Introduction

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Diadenosine polyphosphates (ApnA) act as humoral signal transducers and neurotransmitters in the central nervous system and are coreleased with ATP and catecholamines from the adrenal medulla (Castillo et al., 1992; Sillero et al., 1994). They are also stored in and released from human platelets (Schlüter et al., 1994), and degradation half-time in the blood is considerably longer than that of ATP, the prototype purinergic agonist (Lüthje and Ogilvie, 1988). Depending on the initial vessel tone ApnA induce vasoconstrictions (basal vessel tone) or vasodilations (raised vessel tone). In rat mesenteric resistance arteries ApnA-induced effects are independent of the endothelium or of perivascular innervation (Steinmetz et al., 2000b). The potency of ApnA-induced vasoactivity in the isolated vessel preparations of the rat is dependent on the chain length of the ApnA (n = 3-6) with an optimum at Ap5A (followed by Ap6A, Ap4A, and Ap3A) (Ralevic et al., 1995; van der Giet et al., 1997; Steinmetz et al., 2000b). However, in anesthetized rats, systemically applied (i.e., intravenously infused) ApnA induced a sustained hypotension; herein, Ap4A had the highest potency (followed by Ap6A, Ap5A, and Ap3A), and Ap4A was also used for interrupting hypertensive episodes during anesthesia in humans (Kikuta et al., 1999), which emphasizes the growing importance of these purinergic physiological substances for clinical application. The concentrations of, for example, Ap5A measured systemically in the whole blood certainly do not surpass 1 µM, but regionally after degranulation of thrombocytes due to certain circumstances much higher concentrations will be present in the vascular lumen (Flores et al., 1999).

ApnA, like other purinergic substances, are acting via purinoceptors, with P2X₁ purinoceptors mediating contractile stimuli and P2Y purinoceptors (and probably A₂ purinoceptors) vasodilator stimuli (van der Giet et al., 1998; Steinmetz et al., 2000a). The distribution of purinoceptors in the various regions of the individual resistance arterial tree is not uniform. Depending on the prevalence of P2X, P2Y purinoceptors vasoconstriction or vasorelaxation is predominating (Steinmetz et al., 2000a,b), respectively. Thus, a lot of data concerning ApnA effects in animal arteries are known; however, little is known whether these data derived from animal experiments are comparable with the conditions in humans. Therefore, we evaluated for the first time the ApnA-induced effects in human resistance arteries. To show which role the potential degradation products of ApnA play (Lüthje and Ogilvie, 1988), we also evaluated the effects of ATP, ADP, AMP, and adenosine.

Furthermore, pharmacological studies with purinoceptor antagonists were performed to identify the purinoceptors being activated by ApnA. Numerous antagonists of the different purinoceptors were described: Among those, suramin was found to antagonize P2Y and P2X purinoceptors (Abbracchio and Burnstock, 1994), and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) was reported to act as selective P2X purinoceptor antagonist (Lambrecht et al., 1992; Windscheiff et al., 1994), although there are studies suggesting that PPADS binds to P2Y₁ purinoceptors as well (Communi et al., 1996). ADP3'5' was described as a P2Y₁ purinoceptor antagonist (Boyer et al., 1996). In this study, we antagonized Ap5A- and Ap4A-induced effects with PPADS or ADP3'5' to possibly distinguish between P2X and P2Y purinoceptor activation. Ap5A was chosen because of its high potency and because of detailed experience in its effects in animal resistance arteries, which allows detailed comparisons between the results in human tissue and animal tissue. Ap4A was chosen because of its high potency in various animal studies (e.g., it had the highest hypotensive potency applied systemically in anesthetized rats) and because of its relevance in a study with systemic application of Ap4A in humans (Kikuta et al., 1999). We evaluated the effects of ApnA in human mesenteric resistance-size arteries because they possess a key position in the regulation of precapillary resistance; up to 50% of the precapillary drop of blood pressure occurs in arteries of this size (Mulvany and Halpern, 1977; Mulvany and Aalkjaer, 1990).

In summary, to show the different contractile or vasorelaxing potencies of ApnA in hMRA, concentration-response curves were constructed. The degradation products of ApnA were examined likewise to demonstrate whether the ApnA effects are primary or secondary effects after their degradation. Contractile and vasorelaxing effects of Ap4A and Ap5A were antagonized by different purinoceptor antagonists to determine the responsible purinergic purinoceptors. Finally, a thorough comparison between ApnA effects in hMRA and rat MRA should be able to show whether the varying vasoactivity by ApnA does not only change from one vascular bed to the other (Steinmetz et al., 2000a) but also from one species to the other.

