Reduced L-Carnitine Transport in Aortic Endothelial Cells from Spontaneously Hypertensive Rats

Impaired L-carnitine uptake correlates with higher blood pressure in adult men, and L-carnitine restores endothelial function in aortic rings from spontaneously hypertensive rat (SHR). Thus, endothelial dysfunction in hypertension could result from lower L-carnitine transport in this cell type. L-Carnitine transport is mainly mediated by novel organic cation transporters 1 (Octn1, Na+-independent) and 2 (Octn2, Na+-dependent); however, their kinetic properties and potential consequences in hypertension are unknown. We hypothesize that L-carnitine transport kinetic properties will be altered in aortic endothelium from spontaneously hypertensive rats (SHR). L-Carnitine transport was measured at different extracellular pH (pHo 5.5–8.5) in the absence or presence of sodium in rat aortic endothelial cells (RAECs) from non-hypertensive Wistar-Kyoto (WKY) rats and SHR. Octn1 and Octn2 mRNA relative expression was also determined. Dilation of endothelium-intact or denuded aortic rings in response to calcitonine gene related peptide (CGRP, 0.1–100 nmol/L) was measured (myography) in the absence or presence of L-carnitine. Total L-carnitine transport was lower in cells from SHR compared with WKY rats, an effect due to reduced Na+-dependent (Na+ dep) compared with Na+-independent (Na+ indep) transport components. Saturable L-carnitine transport kinetics show maximal velocity (V max), without changes in apparent K m for Na+ indep transport in SHR compared with WKY rats. Total and Na+ dep component of transport were increased, but Na+ indep transport was reduced by extracellular alkalization in WKY rats. However, alkalization reduced total and Na+ indep transport in cells from SHR. Octn2 mRNA was higher than Octn-1 mRNA expression in cells from both conditions. Dilation of artery rings in response to CGRP was reduced in vessels from SHR compared with WKY rats. CGRP effect was endothelium-dependent and restored by L-carnitine. All together these results suggest that reduced L-carnitine transport (likely via Na+-dependent Octn2) could limit this compound's potential beneficial effects in RAECs from SHR.


Introduction
Essential hypertension is characterized by high blood pressure without an identifiable primary cause [1,2]. Oral administration of L-carnitine, a natural amino acidic compound, in patients with hypertension resulted in an improvement of arterial blood pressure, thus suggesting a beneficial vascular role for this compound in these subjects [3,4]. Along with the well-described role of L-carnitine on fatty acid mitochondrial metabolism [5,6], L-carnitine also increases the metabolic activity in human vascular endothelium [7,8], and improves the bioavailability of nitric oxide (NO) in rat aorta [9] and in fetal lamb pulmonary vasculature [10]. Other studies show that L-carnitine supplementation in healthy subjects improved postprandial flow-mediated dilation after a high-fat meal [11]. Taken together, all these findings suggest that the transport of L-carnitine into the endothelial cells could be an essential, limiting step of its potential beneficial biological effects in hypertension.
The results of this study show that aorta endothelial cells from SHR exhibit reduced maximal L-carnitine transport capacity compared with cells from non-hypertensive animals. Transport of L-carnitine was saturable and mediated by a larger Na +independent, Octn1-like compared with a Na + -dependent, Octn2-like transport activity in these cells from SHR. However, similar Na + -dependent and Na + -independent transport components were found in non-hypertensive rats. It is suggested that the observed endothelial dysfunction in SHR could be due to reduced Na + -dependent transport of L-carnitine, which could limit the potential beneficial effects of this compound in the endothelial function in hypertension.

