Agmatine induced NO dependent rat mesenteric artery relaxation and its impairment in salt-sensitive hypertension

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Nitric Oxide xxx (2013) xxx–xxx

YNIOX 1311 No. of Pages 7, Model 5G

3 September 2013

Contents lists available at ScienceDirect

Nitric Oxide

journal homepage: www.elsevier .com/locate /yniox

Agmatine induced NO dependent rat mesenteric artery relaxationand its impairment in salt-sensitive hypertension q

1089-8603/$ - see front matter � 2013 The Authors. Published by Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.niox.2013.08.005

Abbreviations: ADC, arginine decarboxylase; DFMA, difluoromethylarginine;eNOS, endothelial nitric oxide synthases; L-NAME, L-NG-nitroarginine methyl ester;AR, adrenergic receptor; PTx, pertussis toxin.

q This is an open-access article distributed under the terms of the CreativeCommons Attribution-NonCommercial-No Derivative Works License, which per-mits non-commercial use, distribution, and reproduction in any medium, providedthe original author and source are credited.⇑ Corresponding author. Fax: +1 305 348 6954.

E-mail address: [email protected] (M.S. Joshi).

Please cite this article in press as: T.V. Gadkari et al., Agmatine induced NO dependent rat mesenteric artery relaxation and its impairment in salt-sehypertension, Nitric Oxide (2013), http://dx.doi.org/10.1016/j.niox.2013.08.005

Tushar V. Gadkari, Natalie Cortes, Kumpal Madrasi, Nikolaos M. Tsoukias, Mahesh S. Joshi ⇑Department of Biomedical Engineering, Florida International University, Miami, FL 33174, United States

a r t i c l e i n f o

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Article history:Received 17 June 2013Revised 15 July 2013Available online xxxx

Keywords:AgmatineArginine decarboxylaseL-ArginineNitric oxidea-2 Adreno receptorHypertension

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a b s t r a c t

L-Arginine and its decarboxylated product, agmatine are important mediators of NO production and vas-cular relaxation. However, the underlying mechanisms of their action are not understood. We have inves-tigated the role of arginine and agmatine in resistance vessel relaxation of Sprague-Dawley (SD) and Dahlsalt-sensitive hypertensive rats. Second or 3rd-order mesenteric arterioles were cannulated in an organchamber, pressurized and equilibrated before perfusing intraluminally with agonists. The vessel diame-ters were measured after mounting on the stage of a microscope fitted with a video camera. The geneexpression in Dahl rat vessel homogenates was ascertained by real-time PCR. L-Arginine initiatedrelaxations (EC50, 5.8 ± 0.7 mM; n = 9) were inhibited by arginine decarboxylase (ADC) inhibitor,difluoromethylarginine (DFMA) (EC50, 18.3 ± 1.3 mM; n = 5) suggesting that arginine-induced vesselrelaxation was mediated by agmatine formation. Agmatine relaxed the SD rat vessels at significantlylower concentrations (EC50, 138.7 ± 12.1 lM; n = 22), which was compromised by L-NAME (L-NG-nitroarg-inine methyl ester, an eNOS inhibitor), RX821002 (a-2 AR antagonist) and pertussis toxin (G-proteininhibitor). The agmatine-mediated vessel relaxation from high salt Dahl rats was abolished as comparedto that from normal salt rats (EC50, 143.9 ± 23.4 lM; n = 5). The a-2A AR, a-2B AR and eNOS mRNAexpression was downregulated in mesenteric arterioles of high-salt treated Dahl hypertensive rats. Thesefindings demonstrate that agmatine facilitated the relaxation via activation of a-2 adrenergic G-proteincoupled receptor and NO synthesis, and this pathway is compromised in salt-sensitive hypertension.

� 2013 The Authors. Published by Elsevier Inc. All rights reserved.