	Materials and Methods

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Human Tissue Preparations. Small (ca. 2 cm³) specimens of mesenteric tissue were dissected from resected intestinal human tissues during abdominal surgery at the local surgical clinic. Surgical interventions were necessary because of intestinal tumors, inflammatory bowel diseases, or bowel injuries. Written consent on behalf of the patients was mandatory. The samples of mesenteric tissue were stored in cooled physiological Krebs-Ringer bicarbonate (KRB) solution (see below).

Vessel Isolation. The isolation of vessels was performed immediately after the receipt of the tissue samples from the operation theater. The samples were placed in a Petri dish coated with Sylgard (Dow Corning, Seneffe, Belgium), which was filled with KRB solution. In the mesenteric tissue samples, fatty tissue overlaying and surrounding the arterial structures was carefully removed under a dissection microscope, and 2-mm-long segments of the mesenteric resistance size arteries were isolated.

Tension Measurements. Arteries were mounted on two stainless steel wires (diameter 40 µm) as ring segments in an isometric myograph (Myograph model 410A; J.P. Trading, Aarhus, Denmark), one wire connected to a force transducer (DSC6; Kistler Morse Seattle, WA) for recording of isometric force development (Mulvany and Aalkjaer, 1990), and the other wire connected to a displacement device. With the latter, arteries were stretched to their optimal luminal diameter using an active length tension protocol with 125 mM K⁺ as activating stimulus (De Mey and Brutsaert, 1984). The optimal luminal diameter was established when the force caused by depolarization of the vascular smooth muscle cells with a high K⁺ solution (125 mM K⁺) was maximal. During experimentation the vessels were kept in KRB that was maintained at 37°C and aerated with 95% O₂/5% CO₂.

Experimental Protocols. In previous experiments with animal tissues (Steinmetz et al., 2000b), the considerable desensitizing effect of the purinergic substances was described and studied in detail. In this study, we performed preliminary experiments to find out how much time had to elapse between two experiments to obtain an identical vascular response with the same concentration of the purinergic agent in the same vessel. These time intervals were comparable with those obtained in the animal tissue experiments, consequently the experiments with human arteries were done according to the same pattern; discontinuous concentration-response curves were performed allowing a 30-min interval between each application of the purinergic agents. Effects of vasorelaxing agonists were evaluated during precontraction induced by 0.02 µM endothelin, which provided a stable vasoconstriction of about 90% of the force evoked by high K⁺. In contrast to rat mesenteric resistance arteries, phenylephrine failed to induce a stable and long-lasting precontraction of arteries in human mesenteric resistance arteries, which, however, could be achieved with endothelin. The concentration of 0.02 µM endothelin produced a vasoconstriction of hMRA, which was, compared with a high K⁺-induced vasoconstriction, identical with a constriction by 10 µM phenylephrine in the rat MRA. Inhibitory effects of the purinoceptor antagonists were evaluated after the vessels had been incubated with the respective antagonist for 10 min. Effects of Ap5A and Ap4A were antagonized with two different antagonist concentrations.

Drugs and Solutions. The composition of KRB solution was as follows: 119.1 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 25.0 mM NaHCO₃, 2.5 mM CaCl₂, and 5.5 mM glucose. In high K⁺ solution (125 mM K⁺), all NaCl was replaced by an equimolar concentration of KCl. All agonists and pharmacological tools were obtained from Sigma Chemical (Deisenhofen, Germany) except for PPADS, which was obtained from Sigma/RBI (Natick, MA). Stock solutions were prepared on the day of use in double-distilled water.

Data Analysis. Contractile reactivity was measured as active wall tension (active force divided by twice the vessel segment length) and expressed as the percentage of the tissue response to 125 mM K⁺ at the beginning of the experimental protocols. Whenever possible, concentration-response curves were analyzed in terms of potency (pD₂ = log EC₅₀) determined by least-square sigmoidal curve fitting of individual curves (GraphPad Prism 1.00; GraphPad Software, San Diego, CA). pD₂ values were always calculated if a concentration-response curve reached the maximal effect. This was, however, not always reached with the maximal agonist concentration applied (1 mM). Differences between agonists and between types of vessels were evaluated by analysis of variance followed by t test according to Bonferroni with p < 0.05 denoting statistical significance. Data are shown as mean ± S.E.M.

