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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:1495.)
© 2006 American Heart Association, Inc.

Vascular Biology

Evidence for a Functional Role of Endothelial Transient Receptor Potential V4 in Shear Stress–Induced Vasodilatation

Ralf Köhler; Willm-Thomas Heyken; Philipp Heinau; Rudolf Schubert; Han Si; Michael Kacik; Christoph Busch; Ivica Grgic; Tanja Maier; Joachim Hoyer

From the Department of Internal Medicine-Nephrology (R.K., W.-T.H., P.H., H.S., M.K., C.B., I.G., T.M., J.H.), Philipps-University, Marburg, Germany; and Institute of Physiology (R.S.), University of Rostock, Germany.

Reprint requests to R. Köhler, Department of Internal Medicine-Nephrology, Philipps-University, Baldingerstrasse, 35033 Marburg, Germany. E-mail rkoehler{at}med.uni-marburg.de

	Abstract

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Objective— Ca²⁺-influx through transient receptor potential (TRP) channels was proposed to be important in endothelial function, although the precise role of specific TRP channels is unknown. Here, we investigated the role of the putatively mechanosensitive TRPV4 channel in the mechanisms of endothelium-dependent vasodilatation.

Methods and Results— Expression and function of TRPV4 was investigated in rat carotid artery endothelial cells (RCAECs) by using in situ patch-clamp techniques, single-cell RT-PCR, Ca²⁺ measurements, and pressure myography in carotid artery (CA) and Arteria gracilis. In RCAECs in situ, TRPV4 currents were activated by the selective TRPV4 opener 4-phorbol-12,13-didecanoate (4PDD), arachidonic acid, moderate warmth, and mechanically by hypotonic cell swelling. Single-cell RT-PCR in endothelial cells demonstrated mRNA expression of TRPV4. In FURA-2 Ca²⁺ measurements, 4PDD increased [Ca²⁺]_i by &140 nmol/L above basal levels. In pressure myograph experiments in CAs and A gracilis, 4PDD caused robust endothelium-dependent and strictly endothelium-dependent vasodilatations by &80% (K_D 0.3 µmol/L), which were suppressed by the TRPV4 blocker ruthenium red (RuR). Shear stress–induced vasodilatation was similarly blocked by RuR and also by the phospholipase A₂ inhibitor arachidonyl trifluoromethyl ketone (AACOCF₃). 4PDD produced endothelium-derived hyperpolarizing factor (EDHF)–type responses in A gracilis but not in rat carotid artery. Shear stress did not produce EDHF-type vasodilatation in either vessel type.

Conclusions— Ca²⁺ entry through endothelial TRPV4 channels triggers NO- and EDHF-dependent vasodilatation. Moreover, TRPV4 appears to be mechanistically important in endothelial mechanosensing of shear stress.

Ca²⁺-permeable mechanosensitive channels are believed to be important in endothelial mechanotransduction. The present study provides evidence that the TRPV4 channel, a putatively mechanosensitive member of the TRPV family, mediates wall shear stress–induced vasodilatation in a NO-dependent manner.

Key Words: endothelium-dependent vasodilatation • transient receptor potential • TRPV4 • calcium • shear stress • nitric oxide • 4PDD • rat carotid artery

	Introduction

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Ca²⁺-influx in response to mechanical or humoral stimulation plays a significant role in a variety of endothelial functions and especially in the Ca²⁺-dependent synthesis of endothelium-derived vasodilators such as NO, prostacyclin, or the endothelium-derived hyperpolarizing factor (EDHF).¹ Several members of the transient receptor potential (TRP) superfamily of cation channels² have been identified in endothelial cells (ECs) of humans and other species and may provide Ca²⁺ influx pathways. Within the different subfamilies of TRP channels, ECs express members of the "canonical" TRP subfamily (TRPC), such as TRPC1, TRPC3, and TRPC4, which are primarily believed to serve as Ca²⁺ influx channels after receptor activation or store depletion.^1,3 Regarding the other large TRP subfamilies, the "melastatin" (TRPM) and "vanilloid" (TRPV) subfamilies, TRPM4¹ and TRPM7,^1,4 and TRPV4,⁵ respectively, are also endothelial TRPs. Although TRPV4 was proposed to contribute to EDHF signaling,⁵ the precise roles of TRPV4 and of other TRPs in the mechanism of endothelium-dependent vasodilatation are still undefined.