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Introduction and compartmentalization of intracellular arginine [10–13]. How- 61
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Over the past 2 decades there has been a significant interest inunderstanding the role of NO in vascular relaxation. L-Arginineserves as a substrate for different nitric oxide synthases (NOS).The potential use of exogenous L-arginine as a therapeutic agenthas been widely suggested in various physiological and pathophys-iological processes such as wound healing [1–3], protein synthesisand muscle building [4], endocrine metabolism [5], erectile dys-function [6], and a variety of cardiovascular functions [7–9]. Sup-plying arginine to the endothelial cells and vessels contributes tothe enhanced NO synthesis and vessel relaxation despite its satu-rating cellular levels. Different mechanisms are hypothesized toexplain this arginine paradox including endogenous NOS inhibitors

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ever, there still exists an ambiguity in understanding how exoge-nous L-arginine mediates NO-dependent relaxation [14]. Theapparent benefits of L-arginine supplementation are difficult toreconcile with a purely substrate based mechanism for NO synthe-sis. An alternative proposed by us explains arginine’s NOS sub-strate independent actions via the activation of a-2 adrenergicreceptor (a-2 AR) as demonstrated in cultured endothelial cells[15].

Agmatine [4-(aminobutyl)guanidine] is produced endogenouslyvia decarboxylation of L-arginine by the endothelial arginine decar-boxylase (ADC) [16,17] and it does not act as a substrate for NOS. Abiological function for agmatine was suggested based on the obser-vation that ADC activity transiently increased sevenfold duringcerebral ischemia [18]. In addition, the importance of agmatinehas been highlighted by its discovery as a novel neurotransmitter[19–21] demonstrating its potential to affect multiple biologicaltargets. The presence of agmatine in serum [22] suggests a physi-ological role in the vasculature. Agmatine was shown to serve asa ligand for imidazoline and/or a-2 AR [23] and a-2 AR agonistsmediate endothelium-dependent relaxation in mouse and rat aorta[24]. a-2 ARs (G-protein coupled receptors) play a pivotal role inthe cardiovascular system and influence vascular tone at multiple

nsitive

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3 September 2013

points. These receptors are targets for antihypertensive therapyand their stimulation produces long lasting drop in systemic bloodpressure [25]. However, the signaling mechanisms participating inagmatine-initiated NO synthesis [15,26] and regulation of vasculartone is little understood, and the contribution of a-2 ARs is impli-cated in this process [15]. Compromised NO synthesis and endo-thelial dysfunction has been reported in the hypertensivevasculature including salt-sensitive hypertension [27,28]. How-ever, the factors that are responsible for its impaired synthesisare varied and not clearly understood. Impaired a-2 AR functionhas been documented in several models of hypertension [29–31].However, whether this impairment is a cause or an effect of hyper-tension remains to be elucidated.

Here we show that arginine-mediated arteriolar relaxations aredue to agmatine produced by the actions of ADC and signaling viaGPCR in rat microcirculation. Evidence is also presented document-ing attenuated agmatine-mediated relaxation in Dahl salt-sensitivehypertensive rat mesenteric resistant arterioles, which correlateswith reduced a-2 AR gene expression.

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Materials and methods

Isolated mesenteric arteriole preparation

Resistance mesenteric arterioles of the 2nd or 3rd order (restingdiameter 6150 lm) were isolated from male Sprague-Dawley (SD)and Dahl rats (250–300 g), cleaned of the surrounding tissue andcannulated at both ends on glass cannula. The organ chamberwas maintained constant at 37 �C by superfusion with a modifiedKrebs–Ringer solution containing (mM): NaCl 145, KCl 5, CaCl2

