Receptor-mediated activation of nitric oxide synthesis by arginine in endothelial cells

Mahesh S. Joshi*†, T. Bruce Ferguson, Jr.*, Fruzsina K. Johnson‡, Robert A. Johnson‡, Sampath Parthasarathy§, and Jack R. Lancaster, Jr.¶

Departments of *Surgery and §Pathology, Louisiana State University Health Sciences Center, New Orleans, LA 70112; ‡Department of Physiology, Tulane University Health Sciences Center, New Orleans, LA 70112; and ¶Departments of Anesthesiology, Physiology, and Biophysics and Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35487

Edited by Salvador Moncada, University of London, London, United Kingdom, and approved May 1, 2007 (received for review August 8, 2005) Arginine contains the guanidinium group and thus has structural through caveolae, a subcompartment of the plasma membrane (4, similarity to ligands of imidazoline and ␣-2 adrenoceptors (␣-2 AR). 5). Because of this compartmentalization, NO synthesis in certain Therefore, we investigated the possibility that exogenous arginine cells might require extracellular L-arginine (6), and thus suggesting may act as a ligand for these receptors in human umbilical vein a preferential channeling of extracellular arginine to the site of endothelial cells and activate intracellular nitric oxide (NO) syn- eNOS location (7). thesis. Idazoxan, a mixed antagonist of imidazoline and ␣-2 adre- There is ample evidence documenting the beneficial effects of noceptors, partly inhibited L-arginine-initiated NO formation as exogenous arginine both in animal and clinical studies. Acute and measured by a Griess reaction. Rauwolscine, a highly specific long-term oral administration of L-arginine has been associated antagonist of ␣-2 AR, at very low concentrations completely with a significant improvement in NO-dependent vasodilation in inhibited NO formation. Like L-arginine, agmatine (decarboxylated cholesterol-fed rabbits (8, 9). Benefits of exogenous arginine are arginine) also activated NO synthesis, however, at much lower realized both in healthy humans and in disease conditions. For concentrations. We found that dexmedetomidine, a specific ago- example, in healthy human subjects, i.v. infusion and oral admin- nist of ␣-2 AR was very potent in activating cellular NO, thus istration of L-arginine induced vasodilation (10). Oral arginine also indicating a possible role for ␣-2 AR in L-arginine-mediated NO improved endothelial dysfunction in patients with essential hyper- synthesis. D-arginine also activated NO production and could be tension (11). Plasma and intracellular levels of arginine are altered inhibited by imidazoline and ␣-2 AR antagonists, thus indicating in certain disease states, hypertension, hypercholesterolemia, and nonsubstrate actions of arginine. Pertussis toxin, an inhibitor of G diabetes (12–14). As part of the treatment regimen, L-arginine may proteins, attenuated L-arginine-mediated NO synthesis, thus indi- serve as a safer and more easily administered alternative to inhaled ؉ cating mediation via G proteins. L-type Ca2 channel blocker NO gas or NO donors. nifedipine and phospholipase C inhibitor U73122 inhibited NO Three important metabolic end products of L-arginine, namely formation and thus implicated participation of a second messenger NO, glutamate, and agmatine, are cell-signaling molecules. The pathway. Finally, in isolated rat gracilis vessels, rauwolscine com- decarboxylation of L-arginine, through the action of arginine de- pletely inhibited the L-arginine-initiated vessel relaxation. Taken carboxylase (ADC), forms agmatine and CO2. The finding that together, these data provide evidence for binding of arginine to agmatine is present in serum (15) raises the issue of whether the membrane receptor(s), leading to the activation of endothelial NO amine participates in vascular function. Agmatine was shown to synthase (eNOS) NO production through a second messenger stimulate nitrite formation as well as Ca2ϩ uptake in endothelial pathway. These findings provide a previously unrecognized mech- cells (16). Most recently, a new member of the C family of G anistic explanation for the beneficial effects of L-arginine in the protein-binding receptors, GPRC6A, has been shown to bind a cardiovascular system and thus provide new potential avenues for structurally wide range of L-␣-amino acid analogues of arginine, therapeutic development. lysine, and ornithine as agonists (17). However, its physiological role is unknown. Li et al. (18) and Reis et al. (19) have suggested agmatine ͉ rauwolscine ͉ calcium agmatine as an endogenous ligand for ␣-2 adrenoreceptor (␣-2 AR) and imidazoline receptor (I-receptor). Most imidazolines and struc- ␣ rginine is critical to normal growth and multiple physiological turally related ligands (Fig. 1) bind to both I-receptor and -2 AR. Aprocesses. It serves as a precursor for the synthesis not only of Therefore, these two receptors are always studied together with proteins but also of NO, urea, polyamines, and agmatine. The respect to their mediation in cellular signal transduction mecha- unequivocal demonstration that NO is the product of NO synthase nisms. Endothelial cells have been shown to express a number of (NOS)-catalyzed oxidation of L-arginine led to widespread interest receptors: adrenoceptors (20, 21), imidazoline (22), bradykinin (23, in the actions of L-arginine. The K of L-arginine for endothelial 24), purinoceptor (25, 26), and adenosine A2 receptor (27). I- m ␣ NOS (eNOS) is determined to be 2.9 ␮M (1), and the intracellular receptors are a class of nonadrenergic receptors, and -2 AR L-arginine concentrations are in the range of 0.8–2.0 mM. In other belongs to the class of G protein-coupled receptors (GPCR). These words, cells maintain saturating levels of L-arginine as a substrate receptors are shown to mediate cellular NO formation and re- ␣ for NO synthases. However, an external supply of L-arginine is still laxation. Liao and Homcy (23), using an -2 AR agonist, have required for the cellular production of NO (2). This requirement of exogenous arginine for the cellular NO production is termed Author contributions: M.S.J. and J.R.L. designed research; M.S.J., F.K.J., and R.A.J. per- ‘‘arginine paradox.’’ A number of mechanisms have been proposed formed research; M.S.J., T.B.F., S.P., and J.R.L. analyzed data; and M.S.J. wrote the paper. to address this phenomenon, including endogenous NOS inhibitors The authors declare no conflict of interest. L and compartmentalization of intracellular -arginine. Some have This article is a PNAS Direct Submission. proposed that endogenous NOS inhibitors [e.g., asymmetric di- Abbreviations: ␣-2 AR, ␣-2 adrenoceptor; eNOS, endothelial NOS; HUVEC, human umbilical methylarginine (ADMA)] modulate NO levels by antagonizing vein endothelial cell; I-receptor, imidazoline receptor; IP3, inositol-1,4,5-triphosphate; NOS, intracellular L-arginine (3). An alternative view hypothesizes that NO synthase. L-arginine is compartmentalized within the cell, and part of the †To whom correspondence should be addressed at: Department of Physics, Wake Forest cellular pool of L-arginine is not readily available for eNOS. As part University, Winston-Salem, NC 27109. E-mail: [email protected]. of this hypothesis, it has been proposed that NO signaling occurs © 2007 by The National Academy of Sciences of the USA

