0022-3565/98/2863-1146$03.00/0THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright 1998 by The American Society for Pharmacology and Experimental Therapeutics Evidence against Anandamide as the Hyperpolarizing FactorMediating the Nitric Oxide-Independent Coronary VasodilatorEffect of Bradykinin in the Rat1 Department of Pharmacology and Molecular Cardiobiology Division, Boyer Center for Molecular Medicine, Yale University, New Haven,Connecticut (D.F.) and Department of Cell Biology, UMDNJ-SOM, Stratford, New Jersey (J.Q.) This paper is available online at ABSTRACT
The mediator of nitric oxide-(NO) independent vasodilation at-
nist, SR 141716A (2 ␮M), reduced dose-dependent vasodilator tributed to endothelium-derived hyperpolarizing factor remains responses to anandamide (1–10 ␮g) but was without effect on unidentified although there is evidence for a cytochrome P450- responses to AA (1–10 ␮g), bradykinin (10 –1000 ng) or cro- derived eicosanoid. Anandamide, the ethanolamide of arachi- makalim (1–10 ␮g). Inhibition of voltage-dependent Caϩϩ donic acid and an endogenous ligand for cannabinoid recep- channels with nifedipine (5 nM) attenuated vasodilation to tors, was proposed as an endothelium-derived hyperpolarizing anandamide and arachidonic acid whereas inhibition of Caϩϩ- factor-mediating mesenteric vasodilation to acetylcholine and activated Kϩ channels with charybdotoxin (10 nM) reduced the hypotensive effect of bradykinin. Using pharmacological responses to arachidonic acid but had no effect on vasodilation interventions that attenuate responses to bradykinin, we exam- induced by anandamide. Inhibition of cytochrome P450 with ined the possibility of anandamide as a mediator of the NO- clotrimazole (1 ␮M) greatly reduced vasodilator responses to independent vasodilator effect of bradykinin in the rat perfused bradykinin with less effect on those to anandamide. Finally, the heart by determining responses to anandamide and arachi- time course of the coronary vasodilator responses to anand- donic acid. Hearts were treated with indomethacin to exclude amide and bradykinin were dissimilar. These results argue prostaglandins and nitroarginine to inhibit NO synthesis and against a role of anandamide in the vasodilator effect of bra- elevate perfusion pressure. The cannabinoid receptor antago- The recognition by Furchgott and Zawadski (1980) of the hypothesis (Zygmunt et al., 1996; Edwards et al., 1996; requirement for an intact endothelium for responses to cer- Fukao et al., 1997). Our studies with bradykinin in the rat tain vasodilator agonists led to the identification of NO as heart and/or kidney demonstrate that the NO-independent EDRF. The introduction of inhibitors of NO synthesis under- vasodilator effect of this peptide is susceptible to inhibitors scored the importance of NO to the regulation of vascular PLC and PLA , P450 and Kϩ channels, supporting the con- tone. However, their use also resulted in the realization that cept of a P450-derived eicosanoid as a hyperpolarizing factor NO could not fully account for endothelium-dependent re- (Fulton et al., 1992, 1994, 1995, 1996; Rapacon et al., 1996).
sponses to various agonists including bradykinin and acetyl- Studies using inhibitors of P450 that exhibit differential choline, depending on the vascular bed and the species. Con- activity against epoxygenase vs. w-hydroxylase, i.e., clotrim- sequently, release of an unidentified hyperpolarizing factor, azole vs. 17-ODYA (Fulton et al., 1995), suggest that of the a term first coined by Taylor and Weston (1988), was in- AA metabolites, an EET is the most likely candidate. More- over, GC-MS analysis of coronary perfusates revealed the Currently, there is considerable support for a P450-derived release of EETs but not HETEs. EETs are vasodilator, syn- metabolite of AA as an EDHF (Bauersachs et al., 1994; thesized by the endothelium and stimulate Caϩϩ-activated Hecker et al., 1994; Campbell et al., 1996; Popp et al., 1996) Kϩ channels (Campbell et al., 1996). Our pharmacological although problems with the specificity of inhibitors of P450 studies indicate that, of the four EET regioisomers, only 5,6 have culminated in several recent studies that question this EET can fulfill the criteria for a putative mediator of thecoronary vasodilator effect of bradykinin (Quilley et al., Received for publication January 26, 1998.