2.5, MgSO4 1.2, KH2PO4 1.2, HEPES 20, and Glucose 10.1, pH 7.4[32,33]. Cannulated vessels were axially pre-stretched to removeany bents and to simulate physiological stretch conditions andwere pressurized at 50 mmHg and allowed to equilibrate for60 min before initiating the experiment. To establish a concentra-tion that gave submaximal constriction, we constructed a dose–re-sponse curve to norepinephrine (NE). The vessels werepreconstricted with continuous superfusion of NE and those thatretained a constant pressure and a consistent constriction to NE(Fig. S1a and b), and fully responded to the acetylcholine(Fig. S3a) were included in the study. The presence of functionalendothelium was assessed by the ability of acetylcholine (10 lM)to induce more than 90% relaxation. The vessel reactivity studywas carried out by intraluminal perfusion with various agonistconcentrations. This was achieved by an automated solenoid valvecontrolled pressure driven perfusion system. Diameter measure-ments were tracked in real time by mounting the perfusion cham-ber on the stage of an inverted microscope (Olympus, CenterValley, PA) fitted with a CCD camera (QImaging, Surrey, Canada).Post analysis was performed with IPLAB (BioVision Technologies,Exton, PA) and MATLAB (MathWorks, Natick, MA) software. Chem-icals NE, L-NAME (L-NG-nitroarginine methyl ester), RX821002,agmatine, L-arginine were obtained from Sigma–Aldrich Co. (St.Louis, MO) and pertussis toxin (PTx) were obtained from TocrisBioscience (Ellisville, MO) and SPER-NO from Cayman Chemicals(Ann Arbor, MI).

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Animal model

Male SD and Dahl salt-sensitive (SS/JrHsd) rats and their dietwere purchased from Harlan Laboratories (Madison, WI). SpragueDawley rats were maintained on standard pellet chow (2018Teklad Global) rodent diet whereas the Dahl salt-sensitive ratswere fed 0.49% NaCl diet (Harlan Cat. #TD 96208) or 4% NaCl diet(Harlan Cat. #TD 92034). The animals were housed in temperature

Please cite this article in press as: T.V. Gadkari et al., Agmatine induced NO depehypertension, Nitric Oxide (2013), http://dx.doi.org/10.1016/j.niox.2013.08.005

and humidity controlled rooms with 12 h on/off light cycle at theanimal care facility. All animal studies were performed followingInstitutional Animal Care and Use approved procedures.

After acclimatization for 1 week, 6-weeks old Dahl salt-sensi-tive rats were separated into 2 diet groups; normal salt (NS), fed0.49% NaCl diet and high salt (HS), fed 4% NaCl diet for 5 weeks.Systolic blood pressure was measured weekly by the tail-cuffmethod [34] and HS rats consistently demonstrated sustainedhypertension (BP > 200 mmHg) while NS rats remained normoten-sive (BP < 160 mmHg) (Fig. S2). The rats were euthanized by CO2

inhalation and vascular reactivity assessed on isolated, cannulatedand pressurized mesenteric arterioles as described above.

Determination of plasma nitrite

Blood derived from rats was centrifuged immediately at 5000gfor 5 min and plasma collected. The nitrite analysis was carried outusing iodine/iodide in glacial acetic acid supplemented with 1% v/vantifoam SE-15 (Sigma–Aldrich) using an ozone based chemilumi-nescence analyzer (Sievers, model 280i) as described [35].

Real time-polymerase chain reaction (RT-PCR)

RT-PCR was carried out on mesenteric tissue from Dahl rats[36], cleaned of fat and stabilized with RNAlater (Qiagen, Valencia,CA). The tissue was homogenized (�30 mg) with a sonicator in RLTbuffer (Qiagen), the lysate centrifuged (10,000g) and total RNApurified with reagents from RNeasy� Fibrous Tissue Mini Kit (Qia-gen). A first strand cDNA synthesis was performed using purifiedmRNA by Superscript III RT (Invitrogen, Grand Island, NY) in a ther-mocycler (MJ Research). The new cDNA strand was purified withQIAquick� PCR Purification Kit (Qiagen). Pure cDNA (�10 ng) wasreacted with Power SYBR Green PCR Master Mix reagent (AppliedBiosystems, Mountain View, CA) in RNase-free water in a StepOneRT-PCR system (Applied Biosystems). The relative expressionof a-2A, a-2B AR and eNOS was determined using b-actin as ahousekeeping gene. The primers used were: a-2A; TTT GCA CGTCGT CCA TAG TG (forward) and CAG TGA CAA TGA TGG CCT TG (re-verse). a-2B; AAA CAC TGC CAG CAT CTC CT (forward) and CTG GCAACT CCC ACA TTC TT (reverse). eNOS; CAA CGCTAC CAC GAG GACATT (forward) and CTC CTG CAA AGA AAA GCT CTG G (reverse).b-actin; TCC TAG CAC CAT GAA GAT C (forward) and AAA CGCAGCTCA GTA ACA G (reverse). Standard curves (initial amount ofcDNA versus Ct values) were tested for each set of primers, demon-strating that for the similar range of total cDNA amplification theefficiency of target genes and housekeeping gene (b-actin) wereequal. ‘No reverse transcription control’ was used where the PCRreaction was run in the absence of reverse transcriptase. Expres-sion of the gene of interest was divided by the housekeeping geneand expressed as fold-change compared with the correspondingnormal-salt rat group.