Ethics statement and animals
This investigation strictly conforms to the principles outlined in the European Union Guidelines on the protection of animals used for scientific purposes (DIRECTIVE 2010/63/EU of the European Parliament and of the Council). Protocols were approved by the Committee on the Ethics of Animal Experiments of the University of Sevilla (Spain). Normotensive male Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) aged 8 weeks were obtained from the French Animal Production Center, JANVIER S.A.S. (Saint Berthevin Cedex, France). Rats were housed at a temperature of 22-24uC in individual cages and freely fed (ad libitum) regular pellet diet (12 mm pellet, Harlan Laboratories, Indianapolis, USA) until they were 10 weeks of age (wa). They were divided into two groups of 15 animals each, i.e., WKY (control) and SHR (hypertensive) animals. Characterization of WKY rats and SHR in terms of the diastolic and systolic blood pressures and body weight was determined at arrival of the animals (8 wa) and at the moment of isolation of aorta endothelial cells (i.e., 10 wa) as reported [26]. Diastolic and systolic blood pressure values were not significantly different (P.0.05) at 8 wa (diastolic = 93 6 3 mmHg, systolic = 123 6 6 mmHg) compared with 10 wa (diastolic = 95 6 2 mmHg, systolic = 124 6 5 mmHg) in WKY rats. However, these parameters were elevated (P,0.01) in SHR at 8 wa (diastolic = 190 6 2 mmHg, systolic = 233 6 2 mmHg) and 10 wa (diastolic = 191 6 2 mmHg, systolic = 231 6 1 mmHg) compared with the corresponding systolic or diastolic values in WKY rats, but were not significantly different (P.0.05) between them in SHR.

Cell culture
Aortas from WKY rats and SHR aged 10 weeks were excised and placed in a petri dish containing phosphate-buffered saline (PBS) solution ((mmol/L) NaCl 130, KCl 2.7, Na 2 HPO 4 0.8, KH 2 PO 4 1.4 (pH 7.4, 4uC)). The tissue was rinsed by changing PBS until free of any visible blood, and the aorta was stripped of adventicia as reported [27]. Rat aorta endothelial cells (RAECs) were isolated by scraping of rat aortic lumen in the presence of medium 199 (M199) (Gibco Life Technologies, Carlsbad, CA, USA) containing 5 mmol/L D-glucose, 10% new born calf serum, 10% fetal calf serum (FCS) (Gibco), 3.2 mmol/L L-glutamine and 100 U/mL penicillin-streptomycin (primary culture medium, PCM) (Gibco), and cultured up to passage 3 (37uC, 5% CO 2 ). Twenty-four hours prior to experiments the incubation medium was replaced by M199 containing 2% sera after two rinses with 200 mL in PBS (37uC). Cells were used in passage 3 for most of the experiments. In addition, in some of transport assays confluent freshly isolated cells (i.e., passage 0) or cells in passages 1 or 2 in culture were also used.
Initial rate for TTC, TTC Na + dep and TTC Na + indep components was derived from slopes of lineal phases of 20 mmol/L L-carnitine transport. Values for L-carnitine transport were adjusted to the one phase exponential association equation considering the least squares fit: where v i is initial velocity, V m is mayor velocity at a given time (t) and L-carnitine concentration, and e and k are constants. Data for TTC, TTC Na + dep and TTC Na + indep components at initial rates (i.e., linear uptake up to 30 seconds) was adjusted to the Michaelis-Menten hyperbola plus a nonsaturable, lineal component (mN[Car]) as described [28]: v Vmax~C where v is the initial reaction velocity relative to the maximal velocity (V max ) and apparent Michaelis-Menten parameter (K m ) of transport at a given L-carnitine concentration ([Car]), m represents the slope of transport for the range of 0-80 mmol/L L-carnitine and mN[Car] is the nonsaturable, lineal component of transport in the range of L-carnitine used in this study [28]. Each assay was run in duplicate with transport activity expressed as pmol/mg protein/ minute. After subtracting the mN[Car] component from TTC the remaining transport was defined as total overall saturable transport of L-carnitine (TSC). Transport in Na + -free Krebs was considered as the Na + -independent component of TSC ( TSC Na + indep ) and the Na +component ( TSC Na + dep ) was derived from: The kinetic parameters V max and apparent K m for the TSC, TSC Na + dep or TSC Na + indep components were estimating by fitting the data to the single Michaelis-Menten asymptotic