1 This work was supported by National Institutes of Health Grant 49275 and American Heart Association Grant 940-318.
However, there are major reservations concerning the pro- ABBREVIATIONS: EDHF, endothelium-derived hyperpolarizing factor; AA, arachidonic acid; NO, nitric oxide; P450, cytochrome P450; EET,
epoxide; HETE, hydroxyeicosatetraenoic acid; PLC; phospholipase C; PLA , phospholipase A ; GC-MS, gas chromatography-mass spectrometry.
Anandamide and Vasodilation to BK
posal that a P450-AA metabolite may be a hyperpolarizing n ϭ 4), charydotoxin (10 nM; n ϭ 5) and SR 141716A (2 ␮M; n ϭ 6).
factor. These revolve primarily around the limited specificity Thus, we have previously reported that the coronary vasodilator of the inhibitors that have been used to implicate P450 (Ed- activity of bradykinin is reduced by nifedipine and charybdotoxin wards et al., 1996; Fukao et al., 1997; Ohlmann et al., 1997).
(Fulton et al., 1994) whereas the vasodilator effect of anandamide in As a result, other potential mediators have been sought and the rat mesenteric vascular bed is inhibited by SR 141716A (Randallet al., 1996). The antagonists were added to the perfusate at least 10 Randall et al. (1996) proposed that anandamide, the ethano- min before obtaining responses to anandamide and AA. The concen- lamide of AA and the putative endogenous ligand for canna- tration of SR 141716A was twice that used by Randall et al. (1996) binoid receptors (Devane et al., 1992), may be an EDHF in whereas the concentration of nifedipine and charybdotoxin were the rat. Thus, a product with the chromatographic properties those we had previously shown to inhibit coronary vasodilator re- of authentic anandamide was released from the perfused sponses to bradykinin (Fulton et al., 1994). Three to four prepara- mesenteric vascular bed labeled with 3H-AA and challenged tions per day were completed and at least one served as a control; the with carbachol and the mesenteric vasodilator effect of anan- others were assigned randomly to each of the treatment groups. In damide was greatly reduced in the presence of depolarizing the experiments with SR 141716A, responses to nitroprusside (1 ␮g) concentrations of KCl, suggesting a role for activation of Kϩ were used an index of effects apparently unrelated to antagonism of channels (Randall et al., 1996). This group also reported that cannabinoid receptors. In the experiments with nifedipine and SR141716A which both reduced coronary vascular tone, U46619 was the NO-independent hypotensive effect of bradykinin in the added to the perfusate (10 ng/ml for nifedipine and 0.5–1.0 ng/ml for anesthetized rat was attenuated by pretreatment with a SR 141716A) to restore perfusion pressure to its previous level.
cannabinoid receptor antagonist that also blocked the effect In a second series of experiments, we compared the effects of SR of anandamide in the mesenteric vasculature (Randall et al., 141716A (2 ␮M; n ϭ 4) or vehicle (n ϭ 4) on coronary vasodilator 1996). In contrast to these studies in the rat, Pratt et al. responses to bradykinin (10 –1000 ng) as the hypotensive response to (1998) reported that the vasorelaxant effect of anandamide in bradykinin in anesthetized rats has been reported to be attenuated the bovine coronary artery was independent of cannabinoid by pretreatment with SR 141517A (Randall et al., 1996). Responses receptors but involved the release of AA and its subsequent to cromakalim (1, 3 and 10 ␮g) were used to assess any direct effects conversion to vasodilatory eicosanoids.
