-life and is one of the major and most powerful vasodilator factors present in humans. Some in vitro studies suggest that salicylate treatment could affect NO production in human platelets (O'Kane et al. 2003) but aspirin does not have a significant effect on NO bioavailability in endothelial cells (Madajka et al. 2003) and we did not observe any significant difference in CVC_peak after placebo or aspirin treatment. This supports a role for NO in ACh-mediated vasodilatation in human skin. However, in vitro studies on isolated human and animal vessels reported the participation of a non-NO, non-prostanoid endothelium-dependent hyperpolarizing mechanism in ACh-mediated vasodilatation (Buus et al. 2000; Hoepfl et al. 2002). Further research is necessary to confirm NO as the major factor responsible for the cutaneous response observed.

The late phase of the response was a prolonged vasodilatation which could be due to a neurogenic inflammation and a local axon reflex within the stimulation area, as suggested by Berghoff et al. (2002). ACh can excite unmyelinated C fibres (Douglas & Ritchie, 1960) and induce an axon reflex leading to prolonged vasodilatation in the close area of administration (Berghoff et al. 2002). Özbebit et al. (2004), using mathematical curve analysis, also concluded that the SkBF response to ACh iontophoresis was the sum of two components, one of which (the late component) was abolished after local anaesthesia. This provides further support for the participation of unmyelinated C fibres in the ACh-mediated response. Unmyelinated C fibres contain pools of vasomotor products such as calcitonin gene related peptide, substance P, or PG. It is possible that ACh induces an excitation of C fibres leading to the release of vasomotor products and to the progressive vasodilatation observed following the peak vasodilatation.

We observed that the phase 2 (late) vasodilatation was aspirin sensitive, suggesting that PGs are involved. PGs, whatever their origins, could act as a direct vasodilator agent on the cutaneous vessels. PGI2 (prostacyclin), for example, exhibits these vasorelaxing properties. They could also act as sensitizing agent on C fibres since specific PGs such as the PGE2 were shown to sensitize those fibres (Kindgen Milles, 1995; Minami et al. 1999). A combination of both mechanisms is also possible. Further investigations will be necessary to determine the exact mechanism by which PGs participate in the late phase of ACh-mediated vasodilatation.

If PGs are involved in the vasodilatation following ACh iontophoresis, the origin of the PGs remains unknown. Aspirin irreversibly blocks cyclooxygenase (COX). PG synthesis is restored when COX is resynthesized by nucleated cells. Restoration of COX is variable according to the tissue. For example, in vitro, COX in smooth muscle is restored in 3 h whereas, in endothelial cells, synthesis is altered for up to 24 h (Hla & Bailey, 1989). In an effort to determine the origin of the PGs participating in the ACh-induced vasodilatation, we followed the SkBF response to ACh iontophoresis 2 h, 3 days and 10 days after a single oral dose of 1 g aspirin. Such a dose blocks the whole pool of COX in the human body (Patrono et al. 1998). We observed that the return of the late phase of the vasodilatation following COX inhibition was progressive. A significant inhibition was still observed 3 days after aspirin intake at a time when the endothelial and smooth muscle function COX function should be entirely restored (Hla & Bailey, 1989). This suggests that part of the pool of PGs necessary to obtain the late phase of the vasodilatation to ACh iontophoresis is not of endothelial or smooth muscle cell origin. One possible origin of the PGs may be the nerve (C fibre). In support of this idea, aspirin has been shown to have an inhibitory effect on type 1 vanilloid receptor (VR1), a specific receptor of the non-myelinated C fibre (Szallasi & Blumberg, 1999). In these nucleated cells, due to the necessary axonal transport of molecules, a delay is likely to exist between the synthesis of proteins in the nucleus and the bioavailability at the nerve terminal. However, there is no available information about the time necessary for the VR1 or PG synthesis in the nucleus of C fibres and the delay for the axonal transport of the synthesized COX and/or VR1.

Aspirin may also interfere with the expression of inflammatory factors such as the nuclear factor-kappa B (Kopp & Ghosh, 1994) or interleukin-4 (Cianferoni et al. 2001). Since the non-specific vasodilatation could be due to a neurogenic inflammation from the current application (Berliner, 1997), these products may have been activated during our experiments. However, the possible influence of these factors during iontophoresis experiments remains unknown (Durand et al. 2002c). Could platelets be involved? These cells being devoid of synthesis ability, up to 10 days are necessary to restore the pool of platelet PGs after irreversible blockade of COX through the remaining life of the platelets with 1-g aspirin treatment (Patrono et al. 1998). At day 3, approximately 70% of platelets are still blocked. Further studies are needed to test these possibilities.

Finally, ACh and/or aspirin could have an effect on some other undescribed elements participating directly or indirectly in the vascular response to ACh iontophoresis. Consistent with the suggestion of Berghoff et al. (2002), we propose that ACh administered via iontophoresis, has a dual effect at the microvascular level. ACh could act on endothelial receptors to induce the release of vasodilator mediators responsible for the early vasodilatation. This step would be aspirin insensitive. It could also directly or indirectly excite primary afferent nerve fibres to elicit a prolonged long lasting vasodilator phenomenon (neurogenic inflammation). Contrary to the early phase, this late phase seems to be aspirin sensitive, relying in part on PG.

In summary, the present study results in new considerations about the vascular response to iontophoretically applied ACh and has important clinical implications. This response is biphasic with an early peak and a second, slower vasodilatation, both with different kinetics and likely different mediators. We have demonstrated that PGs are involved in the late phase of the vasodilatation, but not in the early phase. ACh iontophoresis is regularly performed in clinical and scientific settings to assess the endothelium-dependent vasodilatation. When exploring the vascular effect of ACh, the following recommendations should be followed: (1) observations should be of adequate time to monitor both the first phase and the second phase of the ACh-mediated cutaneous vasodilatation, and (2) anti-inflammatory drug treatment, specifically aspirin, should be monitored prior to testing, with testing occurring at a minimum of 3 days or longer following anti-inflammatory drug administration.