hyperbola equation: v Vmax~C ar ½ Kmz Car ½ Cells were exposed to PCM 2% sera for a period of 2 hours before the transport assays were performed. Cell viability was assayed by Trypan blue exclusion and was not significantly altered (,96% of viable cells) in any experimental condition in this study. Rinsing the monolayers with ice-cold Krebs with or without Na + terminated the tracer uptake. Radioactivity in formic acid cell digests was determined by liquid scintillation counting in an automated low activity liquid scintillation analyzer (Tri-Carb 2810TR, PerkinElmer, Santa Clara, CA, USA) with efficiency estimated by converting counts to disintegrations per minute (d.p.m.) [28]. Uptake of L-[ 3  The relative contribution of the hypertension exhibited by SHR to the saturable L-carnitine kinetic parameters (1/F) was estimated from the maximal transport capacity (V max /K m ) values for TSC by: where WKY V max and WKY K m are the kinetics parameters for TSC in cells from WKY rats, and SHR V max and SHR K m are kinetics parameters of transport in cells from SHR. The relative contribution of the TSC Na + dep or TSC Na + indep components to TSC in SHR or WKY rats was estimated from: where X represents the TSC Na + dep or TSC Na + indep components for the kinetics parameters V max and K m of transport compared with TSC values. The relative contribution of the TSC Na + dep transport compared with the TSC Na + indep component to L-carnitine transport in SHR or WKY rats was estimated from:

Extracellular pH dependency
To assay the effect of extracellular pH (pH o ) on TSC, TSC Na + dep or TSC Na + indep components of L-carnitine transport (20 mmol/L Lcarnitine, 3 mCi/mL L-[ 3 H]carnitine, 30 seconds, 37uC) the cells were incubated in Na + -containing or Na + -free Krebs adjusted to a final pH o of 5.5, 6.5, 7.5 or 8.5 as described [20]. The pH o in the Na + -containing Krebs solution was adjusted with 1 N HCl or 1 N NaOH, while the Na + -free Krebs solution was adjusted with 1 N HCl or 1 N KOH. The pH o values were monitored with a pHmeter (Oakton Instrument, Vernon Hills, IL, USA) and tracer uptake was terminated as above.

Isolation of total RNA and reverse transcription
Total RNA was isolated using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). RNA quality and integrity were insured by gel visualization and spectrophotometric analysis (OD 260/280 ), and RNA concentration was determined at 260 nm. Aliquots (1 mg) of total RNA were reversed transcribed into cDNA as described [28].
The relative mRNA level in each group was estimated from the 2 2DDCT method [29]. Data were analyzed using the Light Cycler 480 SW 1.5 relative quantification (delta-delta-Ct) study software (Roche Diagnostic, Barcelona, Spain) and gene expression levels were normalized to GAPDH and given as relative fold change [30]. The GAPDH mRNA level was not significantly altered (P.0.05, n = 15) in all experimental conditions used in this study (not shown).

Rat aorta reactivity
Ring segments of 2-4 mm in length were dissected from rat aorta in cold (4uC) PBS solution. Vessel rings were mounted in a myograph (610M Multiwire Myograph System, Danish Myo Technology A/S, Denmark) for isometric force measurements in a Krebs physiological solution ((mmol/L): NaCl 118.5, KCl 4.7, NaHCO 3 25, MgSO 4 1.2, KH 2 PO 4 1.2, CaCl 2 2.5, D-glucose 5.5, 300 mmol/L L-arginine, pH 7.4). Artery rings were maintained at 37uC and constantly bubbled with a mixture of 95% O 2 /5% CO 2 . The optimal diameter for each vessel was adjusted through the determination of the maximal active response evoked by 62.5 mmol/L KCl [28]. Isometric force was measured in response to calcitonine gene related peptide (CGRP, 0.1-100 nmol/L, 5 minutes) (Peptides International, Inc., KY, USA) in 32.5 mmol/L KCl preconstricted vessels, in the absence or presence of 20 mmol/ L L-carnitine (30 minutes). In some artery rings, the endothelium was removed by gentle abrasion of the intimal surface. Successful removal of this cell layer was determined by a reduction in the vasodilatation to CGRP. Changes in isometric tension were recorded using the software LabChart (LabChart 7 for Windows, ADInstruments, Australia) coupled to a PowerLab (PowerLab 8/ 30 Data Acquisition System, ADIntruments, Australia). The tissue responses are as a percentage of maximal contraction induced by 62.5 mM KCl.