Within the endothelial TRP channels, TRPV4 might be of special interest because this channel provides a significant Ca²⁺ entry pathway because of its moderately high Ca²⁺ permeability.^1,6 Moreover, TRPV4 channels^6–10 have been shown to be opened by diverse physical and chemical stimuli such as cell swelling^6,8,11 and shear stress,^9,12 moderate warmth (>27°C),^13,14 low pH,¹⁵ and pharmacologically by the non–PKC-activating phorbol esther 4-phorbol-12,13-didecanoate (4PDD).⁶ Recent studies suggested that arachidonic acid (AA) and its metabolite 5,6 epoxyeicosatrienoic acid may serve as endogenous activators of TRPV4.⁵ However, thus far, it is unknown how TRPV4 contributes to endothelium-dependent vasodilatation.

The mechanosensitivity of TRPV4 may point to a role of the channel as an endothelial mechanosensor and thus in the mechanisms of flow- or wall shear stress–induced vasodilatation. It is noteworthy that a shear stress–induced TRPV4-mediated Ca²⁺ entry has been shown in heterologous expression systems¹² and renal tubular epithelial cells.⁹ Such flow- and shear stress–induced Ca²⁺ signals have also been observed in ECs in vitro and in the endothelium in intact vessel preparations.^16–18 Although shear stress–activated Ca²⁺ entry channels are presumably encoded by TRP genes,^4,9 the endothelial shear stress–activated channel has not been identified so far. In keeping with the mechanosensitivity of TRPV4, this channel might thus be a molecular candidate for the shear stress–activated Ca²⁺ entry channel in the endothelium.

To elucidate how endothelial TRPV4 channels contribute to endothelial-dependent and especially shear stress–induced vasodilatation, we characterized TRPV4 channels in rat carotid artery ECs (RCAECs) and rat aorta ECs (RAECs) by using in situ patch-clamp techniques and single-cell RT-PCR analysis, FURA-2 measurements, and pressure myography in CAs and small-sized A gracilis. We show that pharmacological activation of endothelial TRPV4 induces robust vasodilatations in a NO- and EDHF-dependent manner. Moreover, we provide evidence that Ca²⁺ influx through TRPV4 is functionally important in the mechanisms of shear stress–induced vasodilatation.

	Methods

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In situ patch-clamp experiments in rat ECs, "multiplex" single-cell RT-PCR, [Ca²⁺]_i measurements, and pressure myography were performed as described previously.^18–21 For detailed methods, please see the online supplement, available at http://atvb.ahajournals.org.

	Results

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Electrophysiological Characterization of TRPV4 in Rat Carotid Endothelium
In whole-cell patch-clamp experiments in electrically uncoupled RCAECs of the endothelium in situ, 4PDD (10^–6 mol/L), the selective TRPV4 opener, activated moderately outward-rectifying currents (Figure 1A through D and 1F). Outward rectification of the current was more pronounced in the presence of extracellular Ca²⁺ ([Ca²⁺]_out; Figure 1A and 1F) than at strongly buffered [Ca²⁺]_out (Figure 1B and 1F). Inward currents were abolished after substitution of Na⁺ by the large nonpermeable cation N-methyl-D-glucamine (NMDG)⁺ (Figure 1B, left and right panels), indicating cation selectivity of this current. 4PDD-activated TRPV4 currents reversed at a potential of &0 mV with 1 mmol/L or 20 nmol/L [Ca²⁺]_out. At high [Ca²⁺]_out of 20 mmol/L, the current reversed at a more positive membrane potential of &10 mV (Figure 1C), demonstrating moderate preference for Ca²⁺ over Na⁺. Calculation of the relative Ca²⁺ permeability ratio (P_Ca/P_Na)⁶ gave a P_Ca/P_Na value of &6, which is similar to P_Ca/P_Na values for cloned human and murine TRPV4.⁶

