Joseph E. Brayden

TRPV4 Forms a Novel Ca2+ Signaling Complex With Ryanodine Receptors and BKCa Channels

Vasodilatory factors produced by the endothelium are critical for the maintenance of normal blood pressure and flow. We hypothesized that endothelial signals are transduced to underlying vascular smooth muscle by vanilloid transient receptor potential (TRPV) channels. TRPV4 message was detected in RNA from cerebral artery smooth muscle cells. In patch-clamp experiments using freshly isolated cerebral myocytes, outwardly rectifying whole-cell currents with properties consistent with those of expressed TRPV4 channels were evoked by the TRPV4 agonist 4&agr;-phorbol 12,13-didecanoate (4&agr;-PDD) (5 &mgr;mol/L) and the endothelium-derived arachidonic acid metabolite 11,12 epoxyeicosatrienoic acid (11,12 EET) (300 nmol/L). Using high-speed laser-scanning confocal microscopy, we found that 11,12 EET increased the frequency of unitary Ca2+ release events (Ca2+ sparks) via ryanodine receptors located on the sarcoplasmic reticulum of cerebral artery smooth muscle cells. EET-induced Ca2+ sparks activated nearby sarcolemmal large-conductance Ca2+-activated K+ (BKCa) channels, measured as an increase in the frequency of transient K+ currents (referred to as “spontaneous transient outward currents” [STOCs]). 11,12 EET–induced increases in Ca2+ spark and STOC frequency were inhibited by lowering external Ca2+ from 2 mmol/L to 10 &mgr;mol/L but not by voltage-dependent Ca2+ channel inhibitors, suggesting that these responses require extracellular Ca2+ influx via channels other than voltage-dependent Ca2+ channels. Antisense-mediated suppression of TRPV4 expression in intact cerebral arteries prevented 11,12 EET–induced smooth muscle hyperpolarization and vasodilation. Thus, we conclude that TRPV4 forms a novel Ca2+ signaling complex with ryanodine receptors and BKCa channels that elicits smooth muscle hyperpolarization and arterial dilation via Ca2+-induced Ca2+ release in response to an endothelial-derived factor.

Nitric oxide hyperpolarizes rabbit mesenteric arteries via ATP‐sensitive potassium channels.

1. Nitric oxide (NO) relaxes vascular smooth muscle (VSM) by mechanisms which are not fully understood. One possibility is that NO hyperpolarizes membranes, thereby diminishing Ca2+ entry through voltage‐dependent Ca2+ channels. In the current study, the effects of NO on membrane potential of rabbit mesenteric arteries were recorded using intracellular microelectrodes. 2. NO, released by 3‐morpholinosydnonimine (SIN‐1, 3 microM), reversibly hyperpolarized arteries by ‐9.5 +/‐ 4.0 mV (means +/‐ S.D., n = 97) from a resting membrane potential of ‐53.1 +/‐ 5.7 mV. The hyperpolarization was blocked by oxyhaemoglobin (20 microM), and only occurred in arteries pre‐treated with N omega‐nitro‐L‐arginine (100 microM) or denuded of endothelium. 3. The effect of SIN‐1 was concentration dependent (EC50 approximately 0.4 microM) and its dose response was shifted to the left by zaprinast (100 microM), an inhibitor of cGMP‐specific phosphodiesterases. 4. The hyperpolarization due to SIN‐1 was modified by changes in extracellular K+ concentration, but not by changes in Ca2+, Na+ or Cl‐. The hyperpolarization was blocked by glibenclamide (IC50 approximately 0.15 microM), but not by apamin (3‐300 nM), barium (5‐150 microM), tetraethylammonium (0.1‐10 mM), or 4‐aminopyridine (5‐500 microM). The hyperpolarization due to lemakalim (0.03‐3 microM), an activator of ATP‐sensitive potassium channels (KATP), displayed the same sensitivities to these K+ channel blocking agents, whereas the endothelium‐derived hyperpolarizing factor, triggered by the addition of acetylcholine (3 microM), caused a hyperpolarization (‐15.3 +/‐ 6.2 mV) that was blocked by apamin, but not by any other agent. 5. These results suggest that NO hyperpolarizes VSM in rabbit mesenteric arteries by activating KATP channels, with the accumulation of cGMP as an intermediate step.

Critical Role for Transient Receptor Potential Channel TRPM4 in Myogenic Constriction of Cerebral Arteries

Local control of cerebral blood flow is regulated in part through myogenic constriction of resistance arteries. Although this response requires Ca2+ influx via voltage-dependent Ca2+ channels secondary to smooth muscle cell depolarization, the mechanisms responsible for alteration of vascular smooth muscle (VSM) cell membrane potential are not fully understood. A previous study from our laboratory demonstrated a critical role for a member of the transient receptor potential (TRP) superfamily of ion channels, TRPC6, in this response. Several other of the approximately 22 identified TRP proteins are also present in cerebral arteries, but their functions have not been elucidated. Two of these channels, TRPM4 and TRPM5, exhibit biophysical properties that are consistent with a role for control of membrane potential of excitable cells. We hypothesized that TRPM4/TRPM5-dependent currents contribute to myogenic vasoconstriction of cerebral arteries. Cation channels with unitary conductance, ion selectivity and Ca2+-dependence similar to those of cloned TRPM4 and TRPM5 were present in freshly isolated VSM cells. We found that TRPM4 mRNA was detected in both whole cerebral arteries and in isolated VSM cells whereas TRPM5 message was absent from cerebral artery myocytes. We also found that pressure-induced smooth muscle cell depolarization was attenuated in isolated cerebral arteries treated with TRPM4 antisense oligodeoxynucleotides to downregulate channel subunit expression. In agreement with these data, myogenic vasoconstriction of intact cerebral arteries administered TRPM4 antisense was attenuated compared with controls, whereas KCl-induced constriction did not differ between groups. We concluded that activation of TRPM4-dependent currents contributed to myogenic vasoconstriction of cerebral arteries.