Statistical analysis
Values are mean 6 SEM, where n indicates the number of different cell cultures (3-4 replicates). Data reported in this study describe a normal standard distribution and comparison between two or more than two groups were performed by means of Student's unpaired t-test and analysis of variance (2-way ANOVA), respectively. If the ANOVA demonstrated a significant interaction between variables, post hoc analyses were performed by the multiple-comparison Bonferroni correction test. The statistical software GraphPad Instat 3.0b and Graphpad Prism 6.0d (GraphPad Software Inc., San Diego, CA, USA) were used for data analysis. P,0.05 was considered statistically significant.

Overall transport of L-carnitine
The TTC for 20 mmol/L L-carnitine was lower (4468%) in RAECs from SHR compared with WKY rats (Fig. 1a). TTC exhibited a TTC Na + dep and a TTC Na + indep component in cells from both SHR and WKY rats. However, in cells from WKY rats the contribution of the TTC Na + dep (5266%) and TTC Na + indep (6067%) components to the TTC were similar (P.0.05), whereas, TTC in cells from SHR resulted from a major contribution of the TTC Na + indep (8466%) compared with the TTC Na + dep (2565%) component of transport (Fig. 1a). Cells in passage 0 (Fig. 1b), 1 (Fig. 1c) or 2 (Fig. 1d) exhibited similar changes in L-carnitine transport compared with cells in passage 3 (Fig. 1a). The TTC was lineal up to 40 seconds incubation (Fig. 2a) with v i values lower in SHR compared with WKY rats (Table 1). In cells from either SHR or WKY rats the v i for the TTC Na + indep compared with the corresponding TTC Na + dep component was higher for 20 mmol/L L-carnitine transport. However, the difference between the v i for the TTC Na + indep compared with TTC Na + dep component was higher (2.160.3 fold) in cells from SHR compared with WKY rats (Fig. 2b).

L-Carnitine transport kinetics
The TTC was semisaturable in RAECs from either SHR or WKY rats (Fig. 3a,b). The K D value for TTC was similar to that for the TTC Na + indep component of transport in cells from WKY rats; however, the K D for TTC Na + dep transport in cells from these animals and all other K D values in SHR were negligible ( Table 1). The TTC Na + dep and TTC Na + indep components derived from TTC were saturable in both groups of cells. The Eadie-Hofstee plot of TTC data was best fitted by an exponential one phase decay equation resulting in a non-linear plot in cells from WKY rats (Fig. 3c). However, the TTC Na + dep and TTC Na + indep components derived from TTC in WKY rats were linear (Fig. 3c), as were TTC, TTC Na + dep and TTC Na + indep components of transport in SHR (Fig. 3d). After subtracting the lineal, non-saturable component of transport data (in the range of concentrations used in this study), the saturable transport for each condition was obtained (Fig. 4a,b). Cells from SHR exhibit reduced V max , but unaltered apparent K m for TSC compared with cells from WKY rats ( Table 1). The V max for the TSC Na + dep , but not for TSC Na + indep components of transport was reduced in cells from SHR compared with the corresponding values in WKY rats. In addition, the V max for the TSC Na + indep component was higher than TSC Na + dep component of transport only in SHR ( Table 1). The Eadie-Hofstee plot of saturable transport data was lineal for all experimental conditions (Fig. 4c,d). The V max /K m values for sturable transport and the Na + dep component were lower in SHR compared with WKY rats, and the value for the TSC Na + dep component was lower than the TSC Na + indep component of transport in cells from SHR or WKY rats (Table 1).  Total (TTC) and saturable (TSC) transport of L-carnitine and the Na + -dependent (Na + dep ) and Na + -independent (Na + indep ) components of transport (0-80 mmol/L Lcarnitine, 3 mCi/mL L-[ 3 H]carnitine, 30 seconds, 37uC, pH 7.4) were measured in cultured (passage 2) rat aortic endothelial cells (RAECs) from normotensive (WKY) or spontaneously hypertensive (SHR) rats as described in Methods. The initial velocity (v i ) was measured for 20 mmol/L L-carnitine up to 30 seconds. v i , initial velocity (pmol/mg protein/second); V max , maximal velocity (pmol/mg protein/minute); K m , apparent Michaelis-Menten constant (mmol/L); V max /K m , maximal transport capacity (pmol/mg protein/minute/(mmol/L)); K D , lineal, non-saturable transport in the range of L-carnitine concentrations used in this study (pmol/mg protein/minute/(mmol/L)). Expression of rOctn1 and rOctn2 mRNA The relative expression of rOctn2 was higher than rOctn1 mRNA in RAECs from SHR or WKY rats (Fig. 5). The relative mRNA expression of rOctn2 was largely lower in SHR when compared with WKY; however, no differences were found in mRNA expression of rOctn1 between cells from these animals.