of SR 141716A on Kϩ channels and unrelated to cannabinoid recep- Consequently, we used pharmacological criteria, based on In a third series of experiments, vasodilator responses to anand- our studies with bradykinin, to examine whether anandam- amide (3 and 10 ␮g) and bradykinin (30 and 100 ng) were compared ide could fulfill the requirements for a putative mediator for in the absence (n ϭ 6) and presence (n ϭ 5) of the P450 inhibitor, bradykinin-induced vasodilation in the isolated heart of the clotrimazole (1 ␮M), as coronary vasodilator responses to bradykinin rat. Thus, we determined coronary vasodilator responses to have been shown to be attenuated by clotrimazole (Fulton et al., anandamide in the presence and absence of nifedipine to 1995) and anandamide has been reported to be a substrate for P450 prevent vasodilation resulting from closure of voltage-depen- (Bornheim et al., 1993). However, if anandamide is the mediator of dent Caϩϩ channels, charydotoxin to inhibit Caϩϩ-activated bradykinin-induced vasodilation, then clotrimazole should be with- Kϩ channels and SR 141716A to antagonize cannabinoid out effect on responses to anandamide although attenuating those to receptors. The effects of SR 141517A on responses to brady- bradykinin. We chose clotrimazole, despite reports of effects on Kϩ kinin were also determined. As anandamide is readily channels, because it is considered to be more specific for epoxygenasethan ␻-hydroxylase. Moreover, at the concentration chosen (1 ␮M), cleaved by an amidase to yield AA, the effects of these inter- we have no evidence for effects on Kϩ channels as clotrimazole did ventions on responses to AA were also examined. We also not affect vasodilator response to cromakalim or SCA 40 (Fulton et compared the effects of a P450 inhibitor, clotrimazole, on al., 1994) which has been reported to stimulate Caϩϩ-activated Kϩ responses to bradykinin and anandamide as this compound channels (Laurent et al., 1993).
is a substrate for P450 (Bornheim et al., 1993). The results Statistics. Vasodilator responses in control and treatment groups
indicate that anandamide is unlikely to be the mediator of were compared by analysis of variance and individual points were bradykinin-induced, NO-independent vasodilation in the rat compared by Neuman-Keuls test. Differences were considered sta- tistically significant when P Ͻ .05.
Materials. Anandamide was obtained from Biomol (Plymouth
Meeting, PA) and was dissolved in ethanol. Indomethacin, nitroargi- nine, bradykinin, nifedipine, cromakalim, clotrimazole and nitro-prusside were purchased from Sigma Chemical Co. (St. Louis, MO).
Male Wistar rats, weight 360 to 460 g, were anaesthetized with Indomethacin was dissolved in 4.2% NaHCO , clotrimazole in etha- pentobarbital, 65 mg/kg i.p., and heparin, 1000 U/kg, was adminis- nol and cromakalim in ethanol before dilution with saline. The other tered i.v. After thoracotomy, the heart with attached aorta was agents were dissolved in distilled water. Charybdotoxin was pur- excised and flushed free of blood with ice-cold Krebs’ buffer. The chased from Peptides International (Louisville, KY) and was dis- heart was then cannulated via the aorta and perfused retrogradely solved in distilled water. SR141716A was a gift from RBI (Natick, with oxygenated Krebs’ buffer at 37°C at a constant flow rate (8 –10 MA) supported by NIMH Chemical Synthesis Program and was ml/min) to obtain an initial basal perfusion pressure of 30 to 40 dissolved in ethanol. U46619 was obtained from UpJohn (Kalmazoo, mmHg. The perfusate contained indomethacin (2.8 ␮M) to inhibit MI) and was dissolved in ethanol and diluted with distilled water.
cyclooxygenase and nitroarginine (50 ␮M) was added to inhibit NO Arachidonic acid (NuChek, Elysian, MN) was dissolved in distilled synthase and elevate perfusion pressure to 130 to 140 mmHg and also to reproduce the experimental conditions that were used toaddress the mechanism of bradykinin-induced vasodilation (Fultonet al., 1994, 1995, 1996).