View larger version (34K): [in this window] [in a new window]   Figure 1. Electrophysiological properties of TRPV4-like currents in RCAECs in situ. A, Left panel, Representative recording of 4PDD (1 µmol/L)-induced currents with 1 mmol/L [Ca2+]out; reversal potential (indicated by arrow) was 1±1 mV (n=5). Note that activation is transient. Right panel, Time course of transient 4PDD (1 µmol/L)-induced TRPV4 currents in the presence of [Ca2+]out (1 mmol/L). B, Left panel, Weaker current rectification with low [Ca2+]out (20 nmol/L). Nonpermeable NMDG abolishes inward currents at negative potentials, proving cation selectivity of this current. Right panel, Stable 4PDD (1 µmol/L)-induced TRPV4 currents in the presence of low [Ca2+]out (20 nmol/L). C, 4PDD (1 µmol/L)-induced Ca2+ currents at 0 mV with high [Ca2+]out (20 mmol/L); reversal potential: 10±2 mV; n=4. D, Inhibition of TRPV4 currents by RuR (1 µmol/L). E, Current oscillations in the continuing presence of 4PDD and with 1 mmol/L [Ca2+]out. F, Normalized 4PDD-induced currents in RCAECs and RAECs (I [pA/pF]) with 20 nmol/L or 1 mmol/L [Ca2+]out. Note the smaller amplitude of inward currents at 1 mmol/L [Ca2+]out, which indicates that outward rectification is more pronounced at physiological [Ca2+]out than at strongly buffered [Ca2+]out ([Ca2+]free; 20 nmol/L). G, Left panel, Representative recordings showing activation of TRPV4 currents by HTS (n=10) and effects of RuR (1 µmol/L; n=5). Middle panel, Suppression of HTS-inducible currents in the presence the PLA2 inhibitor AACOCF3 (4 µmol/L; n=6; middle panel). Right panel, Activation of TRPV4 currents by AA (10 µmol/L; n=6) and inhibition by RuR (1 µmol/L; n=6). Experiments were conducted at very low [Ca2+]out (20 nmol/L) to avoid Ca2+-entry mediated coactivation of Ca2+-activated channels.

View larger version (22K): [in this window] [in a new window]

Ruthenium red (RuR; 1 µmol/L), a blocker of TRPV channels,^6,22 almost completely suppressed inward currents in a voltage-dependent fashion (Figure 1D), although longer exposure times (>20 s) also reduced outward currents. Currents were also blocked by Gd³⁺ (50 µmol/L), a nonselective blocker of TRPs (data not shown). A similar 4PDD-inducible current was observed in freshly isolated RAECs (Figure 1F).

The time course of 4PDD-activated cation currents depended on the presence of [Ca²⁺]_out. Whereas 4PDD-induced currents were stable over time with low [Ca²⁺]_out (20 nmol/L; Figure 1B, right panel), currents showed a rapid decay (within 10 to 20 s), with 1 mmol/L [Ca²⁺]_out (Figure 1A, left and right panels). However, in some RCAECs (n=4), we could observe current oscillation in the continuing presence of 4PDD and with 1 mmol/L [Ca²⁺]_out (Figure 1E). These observations may indicate that Ca²⁺ entry through the channel and the resulting increase of [Ca²⁺]_i lead to channel inactivation. Reactivation of the channel may occur when [Ca²⁺]_i returns to basal levels. This Ca²⁺ inactivation may point to a "negative-feedback" mechanism that has been proposed for cloned TRPV4 previously.^6,23 In contrast to 4PDD, capsaicin (10 µmol/L), an opener of TRPV1, did not produce any currents in RCAECs (data not shown).

