TRPV4-dependent dilation of peripheral resistance arteries influences arterial pressure

Abstract

Transient receptor potential vanilloid 4 (TRPV4) channels have been implicated as mediators of calcium influx in both endothelial and vascular smooth muscle cells and are potentially important modulators of vascular tone. However, very little is known about the functional roles of TRPV4 in the resistance vasculature or how these channels influence hemodynamic properties. In the present study, we examined arterial vasomotor activity in vitro and recorded blood pressure dynamics in vivo using TRPV4 knockout (KO) mice. Acetylcholine-induced hyperpolarization and vasodilation were reduced by ∼75% in mesenteric resistance arteries from TRPV4 KO versus wild-type (WT) mice. Furthermore, 11,12-epoxyeicosatrienoic acid (EET), a putative endothelium-derived hyperpolarizing factor, activated a TRPV4-like cation current and hyperpolarized the membrane of vascular smooth muscle cells, resulting in the dilation of mesenteric arteries from WT mice. In contrast, 11,12-EET had no effect on membrane potential, diameter, or ionic currents in the mesenteric arteries from TRPV4 KO mice. A disruption of the endothelium reduced 11,12-EET-induced hyperpolarization and vasodilatation by ∼50%. A similar inhibition of these responses was observed following the block of endothelial (small and intermediate conductance) or smooth muscle (large conductance) K⁺ channels, suggesting a link between 11,12-EET activity, TRPV4, and K⁺ channels in endothelial and smooth muscle cells. Finally, we found that hypertension induced by the inhibition of nitric oxide synthase was greater in TRPV4 KO compared with WT mice. These results support the conclusion that both endothelial and smooth muscle TRPV4 channels are critically involved in the vasodilation of mesenteric arteries in response to endothelial-derived factors and suggest that in vivo this mechanism opposes the effects of hypertensive stimuli.

the vascular endothelium produces a variety of potent vasoactive factors that are critically important for blood flow and blood pressure regulation. Epoxyeicosatrienoic acids (EETs) are produced by cytochrome P-450 epoxygenase enzymes from arachidonic acid and act as an endothelium-derived hyperpolarizing factor (EDHF) in some vascular beds (3, 24). Vasodilation and smooth muscle cell hyperpolarization in response to EETs are associated with the increased activity of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels. The vanilloid transient receptor potential channel TRPV4 can be activated by EETs, suggesting that the channel can also serve as a cell-surface receptor for these compounds. We recently demonstrated that in cerebrovascular smooth muscle, TRPV4 forms a novel EETs-induced Ca²⁺ signaling complex with ryanodine receptors and BK_Ca channels that elicits smooth muscle hyperpolarization and arterial dilation via Ca²⁺-induced Ca²⁺ release (6). Work from other laboratories shows that EETs activate TRPV4 channels in the endothelium by an autocrine mechanism (30, 31). The relative importance of endothelial cell versus smooth muscle TRPV4 channels in endothelium-dependent vasodilation in response to EETs has not been investigated.

Small arteries and arterioles are the primary site of peripheral vascular resistance, a major determinant of arterial blood pressure. EDHF-type vasodilation appears to have a greater influence on resistance arteries compared with other endothelium-derived factors such as nitric oxide (NO) and prostacyclin. The molecular nature of EDHF remains elusive, and evidence supporting roles for K⁺ ions (8), communication via myoendothelial gap junctions (4), and EETs (3) as mediators of this response has been reported. Further insight into the signaling mechanism responsible for the EDHF-dependent regulation of peripheral resistance vessels will strengthen our understanding of blood flow and blood pressure regulation. Several studies have demonstrated the presence and possible function of TRPV4 in endothelial (31, 34) and vascular smooth muscle cells (6). However, although it is known that TRPV4 channels influence vascular tone in response to EETs, it has not been reported whether this mechanism is related to EDHF-type vasodilation. To test the hypothesis that the TRPV4-dependent vasodilation of peripheral resistance arteries contributes to blood pressure regulation, the EDHF-type and 11,12-EETs-induced vasodilatory response in isolated mesenteric resistance arteries and changes in hemodynamic responses during NO synthase (NOS)-induced hypertension in vivo were compared between wild-type (WT) and TRPV4 gene-deleted knockout (KO) mice. Our findings demonstrate that the TRPV4-dependent regulation of resistance artery tone contributes to arterial pressure regulation during hypertension.