	Results

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The mesenteric arterial side branches were surrounded by abundant fatty tissue and after a short distance of a few millimeters they penetrated the outer intestinal tissue layer. They usually were accompanied by a venous vessel. K⁺ KRB solution (125 mM) induced a stable and lasting vasoconstriction, in contrast to phenylephrine (10 µM), which brought about a merely transient contractile effect. Endothelin was able to cause a stable and lasting vasoconstriction. The period to elapse after washing out the endothelin and the return to the baseline level of vascular tension lasted about 10 to 20 min and, moreover, the necessity to perform discontinuous concentration-response curves to avoid interference with the desensitizing effect of ApnA required repeated precontraction phases. Identical responses to endothelin usually were not reproducible for more than five times. Accordingly, the arterial preparations had to be renewed rather frequently. General tissue characteristics of the hMRA are depicted in Table 1.

Effects of Ap3A, Ap4A, Ap5A, and Ap6A on the Basal Tone of the hMRA. In hMRA all ApnA caused a concentration-dependent, transient contraction (Fig. 1). The maximal contractile force occurred within 10 to 30 s. The contraction was followed by an immediate and complete fading of the active vascular tone. Figure 1 (left) exemplary shows the rapid and brief vasoconstriction by Ap5A (10 µM). The rank order of contractile potency was Ap5A > Ap6A > Ap4A > Ap3A (Fig. 2). Table 2 (left columns) shows the corresponding pD₂ values. The pD₂ values were calculated whenever the maximum of the induced effects was reached within the tested range of concentrations. The maximums of the ApnA-induced vasoconstrictions were about 70 to 80% of the maximal contractile force induced by K⁺ 125 mM KRB. Up to the concentration of 1000 µM the Ap3A-induced contraction did not reach a maximum.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Original records of myograph experiments with hMRA at basal tone (left) showing force development by Ap5A (10 µM). Ap5A causes a highly transient constriction of hMRA. At raised tone (right) Ap5A (10 µM) causes a brief relaxation followed by a further increase in tone which is rapidly followed by a lasting and marked relaxation.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Concentration-response curves for the contractile effects of Ap3A, Ap4A, Ap5A, and Ap6A in hMRA at basal tone. Effects were presented as the percentage of the response to 125 mM K+ and are shown as mean ± S.E.M. (n = 6-9). , denotes statistical significance (p < 0.05).

View this table: [in this window] [in a new window]   TABLE 2 pD2 (log M of EC50) of contractile and relaxing effects by ApnA in hMRA (n = 6-9) and comparison with the effects in rat MRA (Steinmetz et al., 2000b)

Effects of Adenosine, AMP, ADP, and ATP on the Basal Vessel Tone of hMRA. To determine whether the described effects of ApnA in hMRA could be evoked by their degradation products, analog experiments with adenosine, AMP, ADP, and ATP were performed (Fig. 3). In file with the rank order of potency of ApnA, the following results were recorded: Ap5A > Ap6A > Ap4A > Ap3A = ATP  ADP  adenosine > AMP, the latter with no influence on vascular tone at all. Within the concentration range tested (10-1000 µM) among the possible degradation products only the concentration-response curve of adenosine reached its maximum of contractile force. The contractions were transient like those of ApnA. Generally, the potencies of the degradation products of ApnA were lower than those of ApnA; only Ap3A-induced contractions were comparable with those of ATP.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Concentration-response curves for the contractile effects of ATP, ADP, AMP, and adenosine in hMRA at basal tone. Effects were presented as percentage of the response to 125 mM K+ and are shown as mean ± S.E.M. (n = 6-9). , denotes statistical significance (p < 0.05).

Vasorelaxations in the hMRA Induced by Ap3A, Ap4A, Ap5A, and Ap6A. The precontraction of the arteries was initiated by endothelin (0.02 µM). Application of ApnA provided a triphasic vascular response; an initial reduction of vascular tone for seconds was followed by a short-term increase of vascular tone exceeding the level of precontraction and ending in a long-lasting vasorelaxation (Fig. 1). The vasorelaxing potencies of the four ApnA were not significantly different from each other, but Ap3A tended to show a lower potency (Fig. 4).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Relaxing effects of Ap3A, Ap4A, Ap5A, and Ap6A in hMRA at raised tone in percentage of an endothelin (0.02 µM) precontraction. Each point is the mean of five to seven determinations ± S.E.M. , denotes statistical significance (p < 0.05).