Similar to cloned human and murine TRPV4 channels,²⁴ TRPV4-like currents in RCAECs were activated by superfusion with a warmed (37°C) solution (n=4; data not shown) and by hypotonic stress (HTS)-induced cell swelling (Figure 1G, left panel). AA (1 µmol/L), which was reported to mediate HTS-induced TRPV4 activation, but not that by heat or 4PDD,²⁴ effectively activated TRPV4-like current in rat ECs (Figure 1G, right panel). Similar to 4PDD-induced currents, HTS-induced and AA-induced currents were largely inhibited by RuR (Figure 1G, left and right panels). Moreover, inhibition of phospholipase A₂ (PLA₂) by arachidonyl trifluoromethyl ketone ([AACOCF₃] 4 µmol/L), and thus the prevention of AA release, precluded the activation of HTS-inducible TRPV4-like currents (Figure 1G, middle panel).

Subsequent to in situ patch-clamp experiments, single RCAECs were harvested with the patch pipette, and mRNA expression of TRPV channels was verified by "multiplex" single-cell RT-PCR²⁰ (Figure 2). To ensure the selective harvest of RCAECs, cell samples were tested for mRNA expression of endothelial NO synthase (eNOS) as EC marker. Expression of myosin heavy chain (MyHC), as vascular smooth muscle cell marker, was not detectable (data not shown). TRPV4 mRNA was detected in 7 of 10 eNOS-positive and MyHC-negative RCAEC samples. Expression of other closely related members of the TRPV subfamily (ie, TRPV1 through TRPV3) was not detected in any of the cell samples. TRPV4 transcripts were not detected in freshly dissociated and MyHC-positive vascular smooth muscle cell samples (n=14; data not shown).

View larger version (27K): [in this window] [in a new window]   Figure 2. "Multiplex" single-cell RT-PCR analysis of single RCAECs in situ. Ethidium bromide–stained gels of RT-PCR products from representative RCAECs expressing eNOS (top) and TRPV4 (bottom) and no RT-PCR products from a negative control (sample of bath medium).

[Ca²⁺]_i Transients Elicited by TRPV4 Activation in Rat ECs
To determine TRPV4-mediated Ca²⁺ entry in single rat ECs, we switched for technical reasons to freshly isolated RAECs. 4PDD (1 µmol/L) increased [Ca²⁺]_i by &140 nmol/L above basal levels (60±5 nmol/L; n50; Figure 3A). The increase in [Ca²⁺]_i peaked within &10 s and returned thereafter to levels slightly above baseline. After the initial peak, we frequently observed additional peaks of smaller amplitude.

View larger version (22K): [in this window] [in a new window]   Figure 3. A, Increase in [Ca2+]i after activation of TRPV4 by 4PDD (1 µmol/L) in freshly isolated RAECs. B, RuR (1 µmol/L) in the bath solution. C, Strong buffering of [Ca2+]out prevented 4PDD-induced increases in [Ca2+]i. D, Application of 4PDD evoked Mn2+ (1 mmol/L) quenching of FURA-2 fluorescence (n=17), indicating that 4PDD causes Ca2+ influx. As positive control, the Ca2+ ionophore ionomycin (1 µmol/L) caused additional strong quenching. E, ACh (1 µmol/L; n=10) induced increases in [Ca2+]i. F, Calculated mean increases in [Ca2+]i (D[Ca2+]i, nmol/L) above basal levels after stimulation with 4PDD in the absence (n=25) and in the presence of RuR (1 µmol/L; n=3) or at low [Ca2+]out (n=10), with warmth (37°C; n=14), with HTS in the absence (n=10) and in the presence of RuR (1 µmol/L; n=22), with ACh in the absence (n=10) and in the presence of RuR (1 µmol/L; n=6).

4PDD-induced [Ca²⁺]_i transients were not observed in the presence of RuR (1 µmol/L; Figure 3B) or with strongly buffered [Ca²⁺]_out (20 nmol/L; Figure 3C), thus demonstrating that 4PDD-induced increases in [Ca²⁺]_i are elicited by Ca²⁺ entry. This was further supported by our observation that 4PDD application results in Mn²⁺ quenching of the FURA-2 signal (Figure 3D). When compared with TRPV4-mediated increases in [Ca²⁺]_i, acetylcholine (Ach; 1 µmol/L) induced a more pronounced increase in [Ca²⁺]_i by &600 nmol/L above basal levels that peaked more rapidly (within 2 s) and was followed by a prolonged plateau phase (Figure 3E and 3F) as a consequence of inositol triphosphate–triggered Ca²⁺ release from internal stores and subsequent Ca²⁺ entry.¹ Similar to 4PDD, opening of TRPV4 by superfusion with a warmed (37°C) solution and by HTS increased [Ca²⁺]_i (Figure 3F). Similar to 4PDD, HTS-induced Ca²⁺ mobilization was greatly reduced in the presence of RuR. In contrast, ACh-induced Ca²⁺ responses were unaffected by RuR (Figure 3F).