9982–9987 ͉ PNAS ͉ June 12, 2007 ͉ vol. 104 ͉ no. 24 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0506824104 Downloaded by guest on September 30, 2021 N O A B C Idazoxan O N H

Cl H N Moxonidine H3C N N H H3CO

2H H N H N CH N (CH2)3 C L-arginine N HOOC H2

2 Fig. 2. Arginine dose–response and the effect of NOS inhibitor and receptor H H H N N antagonist. (A) Dose-dependent activation of cellular NO synthesis by L- CH N 2 (CH2)3 C Agmatine arginine ( , P Ͻ 0.05 vs. 0; n ϭ 3). (B) Effect of eNOS inhibition with 5 mM N * H2 L-NG-nitroarginine methyl ester (L-NAME) on L-arginine (5 mM) -initiated Ϫ Ϫ ϭ ϭ NO2 /NO3 formation. (*, P 0.08 vs. 5 mM L-arginine; n 3). (C) Effect of Ϫ Ϫ Fig. 1. Structures of guanidinium-containing compounds. Idazoxan and idazoxan (10 ␮M) on L-arginine (5 mM) -mediated NO2 /NO3 formation in moxonidine are the ligands for I-receptor and ␣-2 AR. The guanidinium groups HUVEC cultures (#, P ϭ 0.033 vs. 5 mM L-arginine; n ϭ 3). The experiments were are marked with a dotted box. carried out in 12-well plates at 120,000 cells per well in 95% air/5% CO2 Ϫ Ϫ incubator at 37°C for 30 min. The NO2 /NO3 levels were measured by a Ϫ Ϫ modified Griess reaction that can detect NO2 /NO3 levels in the lower documented the formation of NO in bovine aortic endothelial nanomolar range. cells, which was inhibited by G protein inhibitor pertussis toxin. ␣ Also, -2 AR agonists clonidine and UK14304 are found to mediate Ϫ Ϫ endothelium-dependent relaxation in rat aorta (28). The activation L-arginine and measured NO2 /NO3 formation as stable end of G proteins results in elevated phospholipase C activity, leading products of cellular NO synthesis. L-arginine, at levels present in to hydrolysis of phosphatidylinositol-4,5-biphosphate, which yields normal plasma (0.2 mM), did not activate NO synthesis (Fig. 2A). second messengers, inositol-1,4,5-triphosphate (IP3) and diacyl- However, NO was activated at higher millimolar concentrations. 2ϩ glycerol. IP3 subsequently mediates release of Ca from ER. Then the effect of antagonists on NO formation from L-arginine Cytosolic Ca2ϩ may be the most widely used second messenger in was studied. The eNOS inhibitor, L-NAME, reversed the L- Ϫ Ϫ biology. In endothelial cells, ER accounts for Ϸ75% of the total arginine-mediated NO2 /NO3 formation (Fig. 2B), thus showing 2ϩ 2ϩ Ϫ Ϫ [Ca ]i and IP3, and ryanodine receptors mediate [Ca ]i release that NO2 /NO3 formation is the result of NO metabolism. The from ER. Most functions in these cells depend to various extents on cell cultures were also treated with the hybrid antagonist of 2ϩ changes in the [Ca ]i, including the process of eNOS activation. I-receptor and ␣-2 AR, idazoxan, in the presence and absence of Tsukahara et al. (29) have observed that when human umbilical L-arginine. Idazoxan partially inhibited L-arginine-initiated NO vein endothelial cells (HUVECs) were supplied with L-arginine, formation (Fig. 2C). However, the specific ␣-2 AR antagonist, there was a transient stimulation in NO production, which returned rauwolscine, completely blocked NO formation at very low con- to baseline levels within a few minutes. If exogenous L-arginine were centrations (Fig. 3A). These results show that L-arginine activates BIOCHEMISTRY acting as a substrate, then one would expect a continuous gener- cellular NO production possibly through receptor mediation. ation of NO, because cells have significant levels of L-arginine. This It is also possible that rauwolscine is blocking NO production in observation prompted us to speculate that the stimulatory action of a nonspecific manner at a site downstream from the receptor. To exogenous L-arginine may not be due to its actions as a substrate. rule out this possibility, we treated cells with calcium ionophore Instead, L-arginine may bind to the same or similar receptor(s) as A23187 in the presence and absence of rauwolscine. As shown in agmatine, activating intracellular NOS via signal transduction Fig. 3B, rauwolscine failed to block NO formation from A23187, mechanisms involving second messenger systems and store- 2ϩ thus showing that inhibition in Fig. 3A is due to the specific effect operated Ca channels. There is no evidence in the literature of antagonist on receptor(s). about the receptor-mediated actions of L-arginine to activate intracellular NO synthesis. Understanding of receptor-mediated Agonist of ␣-2 AR Activates NO. If L-arginine is activating NO L-arginine actions will have wide implications in vascular function production by binding to ␣-2 AR on endothelial cell membranes, with respect to the biological actions of L-arginine and NO. This ␣ system will permit further elucidation of the potential importance then a known agonist of -2 AR should activate NO synthesis as well. To explore this possibility, we used dexmedetomidine, a new of L-arginine-receptor transduction mechanisms in regulating NO ␣ production and will potentially provide new avenues for therapeutic specific agonist of -2 AR. Dexmedetomidine dose-dependently development. Here we have investigated the membrane receptor activated NO production at very low concentrations (Fig. 3C). binding of exogenous L-arginine, which resulted in activation of These data show that endothelial cells can produce NO through ␣ NO in HUVECs. The findings show that both L-and D-arginine activation of -2 AR. receptor-dependently activate cellular NO independent of sub- strate effects. D-Arginine Activated NO and Its Inhibition with Antagonists of I- Receptor and ␣-2 AR. The rationale for this experiment is that Results D-arginine is not a substrate for eNOS reaction to form NO and L-N G-Nitroarginine Methyl Ester (L-NAME) and Antagonists of I-Receptor L-citrulline. However, if D-arginine activates NO, then the NO and ␣-2 AR Block L-Arginine-Initiated NO. We first conducted dose– formation must be due to its nonsubstrate effects. Accordingly, the response experiments with increasing concentrations of exogenous cells were treated with D-arginine in the presence and absence of

Joshi et al. PNAS ͉ June 12, 2007 ͉ vol. 104 ͉ no. 24 ͉ 9983 Downloaded by guest on September 30, 2021 1.2 A 0.8 B 2.0 1.0 0.6 * 0.8 * , µM -

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1 µM A23187 + NO 0.8 - 2 5 mM L-arginine

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0.001 0.05 0.5 + 200 ng/ml PTx + 400 ng/ml PTx 5 mM L-arginine