Vasorelaxations in the hMRA Induced by Adenosine, AMP, ADP, and ATP. After precontraction with endothelin (0.02 µM) the ApnA degradation products caused a similar triphasic vascular response as described for ApnA. The vasorelaxing potencies of the mononucleotides were not significantly different from each other (Fig. 5), although ADP had the tendency to display the highest potency, which was significantly higher than that of adenosine. Compared with ApnA-induced vasodilation the concentration-response curve of ADP and Ap3A were quite similar, but the other ApnA were more effective vasorelaxants than their degradation products.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Relaxing effects of ATP, ADP, AMP, and adenosine in hMRA at raised tone in percentage of an endothelin (0.02 µM) precontraction. Each point is the mean of five to seven determinations ± S.E.M. , denotes statistical significance (p < 0.05).

Antagonism of the Contractile Effects of Ap4A and Ap5A in hMRA with PPADS and ADP3'5'. In hMRA, only a high concentration of the antagonist PPADS (100 µM) induced a poorly reproducible vasoconstriction, which was transient within few seconds; however, only inhibitory concentration of up to 10 µM PPADS was used in the experiments. ADP3'5' did not have any effect on the vascular tone. In hMRA, the Ap4A (10 µM)- and Ap5A (10 µM)-induced vasoconstrictions were concentration dependently inhibited by the P2X antagonist PPADS (1 or 10 µM). The antagonism by PPADS of Ap5A-induced vasoconstriction was noncompetitive (Fig. 7). For Ap4A, the inhibitory effect was more marked than with Ap5A (Fig. 6). In presence of the selective P2Y₁ purinoceptor antagonist ADP3'5' (10 or 100 µM), no significant influence on vascular contraction induced by Ap4A or Ap5A was registered (data not shown).

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Contractile effects of Ap4A (10 µM) (left) and of Ap5A (10 µM) (right) in hMRA at basal tone as percentage of the response to 125 mM K+ (control) and contractile effect after preincubation with PPADS (1 and 10 µM). Each column represents the mean of six to nine determinations ± S.E.M. , denotes statistical significance (p < 0.05).

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Concentration-response curve of the contractile effects of Ap5A (0.01-100 µM) in hMRA at basal tone as percentage of the response to 125 mM K+ (control) and concentration-response curves after preincubation with PPADS (1 and 10 µM). Each column represents the mean of four determinations ± S.E.M. , denotes statistical significance (p < 0.05).

Antagonism of the Vasodilating Effects of Ap4A and Ap5A in hMRA with PPADS and ADP3'5'. PPADS or ADP3'5' had no influence on the degree of precontraction induced by 0.02 µM endothelin. Preincubation of the P2X purinoceptor antagonist PPADS (1 or 10 µM) did not inhibit the vasorelaxation induced by Ap5A (10 µM) in hMRA (data not shown). In contrast, the same concentrations of the antagonist could concentration dependently inhibit the Ap4A (10 µM)-induced vasorelaxation in hMRA (Fig. 8). Also, vasodilation induced by Ap4A (10 µM) (Fig. 8) but not by Ap5A (10 µM) (data not shown) in hMRA was concentration dependently inhibited by the P2Y₁ purinoceptor antagonist ADP3'5' (10 or 100 µM).

View larger version (31K): [in this window] [in a new window]   Fig. 8.   Left, relaxing effect of Ap4A (10 µM) in hMRA at raised tone in percentage of an endothelin (0.02 µM) precontraction (control) and relaxing effect after preincubation with ADP3'5 (10 and 100 µM). Right, relaxing effect of Ap4A (10 µM) in hMRA at raised tone in percentage of an endothelin (0.02 µM) precontraction (control) and relaxing effect after preincubation with PPADS (1 and 10 µM). Each column represents the mean of six to nine determinations ± S.E.M. , denotes statistical significance (p < 0.05).

	Discussion

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After gathering detailed knowledge about the ApnA effects on the tone of animal resistance arteries and after studying the impact of intravenously applied ApnA on the rat cardiovascular system (Steinmetz et al., 2000a,b,c), the principal aim of this study was for the first time to examine the effects of the ApnA on mesenteric resistance arteries of humans, i.e., the real focus of medical interest. Additionally, experiments with purinoceptor antagonists should yield hints about the purinoceptors mediating the vascular effects of ApnA in humans. Ap5A was studied at a concentration of 10 µM because at this concentration the most data were available from our own studies with rat MRA. By choosing the same concentrations of Ap5A in human and rat arteries, valid comparisons between rat MRA and hMRA were possible. Ap4A also was studied at a 10 µM concentration, which is close to its EC₅₀ concentration and which allows comparisons with the effects induced by Ap5A. Adenosine, AMP, ADP, and ATP that arise by asymmetric cleavage of ApnA also display purinergic activity (Ogilvie et al., 1996; Fredholm et al., 1998) and were examined likewise.