Extracellular pH dependency on saturable L-carnitine transport
Overall transport of L-carnitine was higher at pH o 8.5 compared with transport at pH o 7.4, but it was unaltered by lower pH o values in cells from WKY rats (Fig. 6a). The TSC Na + indep component of transport at pH o 7.4 was higher than at pH o 8.5, but lower than at acidic pH o values. However, the TSC Na + dep component of transport at pH o 7.4 was lower than at alkaline, but higher than at acidic pH o . The half-maximal stimulatory effect (SE 50 ) of a change in the pH o on overall transport was higher than the SE 50 for the TSC Na + indep (,0.26 pH o units of difference) component (Table 2). A similar effect on TSC was seen for the halfmaximal inhibitory effect (IE 50 ) of a change in the pH o on the TSC Na + dep (,0.53 pH o units of difference) component of transport in cells from WKY rats ( Table 2). In addition, the SE 50 for TSC Na + indep was significantly different from the IE 50 for TSC Na + dep values (,0.24 pH o units of difference) in these cells.
In RAECs from SHR, the TSC and the TSC Na + indep component of transport were lower at alkaline pH o compared with transport at pH o 7.4 (Fig. 6b). However, the TSC Na + dep component of saturable transport was unaltered compared with values at pH o 7.4 in these cell types. The IE 50 values for overall and the TSC Na + indep component of L-carnitine transport (,0.05 pH o units of difference) were not significantly different in cells from SHR (Table 2). However, the alkalization required to reduce the overall transport in cells from SHR was higher (,0.22 pH o units of difference) compared with the alkalization required to increase the transport in cells from WKY rats. In addition, the alkalization required to reduce the TSC Na + indep component of transport in cells from SHR was higher (,0.93 pH o units of difference) compared with cells from WKY rats.

Discussion
We have characterized the kinetics of L-carnitine transport in primary cultures of rat aortic endothelial cells (RAECs) from nonhypertensive WKY rats and contrasted this information with cells from spontaneously hypertensive rats (SHR). Total overall transport of L-carnitine (TTC) was mediated by Na + -dependent ( TTC Na + dep ) and Na + -independent ( TTC Na + indep ) components increased by a lineal, nonsaturable mediated transport. A reduced initial velocity for the TTC and TTC Na + dep , but not the TTC Na + indep component was found in cells from SHR compared with nonhypertensive WKY rats. The kinetics assays for saturable overall transport of this amino acid (TSC) show that maximal transport capacity (V max /K m ) for L-carnitine is lower in cells from SHR compared with WKY rats, a finding paralleled by reduced V max / K m for the TSC Na + dep , but not the TSC Na + indep component. A differential dependency of pH o for TSC, TSC Na + dep and TSC Na + indep transport was seen in cells from SHR compared with WKY rats. These results are the first demonstration that RAECs from SHR exhibit a phenotype characterized by reduced L-carnitine transport compared with cells from non-hypertensive rats. These results are potentially useful for a better understanding of the membrane transport mechanisms of L-carnitine in RAECs from non-hypertensive WKY rats and SHR. Interestingly, a reduced reactivity to an endothelium dependent vasodilator of the aortic rings from SHR compared with WKY rats was seen, an effect that was improved by supplementation of these vessels in vitro with Lcarnitine. However, vasodilation was absent in endotheliumdenuded aortic ring preparations. It is suggested that a reduced uptake of L-carnitine by the endothelium could counteract the reported beneficial vascular effects of L-carnitine supplementation in subjects with hypertension.