MATERIALS AND METHODS

Experimental animals.

Mice that are heterozygous for TRPV4 gene deletion, developed by Liedtke and Friedman (19), were bred to provide the WT and TRPV4 KO mice used in the present study. All animal use procedures were in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee of the University of Vermont. In some experiments, mice were made hypertensive by the addition of the NOS inhibitor N^ω-nitro-l-arginine (l-NNA) to the drinking water (0.5 g/l). Systolic and diastolic blood pressures were recorded by tail-cuff plethysmography (NIPB-2 blood pressure monitor, Columbus Instruments), and mean arterial pressure (MAP) was calculated from these data as we have previously reported (27). The average of three to four measurements on each day of treatment with l-NNA was taken as a representative pressure for each animal.

In vitro studies.

Mice were euthanized by an intraperitoneal injection of pentobarbital sodium (100 mg/kg), followed by a thoracotomy. Second branch order mesenteric resistance arteries (150–200 μm internal diameter) were then isolated and mounted in an arteriograph for measurements of diameter and membrane potential, as previously described (6, 7). In brief, isolated arterial segments were placed in a vessel chamber. One end of the vessel was cannulated with a glass micropipette and secured, the lumen was gently rinsed, and the downstream end of the vessel was then cannulated and secured. In some experiments the endothelium was removed by the passage of an air bubble through the lumen. The vessels were pressurized to 60 mmHg with physiological saline solution and superfused with warmed (37°C) bicarbonate buffered physiological saline solution containing (in mM) 118 NaCl, 4.7 KCl, 24 NaHCO₃, 1.2 KH₂PO₄, 0.026 EDTA, 1.2 MgSO₄, 1.5 CaCl₂, 5 glucose, 0.3 l-NNA, and 0.01 indomethacin (pH 7.4, following aeration with a gas mixture of 21% O₂-5% CO₂-balance N₂). After a 60-min equilibration, vasoconstriction was induced by the administration of phenylephrine (PE, 1 μM) via the superfusate. The inner diameter was monitored using video microscopy and edge-detection software (Living Systems Instrumentation). For the measurement of the smooth muscle cell membrane potential, the arteries were isolated and pressurized and the smooth muscle cells were impaled through the abluminal wall with glass intracellular microelectrodes (tip resistance, 100–200 MΩ). A WPI Intra 767 amplifier was used for recording the membrane potential. Analog output from the amplifier was recorded using Axotape software (sample frequency, 20 Hz). The criteria for the acceptance of the membrane potential recordings were 1) an abrupt negative deflection of potential as the microelectrode was advanced into a cell, 2) a stable membrane potential for at least 1 min, and 3) an abrupt change in potential to ∼0 mV after the electrode was retracted from the cell. The membrane potential was measured once the steady-state diameter responses to various agents were achieved. Changes in membrane potential and vasodilatory responses were measured separately but in the same artery.

Patch-clamp electrophysiology.