Once a stable elevated perfusion pressure was obtained, vasodila- tor responses to increasing doses of anandamide (1, 3 and 10 ␮g) Initial basal perfusion pressures were not different in the were determined followed by responses to increasing doses of AA (1, various groups: vehicle, 37 Ϯ 2 mmHg; SR 141716A, 38 Ϯ 2 3 and 10 ␮g) in the absence (n ϭ 8) and presence of nifedipine (5 nM; mmHg; charybdotoxin, 39 Ϯ 2 mmHg and nifedipine, 42 Ϯ 3 Fulton and Quilley
mmHg. Elevated perfusion pressures were comparable in all Inhibition of voltage-dependent Caϩϩ channels with nifed- the groups except the charybdotoxin group where pressure ipine diminished the vasodilator effects of 3 and 10 ␮g anan- was further increased by inhibition of Caϩϩ-activated Kϩ damide (P Ͻ .05) and AA (P Ͻ .05) to a similar degree without channels to 155 Ϯ 4 mmHg compared to 134 Ϯ 2 mmHg for affecting the responses to the lowest doses of these agents vehicle, 138 Ϯ 3 mmHg for SR 141716A and 131 Ϯ 6 mmHg Inhibition of Caϩϩ-activated Kϩ channels with charydo- In the vehicle control group, 1, 3 and 10 ␮g anandamide toxin did not reduce the coronary vasodilator response to elicited dose-dependent falls in perfusion pressure of 11 Ϯ 2, anandamide (fig. 3), rather, the response to the lowest dose of 24 Ϯ 3 and 40 Ϯ 3 mmHg, respectively (fig. 1). The cannabi- anandamide was slightly increased from 11 Ϯ 2 to 16 Ϯ 2 noid receptor antagonist, SR 141716A, reduced the coronary mmHg (P Ͻ .05). In contrast, the coronary vasodilator effect vasodilator response to the two lower doses of anandamide, of AA was significantly reduced in the presence of charydo- 6 Ϯ 1 and 15 Ϯ 2 mmHg (P Ͻ .05), but was without effect on the highest dose, 36 Ϯ 3 mmHg. In contrast, the dose-depen- In the second series of experiments to determine the effects dent coronary vasodilator response to AA was unaffected by of SR 141716A on vasodilator responses to bradykinin and SR 141716A (fig. 1). SR 141716A did not affect vasodilator cromakalim, basal and elevated perfusion pressures in the responses to nitroprusside, 37 Ϯ 4 vs. 44 Ϯ 7 mmHg for the control and treatment groups were 34 Ϯ 2 and 134 Ϯ 6 Fig. 2. Vasodilator responses to anandamide (upper panel) and arachi-
donic acid (lower panel) in control hearts (solid bars) and in the presence
Fig. 1. Effect of the cannabinoid receptor antagonist, SR 141716A, (2 ␮M;
of nifedipine (5 nM; open bars). Heart were treated with indomethacin open bars) or vehicle (solid bars) on vasodilator responses to anandamide (2.8 ␮M) and nitroarginine (50 ␮M) to inhibit cyclooxygenase and NO (upper panel) and arachidonic acid (lower panel) in the rat isolated synthase and elevate perfusion pressure from 30 – 40 to 130 –140 mmHg.
perfused heart treated with indomethacin (2.8 ␮M) and nitroarginine (50 Nifedipine reduced elevated perfusion pressure that was restored with ␮M) which elevated perfusion pressure from 30–40 to 130–140 mmHg.
Anandamide and Vasodilation to BK
Fig. 4. Vasodilator responses to bradykinin in control hearts (solid bars)
and those treated with SR 141716A (2 ␮M; open bars). The coronary
perfusate contained indomethacin (2.8 ␮M) and nitroarginine (50 ␮M) to
inhibit cyclooxygenase and NO synthase and elevate perfusion pressure
from 30 – 40 to 130 –140 mmHg. SR 141716A reduced elevated perfusion
pressure which was restored with U46619 (0.5–1.0 ng/ml).
sure in the control group was 131 Ϯ 1 mmHg compared to128 Ϯ 2 mmHg in the clotrimazole group.
Figure 5 shows a recording of perfusion pressure from a vehicle-treated heart and the vasodilator responses to brady-kinin and anandamide. The response to bradykinin wasrapid in onset and of short duration whereas the response toanandamide developed more slowly and was of longer dura-tion.