Functional Role of TRPV4 in Endothelium-Dependent Vasodilatation
To understand the functional role of TRPV4 in the mechanism of endothelium-dependent vasodilatation of CAs and small more resistance-like arteries, we conducted pressure myograph experiments in CAs and in small-sized A gracilis, respectively.

Intraluminal application of 4PDD caused robust and dose-dependent vasodilatations of CAs (80% at 1 µmol/L 4PDD; K_D of &0.3 µmol/L; Figure 4A and 4B) that were similar in magnitude to that induced by 1 µmol/L ACh (Figure 4A, left part). The vasodilatory responses to 4PDD were strictly endothelium dependent because vasodilatation was abolished by endothelial inactivation, and extraluminal application of 4PDD had no effect on vessel diameter (data not shown). Intraluminal application of capsaicin (10 µmol/L) or anandamide (10 µmol/L), known to activate TRPV1, did not induce vasodilatation (data not shown). 4PDD-induced vasodilatation was completely antagonized by RuR (1 µmol/L; Figure 4A, left part), whereas ACh-induced vasodilatations were slightly reduced by RuR (Figure 4A, left part). Intraluminal application of RuR alone was without effect (data not shown). 4PDD-induced vasodilatation was also eliminated by chelation of endothelial [Ca²⁺]_i by 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethl ester (BAPTA-AM) (10 µmol/L; intraluminal preincubation for 10 minutes; 3±2% vasodilatation; n=3).

View larger version (38K): [in this window] [in a new window]   Figure 4. TRPV4 and endothelium-dependent vasodilatation. A, Left part, 4PDD (1 µmol/L)-induced vasodilatation (percentage of maximal vasodilatation) in CAs with a functionally active endothelium. 4PDD-induced vasodilatation was suppressed by the TRPV4 blocker RuR (1 µmol/L). For comparison, ACh (1 µmol/L)-induced vasodilatation of similar magnitude is almost RuR insensitive. Middle part, EDHF-type vasodilatation (in the presence of L-NNA [100 µmol/L] and INDO [10 µmol/L]) elicited by ACh (1 µmol/L), the KCa channel opener 1-EBIO (100 µmol/L), and by 4PDD (1 µmol/L). Right part, Shear stress (5% dextran)–induced vasodilatation and inhibition by RuR (1 µmol/L), L-NNA, and INDO, or by L-NNA alone. Effects of inhibition of PLA2 by AACOCF3 (4 µmol/L), of PKC by calphostin-C (Cal-C; 0.5 µmol/L), and of COX by INDO (10 µmol/L) on shear stress–induced vasodilatation. B, Dose-response curve for 4PDD; KD 0.3±0.1 µmol/L (Boltzmann fit). C, Left and middle part, 4PDD (1 µmol/L)-induced and ACh-induced vasodilatation in small myogenically active A gracilis and effects of RuR (1 µmol/L), L-NNA (100 µmol/L), and INDO (10 µmol/L). Note that the error bars (4PDD) are smaller than the line thickness. Right part, Shear stress–induced vasodilatation was suppressed by the TRPV4 blocker RuR (1 µmol/L) or L-NNA (100 µmol/L). Values are given as means±SE; *P<0.05; **P<0.001; t test.