Whole cell patch-clamp experiments were employed to determine whether ion channels with biophysical properties similar to TRPV4 are present in freshly isolated mesenteric artery myocytes. To isolate the vascular smooth muscle cells, second order mesenteric resistance arteries were cut into 2-mm segments and enzymatically digested as previously described for cerebral artery myocytes (6). Patch-clamp experiments were performed using an Axopatch 200B amplifier equipped with a CV203BU headstage (Axon Instruments) as previously described (6). The recording electrodes (resistance, ∼5 MΩ) were pulled from borosilicate glass (outer diameter, 1.5 mm; and inner diameter, 1.17 mm; Sutter Instrument, Novato, CA) and coated with wax to reduce capacitance. The currents were filtered at 1 kHz and digitized at 5 kHz. pCLAMP version 9.0 and Clampfit version 9.0 (Axon Instruments) were used for data acquisition and analysis. The bath solution contained (in mM) 142 NaCl, 10 glucose, 10 HEPES, 2 CaCl₂, 6 KCl, and 1 MgCl₂ (pH 7.4). The pipette solution contained (in mM) 100 Na⁺ glutamate, 20 NaCl, 1 MgCl₂, 1 EGTA, 10 HEPES, and 4 Na₂ATP (pH 7.2). The currents were recorded during voltage ramps from −108 to +92 mV. The ramps were repeated every 20 s for at least 1 min before drug administration and, after administration, for every 20 s until maximal current activation. The cells were held at 0 mV between voltage ramps. For all experiments, the currents were normalized to membrane capacitance and a 12-mV liquid junction potential offset was subtracted during the analysis.

Calculations and statistics.

All data are means ± SE. Values of n refer to number of animals. Comparisons involving two experimental groups were made by unpaired Student's t-tests. Comparisons involving multiple groups were made by one-way or two-way ANOVA. Blood pressure data were compared using a two-way repeated-measures ANOVA. In some cases, individual comparisons were made following ANOVA using the Student-Newman-Keuls post hoc test. A level of P ≤ 0.05 was accepted as statistically significant for all experiments.

RESULTS

Endothelium-dependent vasodilation is impaired in arteries isolated from TRPV4 KO mice.

EDHF responses are characterized by endothelium-dependent dilation in response to receptor activation during NOS and cyclooxygenase inhibition. To determine whether TRPV4 channels are involved in EDHF-type vasodilatory responses, we examined the effects of the muscarinic receptor agonist acetylcholine (ACh) on smooth muscle membrane potential and vascular tone of mesenteric resistance arteries isolated from WT and TRPV4 KO mice. The experiments were performed in the presence of the NOS inhibitor l-NNA (100 μM) and the cyclooxygenase inhibitor indomethacin (10 μM). Arteries were pressurized to 60 mmHg and preconstricted to ∼50% of the resting diameter with the α₁-adrenoreceptor agonist PE. Baseline and maximal luminal diameters and sensitivity to PE-induced constriction did not differ between the WT and TRPV4 KO groups, demonstrating that the agonist-induced vasoconstrictor responses are independent of TRPV4 expression. We found that smooth muscle cell hyperpolarization and vasodilation induced by ACh (3 μM) are reduced by ∼75% in mesenteric arteries from TRPV4 KO versus WT mice (Fig. 1). These findings show that much of the NO- and prostacyclin-independent component of ACh-induced smooth muscle hyperpolarization and vasodilation depends on TRPV4 expression, suggesting a role for TRPV4 in EDHF-dependent responses in these arteries.

Fig. 1.

ACh-induced hyperpolarization and dilation are suppressed in mesenteric arteries from transient receptor potential vanilloid 4 knockout (TRPV4 KO) vs. wild-type (WT) mice. Arteries were pretreated with N^ω-nitro-l-arginine (l-NNA, 100 μM) and indomethacin (10 μM), constricted to 50% of maximum using phenylephrine (PE) and then exposed to a single dose of ACh (3 μM). Initial diameters and membrane potentials were 157 ± 3 μm and −32 ± 2 mV, respectively, for WT arteries and 178 ± 15 μm and −32 ± 1 mV, respectively, for KO arteries. *P < 0.05 vs. WT; n = 7 WT and 6 TRPV4 KO mice.

11,12-EET-induced smooth muscle hyperpolarization and vasodilation are absent in arteries from TRPV4 KO mice.