L-Carnitine transport in normotensive rats
Several reports describe that the natural amino acid L-carnitine could act by improving the high arterial blood pressure in patients with essential hypertension [3,4] and in animal models of hypertension [5,26]. These results complement the possibility that in hypertension the membrane transport of this amino acid is reduced, a phenomenon that is supported by studies showing a correlation between increased L-carnitine plasma level and higher blood pressure in adult men [31]. Plasma membrane transport of L-carnitine is mediated by OCTNs in mammalian cells, and the isotypes Octn1, Octn2 and Octn3 have been cloned from rats [12,32]. Our results show that RAECs exhibit TTC mediated by at least three different components (Na + -dependent ( TTC Na + dep ), Na +independent ( TTC Na + indep ) and lineal, nonsaturable transport) in the range up to 80 mmol/L L-carnitine. These findings agree with the basic characteristics described for Octn-like transport of this amino acid in other cell types [33][34][35]. Since the Eadie-Hofstee representation of the TTC data was not lineal, it is likely that at least two or more transport systems acting in parallel [36,37] will account for L-carnitine transport in RAECs from non-hypertensive rats. In addition, since the TTC Na + dep and TTC Na + indep components of transport were lineal in the Eadie-Hofstee plot, either a single transport system or two or more transport systems with similar kinetic parameters acting in parallel mediate the Na +dependent and the Na + -independent L-carnitine transport in this cell type.
Our results also show that cells from WKY rats exhibit a TSC resulting from a pronounced differential contribution of the TSC Na + dep and the TSC Na + indep components when the relative V max / K m for these components were compared. The relative contribution of the V max /K m for the TSC Na + dep component to TSC is lower (,30%) (from 1/ TSC/Na+-dep F = 0.30) compared with the contribution accounted by the TSC Na + indep component (,73%) (1/ TSC/Na+-indep F = 0.73). Thus, a Na + indep component of Lcarnitine transport predominates in RAECs from non-hypertensive WKY rats.
Since the results of our study show that the apparent K m for the TSC Na + dep and TSC Na + indep components (K m = 21-46 mmol/L) was in the range of values described for this membrane transporter isoform in other cell types [35], the possibility that Octn1 was responsible of the Na + -independent L-carnitine transport in RAECs from WKY rats is supported. However, Octn2-mediated transport of L-carnitine is described as a Na + -dependent transport mechanism with higher affinity (K m = 2-20 mmol/L) compared to Octn1 [16,[18][19][20][21][22]. Octn2 is also expressed in other types of endothelial cells, including human heart and brain capillaries endothelium [23,40]; therefore Octn2 could also account for the Na + -dependent transport of L-carnitine in RAECs. Interestingly, since the plasma concentration of L-carnitine for WKY rats is reported as 20-36 mmol/L [41] it is likely that the lower affinity transport system Octn1 would play a preferential role compared with Octn2, which is likely to be saturated at physiological Lcarnitine plasma concentrations, in maintaining the extracellular physiological concentrations of this amino acid in these animals.
In the present study, both Octn1 and Octn2 mRNA expression was detected in RAECs from WKY rats. Interestingly, Octn2 mRNA relative expression resulted to be ,31 fold higher compared with Octn1 mRNA in these cells. Since the relative contribution of the TSC Na + dep component to the V max /K m for TSC was ,30%, it is likely that not more than ,10 fold change in Octn2 mRNA expression (estimated from the (Octn2 mRNA/ Octn1 mRNA)/(1/ TSC/Na+-dep F) ratio) could sustain a TSC Na + dep component for the TSC in RAECs. The remaining transport mediated via a TSC Na + indep component (,73%) could represents the contribution of a Na + -independent transport activity derived from Octn1 in this cell type. Interestingly, since the contribution for the Na + -dependent Octn2 transport is reported as ,3 fold higher than the transport detected in the absence of extracellular Na + in other cell types [42], an equivalent fractional contribution for these components to L-carnitine transport in RAECs from WKY rats could be expected. However, the latter seems unlikely in this cell type since the relative contribution of the TSC Na + dep component to the V max /K m for TSC was ,30% (1/ TSC/Na+-dep F = 0.3) compared with ,73% (1/ TSC/Na+-indep F = 0.73) for the TSC Na + indep component. Thus, contrasting with other cell types [35,42], these findings further support the possibility that the Na + indep component predominates (,2.4 fold) compared with the Na + dep component regarding their contribution to TSC. This could be interpreted as a major contribution of Octn1 compared with Octn2 to the saturable transport of L-carnitine in RAECs from WKY rats.