Dexmedetomidine, nM

None Fig. 4. Effect of pertussis toxin (PTx) on NO synthesis. The cell cultures (120,000 cells per well) were pretreated with pertussis toxin at 200 and 400 ng/ml for 2 h, followed by L-arginine (5 mM) in the presence and absence of 5 mM D-arginine

+ 50 µM Idazoxan pertussis toxin for 30 min at 37°C in a 5% CO2/95% air incubator. The culture Ϫ Ϫ + 0.2 nM Rauwolscine supernatants were assayed for NO2 /NO3 by modified Griess reaction (n ϭ 3; *, P Ͻ 0.05 vs. ϩ 400 ng/ml PTx; #, P ϭ 0.077 vs. none).

Fig. 3. Effect of ␣-2 AR antagonist on the L-arginine and A23187-mediated NO formation. (A and B) The cell cultures (120,000 cells per well) were treated activation of G proteins may in turn activate phospholipase C, with either 5 mM L-arginine (A)or1␮M A23187 (B) in the presence and yielding IP and diacylglycerol. As shown in Fig. 5A, treatment of absence of 0.2 nM rauwolscine for 30 min at 37°C in a 5% CO2/95% air 3 Ϫ Ϫ incubator. The supernatants were assayed for NO2 /NO3 by modified a Griess cells with phospholipase C inhibitor U73122 in the presence and reaction. (A) n ϭ 4; *, P Ͻ 0.05 vs. 5 mM L-arginine. (B) n ϭ 4; *, P Ͻ 0.05 vs. none. absence of L-arginine resulted in complete inhibition of NO for- (C) Dose–response of ␣-2 AR agonist dexmedetomidine. The cells (180,000 cells mation, thus implying a role for phospholipase C reaction products. per well) were treated with increasing concentrations of dexmedetomidine Ϫ Ϫ for 60 min. The culture supernatants were used for a NO2 /NO3 assay by a Ca2؉ Influx Precedes NO Formation. The cellular NO formation ϭ Ͻ Griess reaction (n 4; *, P 0.05 vs. 0.001 nM dexmedetomidine). (D) The depends on the influx of Ca2ϩ. To test this possibility, we tested the cultures (120,000 cells per well) were treated with 5 mM D-arginine in the 2ϩ presence and absence of 0.2 nM rauwolscine and 50 ␮M idazoxan for 30 min effect of the Ca channel blocker nifedipine. The treatment of cell L in Krebs solution at 37°C in 5% CO2/95% air incubator. The cell supernatants cultures with nifedipine in the presence and absence of -arginine Ϫ Ϫ ϭ ϭ were assayed for NO2 /NO3 by a modified Griess reaction (n 4; *, P 0.009 resulted in complete inhibition of NO formation (Fig. 5A). We also ϩ vs.5mMD-arginine; #, P ϭ 0.007 vs. 5 mM D-arginine; $, P ϭ 0.031 vs. 5 mM analyzed Ca2 influx after treatment with exogenous L-arginine by D-arginine). fluorescence microscopy. The cells were loaded with fluorescence ϩ Ca2 indicator fluo 4 and then treated with L-arginine. As shown in Fig. 5B, there was a Ca2ϩ spike that quickly dissipated with time. antagonists of I-receptor and ␣-2 AR. The results demonstrate that ϩ These data demonstrate that L-arginine mediates influx of Ca2 D-arginine activated cellular NO production, and this NO synthesis through Ca2ϩ channels, thus leading to NO synthesis. could be inhibited by both idazoxan and rauwolscine (Fig. 3D). This finding strongly indicated that arginine may be acting as a ligand on ␣-2 AR Antagonist Attenuates L-Arginine-Initiated Gracilis Vessel membrane receptor(s). D-arginine was shown to act as a vasodilator Relaxation. We wanted to extend the observations made in cell in human forearm when locally infused (30, 31). culture experiments to isolated vessel system to demonstrate the vascular relevance of receptor-mediated actions of L-arginine. We Pertussis Toxin Attenuates L-Arginine-Mediated NO Formation. Be- isolated rat gracilis vessels and treated them with 1 mM L-arginine cause ␣-2 AR is a GPCR, it is possible that L-arginine activates G proteins, leading to the formation of cellular NO. Pertussis toxin is in a vessel chamber superfused with a nonrecirculating system (described in Materials and Methods). This treatment led to signif- a specific inhibitor of heterotrimeric G protein of the type, Gi/o (32, 33). It does so by ADP-ribosylating the ␣ subunit of G protein. icant dilation of spontaneously constricted vessels (Fig. 5C). The Therefore, we pretreated the cell cultures with pertussis toxin and same experiment carried in the presence of very low concentrations ␣ then treated with L-arginine in the presence and absence of of -2 AR antagonist rauwolscine (0.2 nM) resulted in complete indicated doses of pertussis toxin. We observed that pertussis toxin attenuation of vessel dilation (Fig. 5C). These data indicate that dose-dependently inhibited NO formation from L-arginine (Fig. 4). receptor(s) may mediate L-arginine-initiated vessel relaxation. These results indicate participation of G proteins in the actions of extracellular L-arginine. Agmatine Activated NO Synthesis and Its Inhibition with ␣-2 AR Antagonist. Millimolar levels of arginine are required to activate Exogenous L-Arginine Instigates Formation of Second Messengers. cellular NO synthesis. It is very unlikely that arginine will act as a Because L-arginine treatment activates GPCR and G proteins, it is direct receptor agonist at this high concentration. However, it is imperative to know the rest of the signal transduction pathway. The possible that arginine undergoes decarboxylation via arginine de-