Our initial findings (Fig. 1) demonstrate that TRPV4 channels are required for a large portion of ACh-induced vasodilation during NOS and cyclooxygenase inhibition. TRPV4 channels are activated by EETs, which are also endothelium-derived vasodilatory factors in some vascular beds. In the next series of experiments, resistance arteries were studied in isolation to examine the effects of TRPV4 gene deletion on vasodilation and smooth muscle cell hyperpolarization in response to 11,12-EET. Mesenteric resistance arteries from WT and TRPV4 KO mice were cannulated, pressurized to 60 mmHg, and constricted to ∼50% of their initial diameters using PE (1–3 μM). We found that a subsequent administration of 11,12-EET (3 μM) caused smooth muscle cell hyperpolarization and arterial dilation in WT mice (Fig. 2). No additional effect was observed when 11,12-EET was increased to 5 μM, indicating that a concentration of 3 μM is maximally effective for these studies. 11,12-EET-induced hyperpolarization and vasodilation were absent in arteries from TRPV4 KO mice (Fig. 2), demonstrating that TRPV4 mediates smooth muscle cell hyperpolarization and vasodilation in response to 11,12-EET in mesenteric arteries. In further support of a role for TRPV4 channels in the responses to 11,12-EET, we found that ruthenium red (1 μM) fully reversed the 11,12-EET-induced smooth muscle hyperpolarizations of arteries from WT mice [membrane potential (V_m) control,−33 ± 1 mV; V_m with 11,12-EET, −43 ± 1; and V_m with 11,12-EET + ruthenium red, −33 ± 1, n = 4]. To establish the relative importance of smooth muscle versus endothelial cell TRPV4 channels in 11,12-EET-induced responses, experiments were performed following the mechanical disruption of the endothelium. 11,12-EET elicited smooth muscle cell hyperpolarization and vasodilation in endothelium-denuded arteries from WT mice, although to a lesser extent than endothelium-intact vessels (Fig. 2). These findings suggest that ∼50% of the 11,12-EET-induced hyperpolarization and vasodilatation in mesenteric resistance arteries is endothelium dependent.

Fig. 2.

11,12-Epoxyeicosatrienoic acid (11,12-EET)-induced vasodilation and smooth muscle hyperpolarization are absent in mesenteric arteries from TRPV4 KO mice. Top: vasodilation (%reversal of PE-induced constriction) in response to 11,12-EET (3 μM). Bottom: change in smooth muscle membrane potential (in mV) in response to 11,12-EET (3 μM). +E and −E, data from arteries with and without endothelium, respectively. Significance levels are as indicated. NS, no significant difference. Initial diameters and membrane potentials were 143 ± 16 μm and −32 ± 2 mV for WT + E arteries, 137 ± 10 μm and −34 ± 1 mV for WT − E arteries, 181 ± 17 μm and −31 ± 1 mV for KO + E arteries, and 135 ± 10 μm and −30 ± 1 mV for KO − E arteries, respectively; n = 5 for each group.

11,12-EET activates TRPV4-like whole cell currents in mesenteric artery myocytes from WT, but not TRPV4 KO, mice.

EETs activate cloned TRPV4 channels expressed in HEK cells (31) and in TRPV4 channels present in native cerebral artery myocytes (6), suggesting that TRPV4 channels act as an “EETs” receptor. Consistent with this possibility, we found that 11,12-EET (3 μM) activated inwardly rectifying whole cell currents in mesenteric artery myocytes from WT mice, but these currents were absent from cells isolated from TRPV4 KO mice (Fig. 3). These findings demonstrate that 11,12-EET activates a TRPV4-dependent whole cell current in mesenteric artery smooth muscle.

Fig. 3.

11,12-EET-activated whole cell currents are absent in vascular smooth muscle cells from TRPV4 KO mice. A: examples of whole cell currents activated by 11,12-EET (3 μM) in mesenteric arterial smooth muscle cells isolated from WT and TRPV4 KO mice. I, current; V, voltage. B: summary data for 11,12-EET-activated currents (difference currents) in WT (n = 5) and TRPV4 KO (n = 3) mouse mesenteric artery myocytes. *P < 0.05 vs. WT.