Data analysis

Relaxations were expressed as percentage of NE (2 lM) inducedcontraction. The vasodilation was studied by obtaining the maxi-mal response and EC50 values were then calculated by fitting theconcentration–response relationship to a logistic function. Ampli-fied transcripts from RT-PCR were quantified using the compara-tive threshold cycle method (2�DDCt) with b-actin as anormalizer and the corresponding sample from the normal saltfed rat mesentery as internal control.

All data were expressed as mean ± SEM with n representingindependent rat experiments. Statistical significance was testedusing a paired t-test with P < 0.05 considered significant.

ndent rat mesenteric artery relaxation and its impairment in salt-sensitive


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T.V. Gadkari et al. / Nitric Oxide xxx (2013) xxx–xxx 3


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Results

L-Arginine-mediated relaxation is dependent upon ADC activity

As shown in Fig. 1a, L-arginine dose-dependently relaxed thevessel with an EC50 value of 5.8 ± 0.7 mM (n = 9). The requirementof mM levels of arginine prompted us to hypothesize that the ac-tions of arginine may be mediated via its metabolism to agmatineby ADC, which is shown to be localized in the endothelium. Therelaxations to arginine were significantly inhibited in the presenceof ADC inhibitor, DFMA (Fig. 1a: EC50, 18.3 ± 1.3 mM; n = 5). TheEC50 value for L-arginine increased several fold in the presence ofADC inhibitor. DFMA is a specific inhibitor of ADC [37] and its spec-ificity in our system was verified by the absence of any effect onagmatine-mediated vessel relaxation (Fig. 1b). Thus, these experi-

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(c)Fig. 1. Concentration dependant relaxation responses to L-arginine and agmatine. (a) Darterioles and after pre-treatment with ADC blocker, DFMA (0.5 mM) (n = 5); #P < 0.05perfusion of agmatine in rat mesenteric arterioles in the presence and absence of an eNOare mean ± SE with ⁄P < 0.05 vs. agmatine; ⁄⁄P > 0.05 vs. agmatine. (c) Dose response toantagonist, RX821002 (50 nM) (n = 6); ⁄P < 0.05 vs. agmatine. (d) Agmatine relaxation remean ± SE (n = 4); ⁄P < 0.05 vs. agmatine.


ments demonstrated that the arginine’s actions are mediated atleast in part by the formation of agmatine.

Agmatine-induced vessel relaxation

To examine the effect of agmatine treatment on vessel tone, theisolated mesenteric arterioles were subjected to increasing agma-tine concentrations by intraluminal perfusion. Agmatine dose-dependently relaxed the vessel with an EC50 of 138.7 ± 12.1 lM(Fig. 1b; n = 22). Thus, significantly less agmatine was required ascompared to arginine for arteriolar relaxation. As illustrated inFig. 1b, the agmatine-mediated relaxation was partially NO depen-dent as eNOS inhibitor, L-NAME (0.5 mM) did not completelyattenuate the relaxation (EC50, 346.0 ± 19.4 lM; n = 4).