L-Carnitine transport in RAECs from WKY rats was also dependent on the pH o , a characteristic well described for Octn1 [13,32] and Octn2 [43]. Our results show that TSC was increased by ,20% by alkalization of the extracellular medium, an effect resulting from a combined increases in the TSC Na + indep component (,58%) and decreases in the TSC Na + dep component (,29% ] components regarding the change seen in TSC. Based in these findings, a higher alkalization-dependent increase in the TSC could be reached whether these two transport components were equally altered or whether the TSC Na + indep component was unaltered by this environmental condition. Interestingly, the increase of TSC caused by a change of 0.6 units of pH o in RAECs was ,3.3 fold the increase reported for 150 mmol/L tetraethylammonium (TEA) uptake in response to a similar change in pH o units in HEK293 cells expressing the human OCTN1 form [13]. However, contrasting with these results a larger increase in the pH o value (1 unit of pH o ) reduced overall transport of TEA in these cells [32]. Thus, it is likely that OCTNs-like transport is differentially responsive to the degree of alkalization reached in HEK293 cells. Interestingly, a change in ,0.4 units of pH o has been shown to increase the activity of other membrane transport systems, such as the sodium/proton exchanger isoform 1 in MDCK cells [44], suggesting that modulation of Octn1/2 by a similar change in the pH o in RAECs agree with what is reported in other cell types. On the other hand, acidification of the extracellular pH o does not alter TSC, a net effect that result from a proportional reduced TSC Na + dep and increased TSC Na + indep transport components. Interestingly the effect of extracellular acidification was similar for both components, suggesting that these components are equally sensitive to acidification in RAECs from WKY rats. Thus, acidification and alkalization of extracellular medium results in a differential modulation of L-carnitine transport in RAECs from nonhypertensive rats.

Effect of spontaneous hypertension on L-carnitine transport
Cells from SHR exhibit a semisaturable TTC unaffected by a lineal, non-saturable component in the range of L-carnitine concentrations used in this study. This data was best fitted to a first-order regression line in an Eadie-Hofstee plot, suggesting that one or more transport systems with apparent K m values in the same range could mediate L-carnitine transport in RAECs from SHR. The apparent K m values for L-carnitine transport by RAECs in SHR were similar to values detected in non-hypertensive rats, and within the range of the plasma L-carnitine concentration reported in SHR (21-41 mmol/L) [41]. Thus, L-carnitine transport via Octn1/2 could contribute to maintain the physiological plasma concentration of this amino acid in SHR. Our results also show that TTC is mainly mediated by a TTC Na + indep (,75%) with a minor contribution of TTC Na + dep (,25%) components. These findings agree with those obtained by contrasting the relative V max /K m for these components with that for TSC. Since Figure 6. Effect of extracellular pH on saturable transport of L-carnitine, and rat aorta reactivity. Total saturable transport of L-carnitine (Total), and the Na + -dependent (Na + dep ) and Na + -independent (Na + indep ) transport components (20 mmol/L L-carnitine, 3 mCi/mL L-[ 3 H]carnitine, 30 seconds, 37uC) in RAECs cultures from WKY rats (a) or SHR (b) exposed to culture medium with the pH adjusted to different values. (c) Relaxation of 32.5 mmol/L KCl preconstricted endothelium-intact aortic vessel rings (With endothelium) from WKY rats or SHR in response to increasing concentrations of calcitonine gene related peptide (CGRP, 5 minutes), in the absence (-Car) or presence (+Car) of 20 mmol/L L-carnitine (30 minutes). the relative contribution of the V max /K m for the TSC Na + indep component is higher (,75%) (1/ TSC/Na+-indep F = 0.75) compared with the contribution accounted by the TSC Na + dep component (,30%) (1/ TSC/Na+-dep F = 0.30), and considering that similar findings were found for the v i values for these transport components, it is suggested that RAECs from SHR exhibit saturable transport of L-carnitine where the Na + -independent transport predominates by ,2.5 fold compared with the Na +dependent. This result is ,2.1 fold higher compared with cells from non-hypertensive rats supporting the possibility that hypertension could associate with a higher requirement of Na +independent transport of L-carnitine via Octn1/2 activity in RAECs. Thus, it is likely that a deficiency in the TSC Na + dep component results in RAECs dysfunction in SHR. This would not be explained by a lower V max /K m of the TSC Na + dep component, since the relative contribution of this component to TSC in these cells was similar to that in cells from non-hypertensive rat ((1/ TSC/Na+-dep F in WKY rats)/(1/ TSC/Na+-dep F in SHR = 1.01). In addition, the relative contribution of the TSC Na + dep component compared with the TSC Na + indep component to TSC in SHR is also similar to non-hypertensive rats ((1/ Na+-indep/Na+-dep F in WKY rats)/(1/ Na+-indep/Na+-dep F in SHR = 1.03). Thus, reduced overall transport of L-carnitine in RAECs from SHR could be mainly due to reduced expression of the Na + -dependent Octn2 and in a less extend to a reduced expression of the Na + -independent Octn1 membrane transporters. In fact, the Octn2 mRNA expression in cells from SHR is only 2.6 fold compared with Octn1 mRNA expression, a value that is largely minor compared with the 31 fold increase for this mRNA detected in cells from non-hypertensive rats. Thus, a reduced Octn2 expression without alterations in the V max /K m could account for the reduced TSC Na + dep component of L-carnitine transport in RAECs from SHR.