Several studies have provided evidence to support a P450- derived metabolite of AA as an EDHF mediating the NO-independent vasodilator/vasorelaxant response to bradyki- Fig. 3. Effects of charybdotoxin (10 nM; open bars) compared to vehicle
(solid bars) on vasodilator responses to anandamide (upper panel) or
nin and/or acetylcholine (Hecker et al., 1994; Bauersachs et arachidonic acid (lower panel) in the isolated perfused heart treated with al., 1995; Campbell et al., 1996; Popp et al., 1996). Our indomethacin (2.8 ␮M) and nitroarginine (50 ␮M) to inhibit cyclooxygen- studies are consistent with this concept as the coronary ase and NO synthase and elevate perfusion pressure from 30 – 40 to130 –140 mmHg. Charybdotoxin caused a further elevation of perfusion and/or renal vasodilator action of bradykinin is susceptible to inhibitors of PLC and PLA , P450 and Caϩϩ-activated Kϩ channels (Fulton et al., 1992, 1994, 1995, 1996). Of the mmHg, respectively, and 36 Ϯ 2 and 138 Ϯ 5 mmHg, respec- P450-AA metabolites, an EET is considered the most likely tively. SR 141716A did not affect responses to bradykinin as an EDHF as EETs are produced by the endothelium and (fig. 4) but tended to reduce those to cromakalim although are vasodilator, presumably by their ability to activate Kϩ the differences were not significant. In control hearts, 1, 3and 10 ␮g cromakalim decreased perfusion pressure by 6 Ϯ 1,22 Ϯ 3 and 58 Ϯ 7 mmHg, respectively, compared to 5 Ϯ 3,13 Ϯ 4 and 43 Ϯ 4 mmHg, respectively, for hearts treatedwith SR 141716A.
In the presence of clotrimazole to inhibit P450, vasodilator responses to bradykinin were almost abolished, confirmingour previous results (Fulton et al., 1995). Thus, reductions inperfusion pressure to 30 and 100 ng bradykinin were 2 Ϯ 1and 6 Ϯ 2 mmHg, respectively, compared to control values of20 Ϯ 3 and 39 Ϯ 4 mmHg, respectively. Clotrimazole alsoreduced coronary vasodilator responses to 3 and 10 ␮g anan- Fig. 5. Recording of perfusion pressure in response to bradykinin, anan-
damide and cromakalim in the isolated heart treated with indomethacin
damide from 21 Ϯ 2 and 33 Ϯ 2 mmHg, respectively, to 12 Ϯ (2.8 ␮M) and nitroarginine (50 ␮M) to inhibit prostaglandin and NO 1 and 23 Ϯ 1 mmHg, respectively. Elevated perfusion pres- synthesis and elevate perfusion pressure to approximately 130 mmHg.
Fulton and Quilley
channels (Hu and Kim, 1993; Campbell et al., 1996). How- ization, for example. However, the Kϩ channels responsible ever, a role of P450 has been questioned as inhibitors of this for the effects of anandamide and AA must be different, pathway exhibit a variety of actions apparently unrelated to based on the results with charybdotoxin that markedly re- inhibition of P450 and including effects on Kϩ channels (Oye- duced the coronary vasodilator effect of AA but not that of kan et al., 1994; Edwards et al., 1996). Moreover, the admin- anandamide. These observations are good evidence against istration of EETs has been reported to be without effect in anandamide as a source of AA which then exerts a direct some vascular preparations (Zygmunt et al., 1996). Conse- effect or serves as a precursor for the formation of a product quently, alternative mediators have been sought and Randall that elicits vasodilation via a charybdotoxin-sensitive mech- et al. (1996) proposed the ethanolamide of AA (anandamide) anism. The alternative explanation, that AA stimulates an which is the putative endogenous ligand for cannabinoid endothelial Kϩ channel to initiate the release of a vasodilator receptors. We considered anandamide an attractive possibil- is untenable as inhibition of cannabinoid receptors with ity for mediating vasodilator responses to bradykinin be- SR141716A reduced responses to anandamide but failed to cause our previous results would not be inconsistent with influence responses to AA. The effect of SR141716A to reduce this concept; as an analogue of AA, anandamide would pre- responses to anandamide is unlikely to be due to an effect on sumably be stored in phospholipids and released by the ac- Kϩ channels as SR141716A did not affect responses to cro- tions of phospholipases whereupon it could also serve as a makalim and did not alter responses to bradykinin or AA substrate for P450 (Bornheim et al., 1993) to produce a va- which are dependent on activation of Kϩ channels.
sodilator that activates Kϩ channels.