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(d)ose–response to intraluminal perfusion of L-arginine (n = 9) in SD rat mesentericvs. L-arginine. (b) Concentration dependent dose response curve to intraluminal

S blocker, L-NAME (0.5 mM) (n = 4) and ADC blocker, DFMA (0.5 mM) (n = 3). Valuesagmatine in SD rat vessels was obtained in the presence and absence of an a-2 AR

sponse in the absence and presence of G-protein inhibitor, PTx (100 nM). Values are



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3 September 2013

a-2 AR activity in agmatine-mediated relaxation

It has been previously reported that agmatine acts as an a-2 ARligand [23]. To validate if the agmatine-induced relaxation is med-iated via a-2 AR, we treated the vessels with agmatine in the pres-ence and absence of RX821002, a specific antagonist of a-2 AR[38,39]. As shown in Fig. 1c, it partly attenuated agmatine-mediated relaxation (EC50, 1498.0 ± 294.0 lM; n = 6) indicatingthat a-2 AR may be participating in the relaxation process. Thesedata corroborate our earlier observation in cultured HUVECs usinganother selective a-2 AR antagonist, rauwolscine, where it attenu-ated arginine-induced cellular NO synthesis [15].

Inhibition of agmatine-mediated relaxation by pertussis toxin

To narrow down the downstream mechanisms that may be par-ticipating in the agmatine-induced relaxation, we examined thepossible role of G-proteins. We inhibited the G-proteins by pre-treating the vessel with PTx (100 nM) for 60 min at 37 �C. Asshown in Fig. 1d, we observed a significantly reduced relaxationas opposed to that due to agmatine alone while the EC50 value in-creased to 1300 ± 91 lM (n = 4). These data indicated that G-pro-teins may mediate agmatine-induced vessel relaxation. Similarinhibition with PTx was observed in our experiments with culturedHUVECs [15].

Agmatine and arginine-mediated vessel relaxation attenuated in DSrats

Accumulating evidence indicates that endothelium-dependentrelaxation is impaired in salt-sensitive hypertension [40–42].However, the underlying mechanisms have not been clearly delin-eated. We investigated if the agmatine-induced arterial relaxationis affected in Dahl salt-sensitive rats. Six-week old rats were placedon a diet containing either 0.49% NaCl (normal salt) or 4% NaCl(high salt) for 5 weeks and isolated mesenteric arterial relaxationswere recorded in response to agmatine dose. The agmatine-in-duced relaxation was greatly inhibited in high-salt as comparedto normal salt conditions (Fig. 2a; % max. relaxation, 90.4 ± 1.7%(NS); 19.8 ± 2.4% (HS)) with normal salt rats exhibiting relaxation

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(a)Fig. 2. Relaxation response to agmatine and L-arginine in Dahl salt-sensitive hypertensDahl rats maintained on high salt, HS (4% NaCl) and normal salt, NS (0.49% NaCl) for 5 wagmatine.


to physiological agmatine concentration (EC50, 143.9 ± 23.4 lM;n = 5). Similarly, L-arginine-mediated relaxation was impaired inhigh-salt diet rats (Fig. 2b). Ach mediated relaxation was also im-paired in vessels from high salt Dahl rats (Fig. S3b) and thus veri-fying the presence of endothelial dysfunction. However, relaxationdue to an NO donor, SPER-NO was not affected, thereby indicatingan undiminished signaling pathway downstream of NO synthesisin Dahl hypertensive rats (Fig. S4). These data illustrate that agm-atine-mediated signal transduction pathway eliciting vascularrelaxation is severely impaired in salt-induced hypertension.

Attenuated NO synthesis in Dahl rats

Plasma nitrite levels, as a measure of endogenous NO synthesis,were analyzed by ozone based chemiluminescence analyzer. Theresults indicated that NO synthesis was significantly attenuatedin high salt rats as compared to normal salt rats (Fig. 3a). Thesedata are supported by the similar observations made by Fujiiet al. [43].