In the presence of inhibitors of both NO and prostacyclin synthesis or of the NO synthase inhibitor alone, 4PDD induced only weak vasodilatation in CAs (Figure 4A, middle part), whereas ACh or pharmacological opening of endothelial Ca²⁺-activated potassium channels of intermediate (K_Ca 3.1) and small K_Ca 2.3) conductance by 1-ethyl-2-benzylimidazolinone (1-EB10) caused substantial EDHF-type vasodilatation in CAs (Figure 4A, middle part).¹⁹

The reported mechanosensitivity of TRPV4 channels^6–9 may point to a putative role of TRPV4 in endothelial mechanosensing and thus in shear stress–induced vasodilatation. We therefore tested whether vasodilatation in response to an increase in fluid viscosity and thus shear stress is sensitive to the TRPV blocker RuR. As shown in Figure 4A (right part), the increase in shear stress by 3 dyne/cm² elicited substantial vasodilatation by &17%. Similar to 4PDD-induced vasodilatation, the shear stress–induced vasodilatation was reduced greatly in the presence of 1 µmol/L RuR (Figure 4A, right part) or by chelation of [Ca²⁺]_i by BAPTA-AM (3±1%; n=3). Similar to the increase in viscosity, a 2-fold increase in flow rate (n=4) caused vasodilatation by 16±2% (n=4), which was also suppressed by RuR (5±1%; n=4).

The shear stress–induced vasodilatation of CAs was mediated mainly by NO because shear stress–induced vasodilatation was suppressed greatly by either the combination of the NO synthase blocker N-nitro-L-arginine (L-NNA) and the cyclooxygenase (COX) inhibitor indomethacin (INDO) or L-NNA alone (Figure 4A, right part).

In keeping with the idea that TRPV4 channels in RCAECs are activated by AA as a possible endogenous activator of the channel, especially after mechanical stimulation, we tested whether inhibition of PLA₂ and thus prevention of AA generation affect shear stress–induced vasodilatation. After intraluminal preincubation with the PLA₂ inhibitor AACOCF₃ (4 µmol/L), shear stress–induced vasodilatation was almost completely abolished (Figure 4A, right part). Inhibition of PKC by calphostin-C (0.5 µmol/L; Figure 4A, right part) of tyrosine kinases (TK) by genistein (10 µmol/L; data not shown) and of COX by INDO (10 µmol/L; Figure 4A, right part) did not reduce shear stress–induced vasodilatations, indicating that that activation of these pathways is not required for vasodilatation in response to increased shear stress. None of these compounds (at the concentrations used) blocked 4PDD-induced vasodilatation (data not shown). These results indicate that shear stress–induced vasodilatations of CAs require PLA₂ activation. The release of AA may then lead to activation of TRPV4 as also shown in patch-clamp experiments in RCAECs.

In another set of experiments, we tested whether TRPV4-mediated vasodilatation is also present in small arteries that are considered more important in regulating blood pressure. We therefore conducted pressure myograph experiments in small A gracilis with spontaneous myogenic tone (diameter of 200 µm) using the same stimulation protocols. Similar to the vasodilatory response in CAs, intraluminal but not extraluminal application of 1 µmol/L 4PDD induced a robust and almost complete vasodilatation in these small A gracilis (Figure 4C, left part). Furthermore, 1 µmol/L RuR antagonized this vasodilatation effectively. ACh-induced vasodilatations were not significantly reduced by RuR. In the presence of L-NNA or of L-NNA and INDO, 4PDD was still able to cause almost complete vasodilatation in these small vessels (Figure 4C, left part).

Regarding shear stress–induced vasodilatation in these vessels, the increase in shear stress elicited by adding 5% dextran to the perfusion medium resulted in more pronounced vasodilatation by 73% than in CAs (15%). This difference in the magnitude of vasodilatation might be explained by the higher degree of shear stress exerted by 5% dextran (&15 dyne/cm²) in these small vessels than in large-sized CAs (&3 dyne/cm²). Similar to CAs, shear stress–induced vasodilatation in A gracilis was suppressed greatly by 1 µmol/L RuR or 100 µmol/L L-NNA (Figure 4C, right part).

Thus, these findings show that the presumed TRPV4-mediated vasodilatation, induced either pharmacologically or mechanically, is also present in small arteries. Moreover, shear stress–induced vasodilatation is exclusively mediated by NO, whereas pharmacological opening of TRPV4 is capable to produce considerable EDHF responses in small arteries.