Interestingly, as found in cells from non-hypertensive rats, the relative contribution of the TSC Na + dep component to the V max /K m for TSC was ,30%. Thus, a proportional change by Octn2 expression to (i.e., ,1.48 fold) would sustain the TSC Na + dep component of the saturable transport activity in RAECs from SHR. Interestingly, this value is ,85% lower compared with the potential requested change in Octn2 mRNA expression in cells from non-hypertensive rats. Therefore, SHR is a pathological condition that results in lower request of Octn2 mRNA expression compared with RAECs from non-hypertensive rats. The results also show that L-carnitine transport in RAECs from SHR was dependent on the pH o , supporting the possibility that transport was mediated by Octn1/2 in this cell type. In this case, alkalization of the extracellular medium resulted in reduced TSC, which was due to reduced Na + indep component. This finding is different from RAECs from non-hypertensive rats, suggesting that alkalization could result in a differential down-regulation of L-carnitine transport in RAECs from SHR compared with non-hypertensive rats. However, since the Na + indep component of transport was also reduced in cells from non-hypertensive rats, it is likely that sensitivity of this component to a change in the pH o is similar in cells from SHR and WKY rats.
In conclusion, the kinetic parameters of L-carnitine transport in RAECs from SHR and non-hypertensive WKY rats were characterized. The overall saturable transport was mediated by Na + indep and Na + dep components, with the latter being crucial in the reduced maximal transport capacity detected in cells from SHR. The kinetic parameters, pH o -and Na + -dependency of transport suggest that Octn1 and Octn2 are likely responsible for membrane transport of L-carnitine in endothelial cells from the aorta of SHR and WKY rats. These results are the first characterizing the kinetic parameters for the membrane transport mechanisms of Lcarnitine in rat aortic endothelium from non-hypertensive WKY and spontaneously hypertensive rats. Furthermore, since (a) the reactivity of aortic rings to the endothelium-dependent vasodilator CGRP was reduced in preparations from SHR compared with WKY rats, (b) L-carnitine supplementation in vitro restored CGRP vasodilation to values in vessels from normotensive rats, and (c) CGRP was ineffective in endothelium-denuded rat aortic rings, it is suggested that restoration of a functional endothelium could result from bioavailability of L-carnitine to the aorta endothelium in the spontaneously hypertensive animals. Total transport of L-carnitine, and the Na + -dependent (Na + dep ) and Na +independent (Na + indep ) transport components (20 mmol/L L-carnitine, 3 mCi/mL L-[ 3 H]carnitine, 30 seconds, 37uC) in RAECs cultures from WKY rats or SHR exposed to culture medium with the pH adjusted to different values (extracellular pH (pH o ) 5.5-8.5) as described in Methods. The stimulatory (SE 50 ) or inhibitory (IE 50 ) effect of pH o on transport was calculated. ni, not inhibited; ns, not stimulated. *P,0.05 versus Total in WKY rats. Values are mean 6 SEM (n = 12). doi:10.1371/journal.pone.0090339.t002