11,12-EET-induced smooth muscle hyperpolarization and vasodilation are inhibited by Ca²⁺-activated K⁺ channel blockers.

Compelling evidence supporting the hypothesis that the activation of small- and intermediate-conductance Ca²⁺-activated K⁺ channels (K_Ca2.3 and K_Ca3.1) in endothelial cells mediates EDHF-type responses has been reported (16, 26, 27). The activation of these channels can hyperpolarize the endothelial cell membrane, which serves to hyperpolarize the underlying smooth muscle via myoendothelial gap junctional communication. In addition, 11,12-EETs stimulates BK_Ca activity by a mechanism that is dependent on TRPV4 channels and Ca²⁺-induced Ca²⁺ release in cerebral artery myocytes. To explore the potential roles for these pathways in mesenteric resistance arteries, the effects of selective blockers of Ca²⁺-activated K⁺ channels on EETs-induced smooth muscle hyperpolarization and vasodilation were determined. The coadministration of apamin (0.3 μM), a selective inhibitor of small-conductance K_Ca channels, and the K_Ca3.1 blocker 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34, 1 μM) reduced 11,12-EET-induced hyperpolarization and dilation by about 50% (Fig. 4). The administration of the selective BK_Ca channel blocker iberiotoxin (100 nmol/l) alone also diminished 11,12-EET-induced responses by ∼50% (Fig. 4). The coadministration of all three K_Ca channel blockers completely inhibited 11,12-EET-induced hyperpolarization and nearly eliminated the vasodilator response (Fig. 4). Taken together, these results suggest that the activation of K_Ca2.3/ K_Ca3.1 and BK_Ca channels both contribute to 11,12-EET-induced hyperpolarization in mesenteric arteries. The expression of K_Ca2.3 and K_Ca3.1 channels is restricted to the endothelium, whereas BK_Ca channels are expressed primarily in smooth muscle cells (5, 17). Our findings are consistent with the proposal that ∼50% of 11,12-EETs-induced smooth muscle cell hyperpolarization is due to endothelial K_Ca2.3/K_Ca3.1 activity and that ∼50% is due to arterial myocyte BK_Ca activation.

Fig. 4.

11,12-EET-induced hyperpolarizations are inhibited by combined apamin and 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34), or IBTX alone, and are abolished in the presence of all 3 K⁺ channel blockers. The channel blockers were added both to the arterial lumen and the bath solution. Initial diameters and membrane potentials were, respectively, 127 ± 11 μm and −30 ± 1 mV for control arteries, 152 ± 9 μm and −30 ± 1 mV for arteries treated with apamin (0.3 μM) and TRAM-34 (1 μM), 127 ± 7 μm and −31 ± 1 mV for arteries treated with iberiotoxin (IBTX, 100 nM), and 122 ± 7 μm and −31 ± 1 mV for arteries treated with apamin, TRAM-34, and IBTX. *P < 0.05 vs. control; †P < 0.05 vs. control, apamin + TRAM-34, and IBTX; n = 4 mice for each condition.

TRPV4 gene deletion exacerbates NOS inhibition-induced hypertension.

Arteries from TRPV4 KO mice exhibit a diminished responsiveness to endothelium-dependent vasodilators (Fig. 1) that is associated with a decreased sensitivity to the effects of 11,12-EET (Fig. 2). In vivo, a decreased endothelium-dependent vasodilation results in an increased peripheral vascular resistance and elevated arterial pressure. To test the hypothesis that the TRPV4-dependent vasodilation of resistance arteries contributes to hemodynamic responses, we compared MAP between WT and TRPV4 KO mice. In agreement with a prior report (32), we found that MAP did not differ between TRPV4 KO and WT mice under baseline conditions (87.6 ± 2.5 vs. 88.6 ± 4.0 mmHg, n = 8 for each group). To unmask the potential contribution of TRPV4 activity in blood pressure regulation, the NOS inhibitor l-NNA was added to the animals' drinking water. Their heart rates decreased in response to NOS inhibition but did not differ between TRPV4 KO and WT mice (data not shown). Both WT and TRPV4 KO animals became hypertensive after 2 days of l-NNA treatment (Fig. 5). Interestingly, the resulting hypertension was greater in TRPV4 KO mice compared with WT controls (Fig. 5). The peak increases in MAP were observed 3 days after the NOS inhibition protocol was started (105 ± 3.8 for TRPV4 KO vs. 99.3 ± 3.8 mmHg for WT, n = 8 for each group) and returned toward baseline levels by day 5. Although the effect of l-NNA on MAP was transient in both groups, as has been observed in other studies employing mice (20), the present findings clearly indicate that TRPV4 channels can have a moderating effect on MAP during NOS inhibition-induced hypertension.