9984 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0506824104 Joshi et al. Downloaded by guest on September 30, 2021 DAF-FM diacetate. As shown in Fig. 5D, agmatine activated NO A B ␮ 30 at low concentrations (10 M, 10 min) and this could be attenuated

3.0 with rauwolscine (0.2 nM). * 25 In summary, our data demonstrates that exogenous L-arginine 2.5 20 and agmatine activate cellular NO formation through receptor binding. This involves the second messenger pathway of activated 2.0 ϩ

, µM 15 2 -

3 phospholipase C and Ca influx.

1.5 10 + NO - Discussion 2 1.0 5 NO The idea that the substrate of an enzymatic reaction also acts as an

0.5 Units Relative Fluorescence 0 exogenous ligand for a receptor and initiates signal transduction across the cell membrane seems quite intriguing. However, a closer 0.0 -5 0 20406080100120 look at the structures of L-arginine and its decarboxylated product,

None Time, sec agmatine, show that both have a guanidinium group and thus partly resemble the structures of imidazolines (Fig. 1). The compounds

3 µM U-73122 with imidazoline/guanidinium group are known to act as ligands for + 3 µM U-73122 5 µM Nifedipine 5 mM L-arginine + 5 µM Nifedipine both I-receptors and ␣-2 AR. The activation of these receptors initiates cascade of cellular processes with varying functions, in- C D 25 cluding vasodilation, renal sodium excretion, and modulation of 16 * ocular pressure. Agmatine was demonstrated more than a decade * 20 ago to be a ligand for I-receptors and ␣-2 AR in the brain, and it 12 was proposed to be an endogenous ligand for I-receptor (18, 19). 15 Similarly, many biological actions of L-arginine may involve its 8 binding as ligand on membrane receptor(s) and affect the release 10 of NO through signal transduction mechanisms. 4

Max. Dilation, µm We observed that millimolar levels of exogenous L-arginine were 5 required to produce significant cellular NO production, and L- 0 Units Relative Fluorescence arginine at concentrations present in normal human plasma failed 0 to activate NO (Fig. 2A). In vivo studies have demonstrated that -4 plasma arginine levels reached 822 Ϯ 59 ␮M upon i.v. infusion of exogenous L-arginine (6 g). However, this level of arginine had no effect on vasorelaxation. Significant relaxations in humans have only been observed when the plasma arginine levels reach 6.22 Ϯ Untreated 0.4 mM (34–36) upon infusion of significantly higher amounts (30

10 µM Agmatine

1 mM L-arginine g). Our data show that exogenous L-arginine stimulates NO pro- + 0.2 nM Rauwolscine