Finally, we addressed the effect of an inhibitor of P450, To address this possibility, we determined the vasodilator clotrimazole, on the coronary vasodilator action of anandam- activity of anandamide in the presence of pharmacological ide as we have previously shown this agent reduces the interventions that inhibit NO-independent coronary vasodi- coronary and renal vasodilator actions of bradykinin. If lator responses to bradykinin. Under identical experimental anandamide itself is the mediator of the bradykinin effect, conditions of inhibition of prostaglandin and NO synthesis, then inhibition of P450 with clotrimazole should be without coronary vasodilator responses to anandamide were tested effect. Alternatively, if anandamide, after its release in re- after treatment of hearts with nifedipine, charybdotoxin, clo- sponse to bradykinin, requires conversion by P450 for activ- trimazole and SR141716A and compared to those obtained ity, then clotrimazole should inhibit the vasodilator effect of with AA or bradykinin. The results obtained argue against both anandamide and bradykinin to the same degree. Clo- anandamide as the mediator of bradykinin-induced vasodi- trimazole virtually abolished vasodilator responses to brady- lation. First, inhibition of Caϩϩ-activated Kϩ channels with kinin in this series of experiments, consistent with our pre- charybdotoxin at a concentration that almost abolished cor- vious observations (Fulton et al., 1995). In contrast, onary vasodilator responses to bradykinin (Fulton et al., inhibition of vasodilation induced by anandamide was much 1994) was without effect on vasodilator responses to anand- less pronounced, a result that provides further evidence amide. The only explanation for these observations that per- against anandamide as the mediator for bradykinin. How- mits consideration of anandamide as the vasodilator media- ever, the observation that clotrimazole reduced the vasodila- tor activity of anandamide indicates that an intact P450 charydotoxin-sensitive Kϩ channel in the endothelium to system may be required. Thus, anandamide can be a sub- result in the release of the mediator, in this case anandam- strate for P450 (Bornheim et al., 1993) although the activity ide. In this scenario, administration of the mediator, anand- of any products to elicit vasodilation remains to be deter- amide, would by-pass the processes involved in its synthesis mined. It is unlikely that anandamide first releases AA and/or release. Consequently, any intervention that modifies which is then converted by P450 to vasodilatory eicosanoids the response to anandamide should also modify that to the as suggested by Pratt et al. (1998) because charybdotoxin initiating stimulus, i.e., bradykinin. However, the failure of failed to affect dilator responses to anandamide but inhibited the cannabinoid receptor antagonist, SR 141716A, to inhibit those to AA. An alternative explanation for the inhibitory the vasodilator effect of bradykinin although reducing that to effects of clotrimazole on vasodilation induced by anandam- anandamide argues against this possibility regardless of ide is that clotrimazole exerts effects on Kϩ channels or even whether the effect of SR 141716A is via inhibition of canna- the cannabinoid receptor in addition to inhibiting P450.
binoid receptors or an alternative mechanism. Thus, the in- The results from this study, therefore, do not support the hibitory effect of SR 141716A on responses to anandamide hypothesis proposed by Randall et al. (1996) that anandam- was not pronounced and may reflect functional antagonism ide is an EDHF in the rat. However, they used the perfused (White and Hiley, 1997). Nonetheless, if anandamide is the mesentery and studied vasodilation to acetylcholine which mediator of bradykinin-induced vasodilation, then SR was inhibited by SR141716A as was the endothelium-inde- 141716A should also attenuate the response to bradykinin pendent vasodilator effect of anandamide, suggesting a role for CB receptors. This is in contrast to our studies where the The possibility that anandamide yields AA that then un- effects of bradykinin in the heart were addressed and which dergoes transformation by P450 to generate a vasodilator provides a possible explanation for the different results.
product was also addressed in this study. Thus, the relatively Thus, depending on the tissue and the agonist, different slow onset of vasodilation to anandamide compared with hyperpolarizing factors may be involved. However, the obser- bradykinin is consistent with conversion to an active product.
vation of Randall et al. (1996) that the NO-independent hy- The observation that nifedipine reduced the coronary vaso- potensive effect of bradykinin in the rat is also attenuated by dilator effects of both anandamide and AA is consistent with SR141716A, suggesting a mechanism operating through can- a common vasodilator mechanism that involves closure of nabinoid receptors, is not supported by our study.
voltage-dependent Caϩϩ channels in response to hyperpolar- In summary, our observations, when viewed collectively, Anandamide and Vasodilation to BK
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