Fig. 5.

Nitric oxide synthase inhibition-induced hypertension is greater in TRPV4 KO vs. WT mice. Mean arterial pressure in TRPV4 KO (n = 5–8) and WT (n = 7 to 8) mice before and after the nitric oxide synthase inhibitor l-NNA was added to drinking water. *P ≤ 0.05 vs. WT.

DISCUSSION

The major findings of this study are as follows: 1) smooth muscle cell hyperpolarization and vasodilation of mesenteric resistance arteries induced by ACh or the putative EDHF 11,12-EET are greatly attenuated in arteries from TRPV4 KO mice; 2) ∼50% of 11,12-EET-induced vasodilation is endothelium dependent, and about 50% is due to direct effects on the vascular smooth muscle cells; 3) 11,12-EET activates a TRPV4-like current in mesenteric arterial smooth muscle cells from WT, but not TRPV4 KO, mice; 4) 11,12-EET-induced smooth muscle hyperpolarization in intact arteries is mediated by small-, intermediate-, and large-conductance Ca²⁺-activated K⁺ channels; and 5) hypertension induced by the inhibition of NOS activity is enhanced in TRPV4 KO mice versus WT controls. These results indicate that TRPV4 channels in both endothelial and smooth muscle cells are important contributors to endothelium-dependent hyperpolarization and vasodilation of mesenteric resistance arteries. Furthermore, these studies suggest that vascular TRPV4 channel activity may be an important mechanism that normally opposes hypertensive stimuli in vivo.

Agonist-induced, endothelium-dependent vasodilation in the absence of NO and prostacyclin production defines the EDHF response. The present study demonstrates that >75% of ACh-induced dilation of mesenteric resistance arteries during NOS and cyclooxygenase inhibition is dependent on the expression of TRPV4 channels. This finding is consistent with previous reports on muscarinic receptor-mediated vasodilation of the superior mesenteric artery (25) and its branches (34) but contrasts with the apparent lack of involvement of TRPV4 channels in ACh-mediated vasodilation of the carotid artery (15), a conduit vessel. This could be explained by the general observation that the contribution of EDHF to the regulation of vascular tone is inversely proportional to arterial diameter, with the greatest involvement in resistance arteries (14). In any case, our findings imply a critical contribution of TRPV4 channels in EDHF-mediated hyperpolarization and vasodilation in the resistance vasculature.

In a number of vascular beds (3), including the mesenteric circulation (7), cytochrome P-450 metabolites, in particular certain isoforms of EETs, have been identified as likely EDHFs. The current results indicate that TRPV4 channels present in vascular smooth muscle cells contribute to the mechanisms of vasodilation associated with EET activity in peripheral resistance arteries. TRPV4 channels have been reported to be present in cerebral, aortic, pulmonary, and submucosal mesenteric vascular smooth muscle cells (1, 6, 21, 23, 33). These channels are activated by EETs in various cell types (6, 31), and significant Ca²⁺ entry through the activated TRPV4 channels occurs (29). We have found that Ca²⁺ entry through TRPV4 channels, activated by 11,12-EET in rat cerebral artery myocytes, increases localized Ca²⁺ release events (“Ca²⁺ sparks”) from the sarcoplasmic reticulum, which then increases the activity of BK_Ca channels (6). The associated membrane hyperpolarization decreases the activity of voltage-dependent Ca²⁺ channels, which leads to vasodilation. Here we find that TRPV4 channels are present in mesenteric arterial smooth muscle and are activated by EETs and that the activation of these channels in smooth muscle cells leads to hyperpolarization and dilation via a mechanism that involves BK_Ca channels. Thus this mechanism of the action of a probable EDHF is potentially important not only regarding regulation of cerebral blood flow but also in the peripheral circulation where a significant role in the regulation of vascular resistance and blood pressure is possible.