	Discussion

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In the present study, we investigated the function and expression of the TRPV4 channel in rat endothelium and its functional role in the mechanisms of endothelium-dependent vasodilatation. We provide evidence that pharmacological activation of endothelial TRPV4 and subsequent Ca²⁺ influx triggers NO-mediated vasodilatation. Moreover, we propose that mechanical activation of TRPV4 by shear stress might be an important component of endothelial mechanotransduction and thus of shear stress–induced vasodilatation.

We obtained the following evidence that the cation current described here is mediated by TRPV4: (1) The current was activated by 4PDD, which is considered a selective opener of the TRPV4 channels,⁶ and does not affect the function of any other TRP or other channel and enzymes as known so far. Moreover, in mice lacking TRPV4, 4PDD-induced currents as well as Ca²⁺-entry is absent,²⁵ which further supports the selectivity of 4PDD. (2) The endothelial TRPV4-like current in RCAECs exhibited a Ca²⁺-dependent outward rectification and Ca²⁺-dependent inactivation similar to cloned TRPV4.^8,23 (3) The endothelial TRPV4-like current exhibited moderate Ca²⁺ selectivity with a P_Ca/P_Na of &6, similar to cloned TRPV4.^6,8 (4) Endothelial TRPV4-like current was sensitive to RuR, which is considered a fairly selective blocker of TRPV channels within the TRP gene family. (5) Similar to cloned human and murine TRPV4, the TRPV4-like current in RCAECs was activated by moderate warmth,^13,14 mechanically by cell swelling,^6,8,11 and directly by AA as the potential endogenous mediator of mechanical TRPV4 activation.²⁴ (6) Activation of rat TRPV4 by either pharmacologically or other physical stimuli caused significant Ca²⁺ entry.

Moreover, our in situ single-cell RT-PCR analysis revealed mRNA expression of TRPV4 in RCAECs but of none of the other closely related members of this TRPV subfamily.

Regarding the functional role of TRPV4 in endothelium-dependent vasodilatation, our myograph experiments revealed that pharmacological opening of TRPV4 caused a robust vasodilatation in both small-sized (A gracilis) and large CAs. It is noteworthy that this 4PDD-induced vasodilatation was almost as large as that achieved by physiologically relevant concentrations of ACh. The 4PDD-induced vasodilatation required a functionally intact endothelium, which indicates that the 4PDD-induced vasodilatation is indeed caused by opening of endothelial TRPV4. Moreover, 4PDD elicited vasodilatation with a K_D of &0.3 µmol/L, which is comparable to the K_D reported for TRPV4 activation.⁶ 4PDD-induced vasodilatation was prevented by buffering endothelial [Ca²⁺]_i and by the TRPV4 channel blocker RuR, indicating that 4PDD exerts its vasodilating effect by inducing Ca²⁺ influx and subsequently synthesis of endothelial vasodilators. RuR modestly reduced ACh-induced vasodilatation, indicating that endothelial TRPV4 does not contribute substantially to agonist-induced Ca²⁺ signaling and vasodilatation. Perfusion with RuR was without effect on basal vessel diameter, which suggests that TRPV4 is not involved in the basal control of vascular tone. This may also indicate that the rather nonselective blocker RuR does not exert gross unspecific effects or other effects caused by blocking ryanodine-sensitive Ca²⁺ release channels in smooth muscle.²⁶

In CAs with a functionally active endothelium, inhibition of NO synthase alone or in combination with blockade of prostacyclin synthesis almost completely suppressed TRPV4-mediated vasodilatation, suggesting that this type of vasodilatation largely relies on the Ca²⁺-dependent synthesis and action of NO after TRPV4-mediated Ca²⁺-influx, whereas the other 2 major vasodilator systems (ie, the prostacyclin system or the EDHF system) do not seem to make a significant contribution in this CA. Regarding EDHF-mediated vasodilatation, Ca²⁺-dependent activation of endothelial K_Ca channels of the IKCa1 and SKCa3 type and subsequent endothelial hyperpolarization have been considered a prerequisite for the generation of the EDHF signal in many vessels²⁷ and species including rat CAs.¹⁹ Moreover, EDHF-type vasodilatations have been shown to become more important when vessel size decreases.²⁸ In the large CAs, in which the EDHF system is apparently less important than the NO-system, 4PDD-induced TRPV4 activation did not cause major EDHF-mediated vasodilatation. In contrast, 4PDD was able to produce EDHF-mediated vasodilatation in small-sized A gracilis. Therefore, pharmacological opening of TRPV4 appears to be sufficient to induce EDHF-type vasodilatation in small-sized arteries, in which EDHF plays a significant role.