The experiments were performed with the biometric method of microvessel myography in small resistance arteries being crucial for the regulation of the systemic blood pressure (Mulvany and Aalkjaer, 1990).

The ApnA produced transient vasoconstrictions in hMRA. The rank order of potency was Ap5A > Ap6A > Ap4A > Ap3A. The degradation products of ApnA had less contractile potency. The Ap4A- and Ap5A-induced vasoconstrictions were not inhibited by the P2Y₁ purinoceptor antagonist ADP3'5' (Boyer et al., 1996); however, they were inhibited by the putative P2X purinoceptor antagonist PPADS (Lambrecht et al., 1992; Humphrey et al., 1998). PPADS turned out to be a noncompetitive antagonist.

The identical effects of ApnA and the same pharmacological pattern of antagonism in human and rat MRA suggest a similar mechanism of vasoconstriction in rat and human. Activation of P2X receptors seems to be responsible for Ap4A- and Ap5A-induced vasoconstriction in hMRA. Vascular tissue exposed to PPADS is easily impregnated by this orange substance, which possibly only pretends a noncompetitive antagonism. The fact that the degradation products of ApnA had lower potencies than the original nucleotides excludes that the observed contractile effects of ApnA are primarily due to their degradation products.

The rank order of contractile potency of ApnA in hMRA is comparable with rat MRA (Steinmetz eat al., 2000b) and principally also with the perfused rat mesenteric artery (Ralevic et al., 1995), where a pivotal role of the phosphate chain length for the pharmacological potency of the ApnA was postulated. These observations are also compatible with Gabriels et al. (2000), who found stronger vasoconstrictions by Ap5A than by Ap3A in interlobar arteries of hydronephrotic rat kidneys. In perfused rat kidneys, van der Giet et al. (1997) reported a rank order of potency of ApnA increasing the perfusion pressure, which was the same as for the hMRA. The higher contractile potency of ApnA than their degradation products may be reflected by their considerably higher stability in the blood than, for instance, ATP, which is cleaved into adenosine and phosphate within seconds (Lüthje and Ogilvie, 1988; Lüthje, 1989). Although vasocontractile effects of ApnA on the local vascular level are well established, it was repeatedly confirmed that systemically applied ApnA only induce hypotensive effects (Kikuta et al., 1994; Khattab et al., 1998; Steinmetz et al., 2000c).

Various studies with animal vascular preparations showed that the purinergically mediated vasoconstrictions are P2X purinoceptor-dependent (Bo and Burnstock, 1993; van der Giet et al., 1998; Steinmetz et al., 2000a,c). Selectivity of antagonists still is a critical item in purinoceptor research, like in this microvessel myography bioassay. However, the pharmacological profile of antagonism by different agents allows to draw conclusions about the receptors involved. Generally, in experiments with isolated organs, the concentrations of agonists and antagonists necessary to induce purinergic effects are higher than in experiments with single cells or cell cultures (Evans and Kennedy, 1994) because the potency of some agonists and antagonists is greatly decreased by breakdown by ectonucleotidases and other enzymes (Kennedy and Leff, 1995). PPADS has apart from P2X-antagonistic qualities probably P2Y inhibitory effects, too (Communi et al., 1996; Vigne et al., 1998). But that vasoconstriction in hMRA is not inhibited by the P2Y₁ antagonist ADP3'5', that the extremely rapid course of vasoconstriction in hMRA suits very well an ionotropic process at the ion channel-gated P2X receptor (in contrast to the slow metabotropic process of vasorelaxation mediated by the G protein-coupled P2Y receptors), and that there is no other study reporting on P2Y-mediated vasoconstriction clearly justify our conclusion that ApnA-induced vasoconstriction in hMRA is P2X purinoceptor-dependent. Different P2X purinoceptor subtypes with varying pharmacological qualities potentially mediate smooth muscle constriction (Bianchi et al., 1999). For example, the P2X₁ purinoceptor of the human urinary bladder is known for a rapid desensitization, whereas the rat P2X₂ purinoceptor desensitizes very slowly (Evans et al., 1995). Between homologous purinoceptor subtypes of human and rat there are significant differences; the human P2X₄ purinoceptor shows a sensitivity for antagonism by suramin and PPADS but the P2X₄ purinoceptor of the rat does not (Garcia-Guzman et al., 1997).