Numerous studies demonstrate that EDHF-type vasodilatory responses involve small- and intermediate-conductance Ca²⁺-activated K⁺ channels (2, 5, 9). We report here that ∼50% of 11,12-EET-induced hyperpolarization and the dilation of mesenteric resistance arteries is endothelium dependent and appears to involve TRPV4 as well as K_Ca2.3 and K_Ca3.1 channels present in endothelial cells. Previous studies have demonstrated that TRPV4 channels are present in arterial endothelial cells (21) and that the stimulation of these cells by EETs promotes Ca²⁺ entry and endothelial cell hyperpolarization (11). Our study is the first to demonstrate that the activation of endothelial TRPV4 channels by EETs induces the hyperpolarization and relaxation of resistance artery smooth muscle and that this response involves the activation of K_Ca channels, presumably located in the endothelium. The small- and intermediate-conductance K_Ca channel-mediated endothelial hyperpolarization is likely transmitted to the vascular smooth muscle cells via gap junctional communication (10, 16).

Previous studies demonstrated that TRPV4 KO mice have reduced responsiveness to pressure sensations (19, 22), disturbed osmotic regulation (19), altered thermosensitivity (18), and abnormal urinary bladder-voiding function (13, 28). We have found that elevations in blood pressure to a hypertensive challenge, although modest and transient, are significantly larger in TRPV4 KO compared with WT mice. This observation indicates that TRPV4 channels are involved in a negative feedback mechanism that limits the response to hypertensive stimuli. The suggestion that the activation of TRPV4 channels could have a significant impact on blood pressure regulation is supported by recent studies demonstrating that ACh-induced reductions in blood pressure are substantially less in TRPV4 KO versus WT mice (34) and that TRPV4-channel activation substantially reduces blood pressure in normotensive and salt-loaded hypertensive rats, an effect that is attenuated by the suppression of TRPV4 channel expression in vivo (12). Blood pressure control is extremely complex and involves numerous factors such as the central nervous system-mediated release of autonomic neurotransmitters, the hormone-dependent regulation of blood volume, and the local control of resistance artery tone. The antihypertensive effects of TRPV4 expression may encompass any or all of these systems. Nevertheless, our observations demonstrating a critical vasodilatory role for TRPV4 channels in the resistance vasculature suggests that at least a portion of this effect occurs at the level of the vascular smooth muscle and endothelial cell membrane via the regulation of membrane potential and arterial tone.

Our findings demonstrate that TRPV4 channels are essential for agonist-dependent vasodilation of resistance arteries during NOS and cyclooxygenase inhibition. This work also clearly implies a linkage between TRPV4 channels and small-, intermediate-, and large-conductance channels in the mechanisms of vasodilation mediated by EDHF. Furthermore, we show that TRPV4 KO mice are more sensitive to NOS inhibition-induced hypertension. Collectively, this study suggests that the localized control of resistance artery tone involving TRPV4 and K_Ca channel activity could contribute to the regulation of vascular tone during hypertension. Clearly, animal models with a cell type-specific deletion of TRPV4 channels would be required to verify this proposal. Nevertheless, TRPV4 channels in the vasculature represent a promising new target for antihypertensive drug therapy.

GRANTS

This work was supported by American Heart Association Grant AHA0535226N; National Heart, Lung, and Blood Institute Grants R01-HL-091905 (to S. Earley) and RO1-HL-58231 (to J. E. Brayden); and the Totman Medical Research Trust.