+ 0.2 nM Rauwolscine duction in HUVECs, and this could be attenuated by hybrid ␣ ␣ ϩ I-receptor and -2 AR antagonist idazoxan and specific -2 AR Fig. 5. Ca2 dependence of L-arginine activity and agmatine-mediated L cellular NO synthesis. (A) Effect of Ca2ϩ channel blocker and phospholipase C antagonist rauwolscine. This result indicates that -arginine may be inhibitor on NO formation. The cell cultures were treated with 5 mM L- acting by way of receptor(s). Rauwolscine was more potent that arginine in the presence and absence of 5 ␮M nifedipine and 3 ␮M U73122. idazoxan in attenuating L-arginine effects. Thus, it appears that ␣-2 The experiments were conducted at 37°C for 30 min in a 5% CO2/95% air AR is the more likely target for L-arginine binding. These obser- Ϫ Ϫ incubator. The culture supernatants were assayed for NO2 /NO3 by using a vations from cell cultures were extended to isolated vessels where modified Griess reaction (n ϭ 3; *, P Ͻ 0.05 vs. none). (B) Fluorescence we find potent antagonistic effects of rauwolscine on L-arginine- recording of fluo-4-loaded cells (1 ␮M in a six-well plate; 150,000 cells per well) initiated dilation (Fig. 5C). The role of ␣-2 AR in the vascular BIOCHEMISTRY with excitation at 488 nm and emission at 526 nm. A 10 mM aliquot of relaxation was previously demonstrated in isolated rings of rat 2ϩ L-arginine was added (at arrow), and a time course of Ca fluorescence was superior mesenteric arteries (37). In these studies, rauwolscine recorded using a Leica deconvolution microscope with a Xenon light source. Digitized images were captured with Slidebook software, and this software is effectively blocked agonist-mediated relaxation. used for time lapse and excitation of the fluorophore probe. The data are The observation that millimolar levels of arginine are required representative of three similar recordings. (C) Effect of ␣-2 AR antagonist on for the activation of cellular NO production raises the possibility L-arginine-mediated dilation. The gracilis anticus muscle segments of the that either arginine is binding to a low affinity binding site on cell first-order gracilis muscle arterioles were isolated as described in Materials membrane or it is metabolizing to form a product that in turn acts and Methods. The individual arteriolar segments were cannulated at both as a direct ligand on the receptor. Agmatine could be one such ends in a water-jacketed vessel chamber. The distal micropipette was con- product because endothelial cells do possess arginine decarboxylase nected to a stopcock, and the proximal micropipette was connected to a (ADC) activity (38). We found that agmatine at low micromolar reservoir, the height of which was adjusted to achieve 80-mmHg intraluminal concentrations activated NO synthesis and could be attenuated with pressure. Test compounds (L-arginine and rauwolscine) were included in the rauwolscine (Fig. 5D). Morrisey and Klahr (16) have similarly superfusion buffer (*, P Ͻ 0.05 vs. ϩ 0.2 nM rauwolscine; n ϭ 3). (D) Effect of ␣-2 AR antagonist on the agmatine-mediated NO formation. The 90% con- demonstrated endothelial NO synthesis in response to agmatine fluent cells preloaded with 5 ␮M DAF-FM diacetate were treated with 10 ␮M exposure. This could be inhibited by idazoxan (16), thus suggesting agmatine in the presence and absence of 0.2 nM rauwolscine for 10 min as possible involvement of receptor(s) in the actions of agmatine. But described in Materials and Methods. The fluorescence as a measure of NO a detailed study of this agmatine binding and the signal transduction synthesis was measured using a UV confocal microscope (n ϭ 3; *, P Ͻ 0.05 vs. pathway that leads to the activation of cellular NO production are untreated). lacking. Also, histamine, acetylcholine, adenosine, and clonidine have been demonstrated to enhance endothelium-dependent va- sorelaxation via specific membrane receptors (39). Thus, there is carboxylase (ADC) to form agmatine. This agmatine in turn will precedence for agonist activation of various receptors in endothelial bind to the membrane as agonist and activate NO synthesis. We cells resulting in cellular NO synthesis. A nonsubstrate role for tested this hypothesis by exposing the cell culture to exogenous arginine was supported by the observation that NO was stimulated agmatine and measured NO synthesis by using fluorescent dye, by exogenous D-arginine and was attenuated by idazoxan and