Down-regulation of mesenteric artery a-2 AR mRNA expression inDahl rats

Since agmatine is proposed to act via a-2 AR binding, we carriedout mRNA expression analysis of a-2 AR and eNOS in Dahl rats byreal time RT-PCR. The mRNA expression of a-2A, a-2B AR and eNOSwere attenuated in high-salt Dahl rat mesenteric arteries as com-pared to normal-salt arterioles (Fig. 3b) indicating that mRNAexpression levels of these genes were reversed during the develop-ment of salt-sensitive hypertension. Among the 3 genes analyzed,there was a 10-fold under-expression of a-2A AR mRNA.

Discussion

Although the beneficial effects of arginine in vascular relaxationare well documented and appreciated, the mechanism of its actionis not clearly understood. Arginine serves a major role by acting asa substrate for NOS and synthesizing NO, which in turn partici-pates in signal transduction of vascular relaxation. Since millimolaramounts of arginine are required to produce measurable NO levels

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(b)ion. (a) Agmatine and (b) L-arginine-mediated relaxation observed in salt-sensitive

eeks, (a: n = 5, b: n = 3). Values are mean ± SE, #P < 0.05 vs. L-arginine, ⁄P < 0.05 vs.



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(b)Fig. 3. Compromised nitric oxide activity in Dahl salt-sensitive hypertension. (a) Plasma nitrite concentrations in HS and NS fed Dahl rats (n = 3). (b) Real time PCR data for a-2A AR, a-2B AR and eNOS mRNA expression. Mesenteric artery tissue was harvested and total RNA isolated from NS and HS rats. The semi-quantitative RT-PCR was carried outusing SYBR Green technology in a StepOne RT-PCR system. Values are mean ± SE and expressed as fold change in mRNA expression in HS rats as compared to that in NS rats(n = 4).



3 September 2013

[44], it was speculated that arginine’s metabolic products could becontributing to its observed effects including vasodilation [15]. Wehave proposed that some of the observed effects of arginine couldbe attributed to its conversion to agmatine by ADC.

Our important finding that the inhibition of ADC with DFMAsignificantly attenuated arginine-initiated mesenteric arterial dila-tion, thereby pointing toward an arginine-to-agmatine conversionfor the observed relaxation. The endothelium is known to possessADC activity [16], which was documented to mediate an increasedvasodilation and glomerular filtration rate following arginine infu-sion into animals [45,46]. In macrophages under inflammatoryconditions, ADC was shown to modulate agmatine levels, whichin turn down-regulated iNOS catalyzed NO synthesis [47]. Higherlevels of ADC are present in the brain [48]and hence it can be spec-ulated that agmatine synthesized may have biological role in thedistant organs in particular the vascular function. We observedthat agmatine initiated the relaxation at a significantly lower con-centration (EC50, 138.7 ± 12.1 lM; n = 22) as compared to arginine.The increased vessel relaxation with lM agmatine is physiologi-cally relevant since the plasma agmatine levels have been reportedin the lM range [49,50]. Agmatine was shown to activate NO syn-thesis in endothelial cells but the mechanism of activation was notelucidated [26,51]. The partial attenuation of agmatine-initiatedrelaxation by L-NAME indicated that agmatine partly triggeredthe relaxation via NO independent, possibly EDHF mediated path-way. Binding of agmatine to imidazoline or a-2 AR is well docu-mented in the mammalian system. Our hypothesis that agmatineacts by receptor binding, possibly via a-2 AR (Fig. 4), was examinedby using an a-2 AR antagonist. The observation that RX821002 didnot completely attenuate agmatine-triggered relaxation impliedthat agmatine affecting relaxation partly via a-AR independentpathway. Recently, agmatine’s relaxing effects in rat aorta are doc-umented to be via small conductance Ca2+-activated K+ channeland ATP-sensitive inward rectifying K+ channels, and idazoxanfailed to inhibit these relaxations [52]. It appears that rat aorta isdevoid of I-receptors, as Musgrave et al. [53] have reported theabsence of I-receptor antagonist effects, and thus it is likely thatagmatine is using alternative pathways of aortic relaxation.