Concerning vasorelaxation of hMRA, pharmacological precontraction of the arteries is necessary to quantify vasorelaxation with small vessel myography. In contrast to rat MRA in which a stable precontraction with phenylephrine can be induced in the hMRA, phenylephrine only provides a poorly reproducible and unstable vasoconstriction. Therefore, in the hMRA, endothelin was chosen to provide the necessary stable arterial precontraction. In precontracted hMRA, ApnA caused a triphasic vascular response with a terminal long-lasting vasorelaxation. There was no significant difference between the different ApnA concerning their vasorelaxing potency in the hMRA. The ApnA degradation products generally had a lower potency than ApnA. Only vasodilation induced by Ap4A but not by Ap5A was concentration dependently inhibited by the P2Y₁ purinoceptor antagonist ADP3'5'. Furthermore, the antagonist PPADS inhibited the Ap4A-induced vasorelaxation in hMRA, whereas the same antagonist concentration did not inhibit the Ap5A-induced vasorelaxation. Thus, the Ap4A-induced vasorelaxation in hMRA is P2Y₁ purinoceptor-mediated and Ap5A evokes vasorelaxation via another mechanism than Ap4A.

In contrast to the rat MRA, there was no significant difference between the different ApnA concerning their vasorelaxing potency in the hMRA. Similar lack of difference was reported for experiments to evaluate the vasorelaxing potency of Ap3A and Ap5A in interlobar arteries of hydronephrotic rat kidneys (Gabriels et al., 2000). However, in the precontracted rat mesenteric resistance artery (Steinmetz et al., 2000b), a clear rank order of potency (Ap6A > Ap5A > Ap4A > Ap3A) was observed. The weaker potencies of the ApnA degradation products suggest that the observed ApnA effects are primary effects. Interestingly, adenosine, which was almost inert in rat MRA, induced vasorelaxations in hMRA at higher (100 µM) concentrations. However, adenosine-induced vasorelaxation of larger rat mesenteric arteries was described previously (Prentice et al., 1997).

Purinergic vasodilation was shown to be mediated via P2Y purinoceptors (Ralevic et al., 1995). Ap5A-induced vasorelaxation in rat MRA was inhibited by P2Y receptor blockade (Steinmetz et al., 2000a). In contrast to these studies with rat arteries, in our study with hMRA, only vasodilation induced by Ap4A but not by Ap5A was inhibited by the P2Y₁ purinoceptor antagonist ADP3'5', and the purinoceptor antagonist PPADS inhibited the Ap4A- but not Ap5A-induced vasorelaxation. The nonantagonism of Ap5A-induced vasorelaxation by these P2X and P2Y antagonists in hMRA is surprising because this finding stands in contrast to those in the rat MRA where a competitive inhibition of Ap5A-induced vasorelaxation by ADP3'5' and a noncompetitive inhibition by PPADS was demonstrated, suggesting a role for P2Y₁ receptors. Apparently in humans Ap5A activates other purinoceptors, leading to vasorelaxation. The inhibition of the Ap4A-induced vasorelaxation by PPADS may reflect the limited P2X purinoceptor selectivity of PPADS, which was suggested previously (Harden et al., 1998; Vigne et al., 1998). The selectivity of ADP3'5' at P2Y₁ purinoceptors was repeatedly proved (Boyer et al., 1996; von Kügelgen and Wetter, 2000). ADP3'5' was used as P2Y₁ antagonist in the concentration range used in this study in several other studies (Fagura et al., 1998; Choi et al., 2001; Turner et al., 2001).

The results of this study show that experimental findings in the animal model cannot be transferred unrestrictedly to human conditions. Therefore, it is desirable to derive data relevant for the human physiology and therapy first and foremost from experiments with human tissues wherever possible. Ap4A was already used for treatment of high blood pressure in humans (Kikuta et al., 1999), offering the advantage to apply a physiologically existing and easily metabolized pharmacological agent. Therefore, it is of importance to retrieve further knowledge and deeper insight into the pharmacology of ApnA in the human cardiovascular system. Regarding the fact that there exists a heterogeneous distribution of purinoceptor subtypes in different species and different vascular beds and that the vasoactivity of ApnA seems to depend on the underlying vascular tone makes it conceivable to provide a selective and differentiated pharmacological regulation of the blood pressure with the help of ApnA.