Joshi et al. PNAS ͉ June 12, 2007 ͉ vol. 104 ͉ no. 24 ͉ 9985 Downloaded by guest on September 30, 2021 rauwolscine (Fig. 3C). Several in vivo studies corroborate this agonist and activating eNOS (and thus relaxation) through signal observation. D-arginine was found to increase venous diameter in transduction, as proposed here. humans and hypotension in rats (30, 40). Rhodes et al. (31) have In conclusion, this investigation using endothelial cells shows that found that D-isomer of arginine was more potent than L-isomer as exogenous arginine or agmatine can act as an agonist for membrane vasodilator of human forearm. However, mediation of NO in these receptor(s). This binding of arginine activated a cascade of signal studies was not clearly documented, which raises the important transduction involving G proteins, phospholipase C, and mobiliza- ϩ question about whether other structural analogs of L-arginine may tion of intracellular Ca2 , resulting in activation of eNOS to also exogenously activate endothelial NO. produce NO. These findings could alter many fundamental aspects To further clarify the receptor type, we tested the effect of of cardiovascular physiology by revealing that various effects of specific ␣-2 AR agonist, dexmedetomidine. If arginine is stimulat- exogenous arginine are due to its actions on receptor(s), either ing NO synthesis by binding to ␣-2 AR as an agonist, then directly as agonist or via catalytic conversion to agmatine. This dexmedetomidine must also activate NO. We found that dexme- finding may lead to the development of new areas of therapeutic detomidine dose-dependently stimulated endothelial NO produc- strategies in a variety of cardiovascular diseases. tion (Fig. 3C). Other ␣-2 AR agonists also have been shown to stimulate NO synthesis in endothelial cell cultures (41) and to Materials and Methods stimulate NO-dependent vasodilation in animals (28). However, Cell Culture and Reagents. HUVECs were obtained from American L-arginine targeting receptor(s) other than ␣-2 AR cannot be Type Culture Collection (Manassas, VA). The cells were cultured discounted, because rauwolscine is also known to act on serotonin in media consisting of modified Kaigan’s F-12 (American Type [5-hydroxytryptamine (5-HT)] receptors (42, 43). Culture Collection) supplemented with 10% FBS (American Type If L-arginine is binding to the membrane receptor(s) and acti- Culture Collection), 0.1 mg/ml heparin (Sigma Chemical, St. Louis, vating intracellular NO production, then it is imperative to know the MO), 0.03 mg/ml endothelial cell growth supplement (Upstate, signal transduction mechanism involved. Pertussis toxin, which Lake Placid, NY), 100 units/ml penicillin, and 100 ␮g/ml strepto- inhibits G proteins by ADP-ribosylation of ␣ subunit, attenuated mycin in a humidified chamber at 37°C in a 5% CO2/95% air NO synthesis from L-arginine (Fig. 4). Thus, our data showed atmosphere. The cells were cultured in T75 flasks and 12-well tissue mediation of G proteins in the actions of L-arginine, which is culture plates. For these studies, the cells were used for up to eight consistent with the receptor being a GPCR. The sensitivity to passages. pertussis toxin indicates that the heterotrimeric G protein may be The experiments were conducted by preincubating cultures in of the type Gi. In endothelial cells, ␣-2 AR and other receptors are Krebs solution for 30 min. The cells were then treated with arginine believed to couple selectively to heterotrimeric Gi proteins (23). A and/or various compounds for another 15–30 min in a humidified similar observation was made with UK14304, an agonist of ␣-2 AR, chamber at 37°C in a 5% CO2/95% air atmosphere. Both L-arginine wherein pretreatment with pertussis toxin attenuated agonist- and D-arginine (Sigma Chemical) were in free acid form and initiated NO synthesis in bovine aortic endothelial cells (41) and prepared in Hepes buffer, pH 7.4. L-arginine had undetectable relaxations in porcine coronary arteries (24). Signaling via G levels of D-isomer as contaminant, and vice versa with D-arginine. proteins leads to a variety of cellular responses, including activation of phospholipase C, yielding