In keeping with the proposed mechanosensitivity of TRPV4,^1,7–9 we speculated that TRPV4 activation and Ca²⁺ entry may occur by mechanical stimulation of the endothelium by increased fluid viscosity and thus shear stress. In this regard, shear stress– or flow-induced increases in [Ca²⁺]_i attributable to both Ca²⁺ influx and Ca²⁺ release from internal stores have been observed in cultured ECs^17,18 as well as in endothelium of perfused vessels.¹⁶ It is noteworthy that such a shear stress–induced increase in [Ca²⁺]_i is prevented by strongly buffering extracellular Ca²⁺ or by the mechanosensitive cation channel and TRP blocker Gd³⁺¹⁸, indicating that an increase in shear stress activates a "directly" or "indirectly" mechanosensitive Ca²⁺ entry channel.

In the present study, we found that an increase in shear stress caused vasodilatation of rat CAs and of small-sized A gracilis in a strictly NO-dependent fashion, which is in agreement with findings in arteries of humans²⁹ and other species;³⁰ whereas in mice, both prostaglandins as well as NO mediate this type of vasodilatation.³¹ Similar to 4PDD-induced vasodilatation, shear stress–induced vasodilatation in rat CAs was greatly blocked by the TRPV4 inhibitor RuR as well as by buffering endothelial [Ca²⁺]_i with BAPTA-AM, suggesting an involvement of TRPV4 in this response. Inhibition of PKC and of TK was without effect, suggesting that protein phosphorylation or potential TRPV4 phosphorylation by one of these kinases does not seem to play a major role in shear stress–induced vasodilatation or activation.

Importantly, shear stress–induced vasodilatation was prevented by inhibition of PLA₂ and thus AA release in rat CAs. Release of AA and production of AA metabolites in response to flow is well documented in cultured ECs,³² and a role of AA metabolites in flow-induced vasodilatation has been proposed previously.³¹ With respect to TRPV4, exogenously applied AA has been shown to activate rat TRPV4 as shown here and cloned TRPV4 previously,⁵ and endogenously produced AA mediates mechanical activation of TRPV4 by cell swelling.²⁴ These roles of AA in both shear stress–induced vasodilatation and TRPV4 activation tempted us to speculate that PLA₂-mediated AA release after shear stress stimulation mediates TRPV4 activation. This interpretation also implies that TRPV4 is unlikely to be the mechanosensor per se. Nonetheless, AA-dependent activation of TRPV4 might be an essential component in the signal transduction mechanism of endothelial mechanotransduction.

Collectively, this set of data strongly suggests that Ca²⁺ entry through endothelial TRPV4 channels triggers NO-dependent vasodilatation in endothelium of rat CAs and NO- and EDHF-dependent vasodilatation of small-sized A gracilis (more resistance-like artery). Moreover, we provide evidence that endothelial TRPV4 channels are involved in endothelial mechanosensing of shear stress–induced vasodilatation. Thus, among the numerous TRP channels expressed in endothelium, a role of TRPV4 might be specifically assigned to endothelial mechanotransduction. Moreover, because pharmacological opening of TRPV4 causes robust vasodilatation, endothelial TRPV4 may represent a novel pharmacological target for the treatment of hypertension.

	Acknowledgments

 
Sources of Funding

This work was supported by the Deutsche Forschungsgemeinschaft (FOR 341/7, HO 1103/2-4, GRK 865 [R.K., J.H.] and SCHU 805/7-1 [R.S.]).

Disclosures

None.

	Footnotes

 
Original received December 12, 2005; final version accepted April 18, 2006.

	References

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