Since a-2 AR belongs to GPCR family, we tested the effects ofG-protein inhibition. PTx, a potent G-protein inhibitor, consider-


ably attenuated agmatine-initiated relaxation. The inhibitoryeffects of a-2 AR antagonist (RX821002) and G-protein inhibitor(PTx) together provide stronger evidence for the possible participa-tion of GPCR in the vasodilatory actions of agmatine. Using 2knockout mouse models, Shafaroudi et al. [24] have demonstratedthe mediation of endothelial a-2A AR in vasodilation viaNO-dependent mechanisms. These receptors may play importantphysiological functions as they are activated by the endogenous li-gand NE. In addition, there is evidence for their participation invarious pathophysiological conditions. For example, blood pres-sure was elevated in transgenic a-2A AR knockout mice on a nor-mal salt diet [54], showing that a-2A AR activation with specificagonists may serve to alleviate hypertensive conditions.

Dahl hypertensive rats are reported to exhibit endothelial dys-function [55] and agmatine-mediated vascular relaxation couldbe impaired in this model. Small size arterioles (100–500 lmdiameter) are proposed to contribute significantly to the vascularresistance [56]. Our measurements of agmatine-initiated vesselrelaxation showed that relaxation was severely impaired in highsalt as compared to normal salt diet fed Dahl rats. This observationsupports our postulation that a-2A AR activity and associated sig-naling pathway is down-regulated in high salt treated rats leadingto impairment of agmatine-mediated relaxation. Secondly, mRNAanalysis by real time PCR showed suppression of a-2A and a-2B

AR expression in the mesenteric artery isolated from high saltfed Dahl rats as compared those from normal salt fed rats. Therewas noticeably a robust 10-fold down-regulation of a-2A AR, whichcould be the predominant factor responsible for the observed abol-ishment of vessel relaxation in HS rats. Hirano et al. [57] have alsoobserved attenuated pre- and post-synaptic a-2A AR reactivity inDahl rats fed high salt diet. The attenuated L-arginine and agma-tine-mediated relaxation could be also due to the observeddown-regulation of eNOS expression. Similarly, down-regulationof eNOS protein expression was observed in kidney and aorta fromDahl salt-sensitive rats [58]. Our finding that substantial depletionin plasma nitrite levels in hypertensive rats could be the result ofeither decreased L-arginine/agmatine levels and/or reduced a-2A

AR and eNOS expression. Further investigations are warranted todelineate the impaired agmatine pathway as the underlying causeof endothelial dysfunction in hypertension.



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Fig. 4. Schematic representation of the proposed receptor-mediated hypothesis for arginine/agmatine stimulation of vascular NO synthesis.



3 September 2013

In summary, we have demonstrated the mediation of ADC inthe arginine-initiated stimulation of rat mesenteric artery relaxa-tion. Physiological concentrations of agmatine activated vesselrelaxation via a-2 AR and G-proteins and this was partially depen-dent on vascular NO synthesis. Agmatine relaxed the vessels at 100times lower concentration as compared to arginine and thus mak-ing it a physiologically relevant agonist at a-2 AR binding site.Most importantly, our investigations have provided a novel mech-anistic explanation for the observed beneficial effects of L-arginine-initiated vessel relaxation via agmatine formation. Lastly, a-2 ARactivity, mRNA expression, and agmatine-mediated relaxationsare impaired in vessels isolated from high salt loaded Dahl hyper-tensive rats. These findings may help define fundamental aspectsof cardiovascular physiology and pathophysiology by demonstrat-ing that many effects of arginine may occur due to its metabolismto agmatine.

Acknowledgments

This work was supported by the National Institutes of Healthgrant (NMT). We wish to thank Deepak Balasubramanian and KalaiMathee for their valuable help in carrying out RT-PCR experimentsand analysis of data.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.niox.2013.08.005.


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Agmatine induced NO dependent rat mesenteric artery relaxation and its impairment in salt-sensitive hypertension