second messengers, diacylglycerol and Nitrate/Nitrite Determination. At the end of incubations, the culture IP3. Activation of several receptor systems that lead to endothelial supernatants were collected by centrifugation (10,000 ϫ g for 10 NO synthesis has been shown to involve G protein-coupled signal- min) in a Centra MP4R centrifuge (International Equipment, ing via phospholipase C activation (44–46). A similar pathway may Needham Heights, MA). The supernatants were used for deter- be responsible for L-arginine-mediated NO signaling. The activa- mination of nitrate/nitrite by a modification of Griess reaction (52). tion of phospholipase C ultimately results in initiation of Ca2ϩ Briefly, nitrate in the samples was first converted into nitrite with signaling that plays important second messenger roles. Our results nitrate reductase. After an incubation of 45 min, the samples were ϩ show that L-arginine treatment yielded a spike in Ca2 as measured mixed with a solution consisting of equal volumes of 14 mM by Ca2ϩ indicator dye, fluo 4 (Fig. 5B). This observation prompted dapsone (4, 4Ј-diamino-diphenylsulpfone) and 4 mM N-(1- an important question about the interdependence of L-arginine- Naphthyl)ethylenediamine. The color was allowed to develop for 5 mediated NO synthesis and Ca2ϩ spike. The Ca2ϩ channel blocker, min, and absorbance was read at 550 nm in a Bio-Rad (Hercules, nifedipine, and the phospholipase C inhibitor, U73122, both atten- CA) plate reader. The nitrate/nitrite was also determined using a uated L-arginine-initiated NO production (Fig. 5A), thus showing chemiluminescence technique (53). The nitrate in the samples was that NO synthesis depends on intracellular Ca2ϩ mobilization. converted to nitrite with nitrate reductase and then analyzed by In atherosclerosis or hypercholesterolemia, impaired NO syn- chemiluminescence. The nitrite was determined as the difference thesis is believed to be a major contributor to endothelial dysfunc- between in the presence and absence of 0.5% sulfanilamide in 0.1 tion (47–49). Interestingly, bradykinin- and A23187-mediated re- M HCl. laxations are not affected, but that relaxation due to acetylcholine is impaired. The prevailing hypothesis is that receptor-mediated NO Measurements with DAF-FM Diacetate. The fluorescence mea- relaxation via Gi proteins is affected in this condition (50). Whereas surements were made according to published procedures (54). In acetylcholine-mediated NO formation is via Gi proteins, that due to brief, the cells grown on a chambered borosilicate coverglass system bradykinin is not coupled to Gi. Thus, the endothelial dysfunction were incubated with serum-free and phenol red-free M 199 me- is not a general phenomenon in atherosclerosis/hypercholesterol- dium for 60 min. These cells were then loaded with 5 ␮M DAF-FM emia. In attempts to correct the endothelium dysfunction, i.v. diacetate (Invitrogen, Carlsbad, CA) for 30 min at 37°C. After L-arginine injections were able to restore acetylcholine-mediated loading, the cells were washed with the media and then treated with relaxations (51). Unfortunately, the plasma L-arginine levels in agmatine for 10 min in the presence and absence of rauwolscine at hypercholesterolemia were not determined. However, we hypoth- room temperature. The cellular fluorescence was measured using esize that exogenous L-arginine restored the depleted substrate a Leica (Exton, PA) DMIRBE inverted epifluorescence /Nomarski levels occurring during the development of hypercholesterolemia. If microscope outfitted with Leica TCS-NT/SP1 laser confocal optics this argument were true, then bradykinin- and A23187-mediated and appropriate filters for fluorescence microscopy. relaxation should also be affected in hypercholesterolemia. There- fore, the availability of L-arginine as substrate for eNOS may not be Ca2؉ Measurements. The fluorescence recording of fluo-4-loaded the limiting factor. The L-arginine reversal of hypercholesterolemic cells (1 ␮Mina35ϫ 10-mm plate; 150,000 cells per plate) was dysfunction may, however, be due to L-arginine acting as a receptor monitored with excitation at 488 nm and emission at 526 nm. A 10

9986 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0506824104 Joshi et al. Downloaded by guest on September 30, 2021 ϩ mM aliquot of L-arginine was added, and a time course of Ca2 with a video camera leading to a video caliper. Mounted vessels fluorescence was recorded using a Leica deconvolution microscope were allowed to stabilize for 60 min before initiation of dilation with a Xenon light source. Digitized images were captured with experiments. During the stabilization period, the internal diameter Slidebook software, and this software was used for time-lapse and of the vessels decreased spontaneously from 193.5 Ϯ 22.92 ␮mto excitation of the fluorophore probe (55). 135.92 Ϯ 10.38 ␮m(n ϭ 4). Only those vessels that developed an active tone during the stabilization period were used for the studies. Isolated Rat Gracilis Vessel Studies. The gracilis anticus muscles were Test compounds (L-arginine and rauwolscine) were included in the removed from rats (male, Sprague–Dawley), and segments of the superfusion buffer. first-order gracilis muscle arterioles were isolated as described in Ϯ ref. 56. The individual arteriolar segments were cannulated at both Statistical Analysis. Data are expressed as mean SD values for the number of culture preparations indicated in the figure legends. ends in a water-jacketed vessel chamber. The distal micropipette Ͻ was connected to a stopcock, and the proximal micropipette was Statistical significance was evaluated by one-way ANOVA, and P n connected to a reservoir, the height of which was adjusted to 108.8 0.05 is considered significant. The values represent the number of independent experiments. cm to achieve 80 mmHg intraluminal pressure. The vessel chamber was superfused continuously via a nonrecirculating system with We thank Dr. Daniel Kim-Shapiro for providing laboratory space and oxygenated (14% O2/5% CO2-balance N2) modified Krebs buffer facilities to carry out part of this work. This work was supported in part (5 ml/min) at 37°C. For internal diameter measurements, the vessel by National Institutes of Health Grants HL074391 and HL71189 (to chamber was mounted on the stage of a microscope that was fitted J.R.L.).

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Joshi et al. PNAS ͉ June 12, 2007 ͉ vol. 104 ͉ no. 24 ͉ 9987 Downloaded by guest on September 30, 2021