Functional Roles Of KATP Channels In Vascular Smooth Muscle

1. ATP‐sensitive potassium channels (KATP) are present in vascular smooth muscle cells and play important roles in the vascular responses to a variety of pharmacological and endogenous vasodilators.

Calcitonin gene‐related peptide activated ATP‐sensitive K+ currents in rabbit arterial smooth muscle via protein kinase A.

1. Whole‐cell K+ currents activated by calcitonin gene‐related peptide (CGRP) in smooth muscle cells enzymatically isolated from rabbit mesenteric arteries were measured in the conventional and perforated configurations of the patch clamp technique. The signal transduction pathway from CGRP receptors to activation of potassium currents was investigated. 2. CGRP (10 nM) activated a whole‐cell current that was blocked by glibenclamide (10 microM), an inhibitor of ATP‐sensitive K+ channels. Elevating intracellular ATP reduced glibenclamide‐sensitive currents. CGRP increased the glibenclamide‐sensitive currents by 3‐ to 6‐fold in cells dialysed with 0.1 mM ATP, 3.0 mM ATP or in intact cells. The reversal potential of the glibenclamide‐sensitive current in the presence of CGRP shifted with the potassium equilibrium potential, while its current‐voltage relationship exhibited little voltage dependence. 3. Forskolin (10 microM), an adenylyl cyclase activator, Sp‐cAMPS (500 microM) and the catalytic subunit of protein kinase A increased glibenclamide‐sensitive K+ currents 2.1‐, 3.3‐ and 8.2‐fold, respectively. 4. Nitric oxide and nitroprusside did not activate glibenclamide‐sensitive K+ currents. 5. Dialysis of the cells interior with inhibitors of protein kinase A (synthetic peptide inhibitor, 4.6 microM or H‐8, 100 microM) completely blocked activation of K+ currents by CGRP. 6. Our results suggest the following signal transduction scheme for activation of K+ currents by CGRP in arterial smooth muscle: (1) CGRP stimulates adenylyl cyclase, which leads to an elevation of cAMP; (2) cAMP activates protein kinase A, which opens ATP‐sensitive K+ channels.

Chloride channel blockers inhibit myogenic tone in rat cerebral arteries

1 We have investigated the role of chloride channels in pressure‐induced depolarization and contraction of cerebral artery smooth muscle cells. 2 Two chloride channel blockers, indanyloxyacetic acid (IAA‐94) and 4,4′‐diisothiocyanato‐stilbene‐2,2′‐disulphonic acid (DIDS), caused hyperpolarizations (10–15 mV) and dilatations (up to 90 %) of pressurized (80 mmHg), rat posterior cerebral arteries. Niflumic acid, a blocker of calcium‐activated chloride channels, did not affect arterial tone. 3 Dilatations to IAA‐94 and DIDS were unaffected by potassium channel blockers, but were prevented by elevated potassium. IAA‐94 and DIDS had no effect on membrane potential or diameter of arteries at low intravascular pressure, where myogenic tone is absent. Reduction of extracellular chloride (60 mm Cl−) increased the pressure‐induced contractions. Removal of extracellular sodium did not affect the pressure‐induced responses. 4 Our results suggest that intravascular pressure activates DIDS‐ and IAA‐94‐sensitive chloride channels to depolarize arterial smooth muscle, thereby contributing to the myogenic constriction.

Apamin‐sensitive K+ channels mediate an endothelium‐dependent hyperpolarization in rabbit mesenteric arteries.

1. Vascular endothelial cells release a variety of substances which affect the membrane potential and tone of underlying vascular smooth muscle. In the presence of N omega‐nitro‐L‐arginine to inhibit nitric oxide synthase and indomethacin to inhibit cyclo‐oxygenase, acetylcholine (ACh; EC50 approximately 1 microM) elicited the release of an endothelium‐derived hyperpolarizing factor (EDHF) in rabbit mesenteric arteries. 2. The hyperpolarization due to EDHF was blocked by apamin (IC50 approximately 0.3 nM), and by other inhibitors of the apamin‐sensitive K+ channel (10 nM scyllatoxin, 100 microM d‐tubocurarine, 300 microM gallamine) in the presence of indomethacin and N omega‐nitro‐L‐arginine. The hyperpolarization was not blocked by glibenclamide (5 microM), iberiotoxin (10 nM), tetraethylammonium (1 mM), barium (500 microM), 4‐aminopyridine (500 microM), ouabain (10 microM), bumetanide (10 microM), or nimodipine (100 nM). 3. In the presence of apamin and N omega‐nitro‐L‐arginine, but the absence of indomethacin, ACh triggered a hyperpolarization that was blocked by glibenclamide, an inhibitor of ATP‐sensitive K+ (KATP) channels. A similar glibenclamide‐sensitive hyperpolarization was caused by Iloprost, a stable analogue of prostacyclin. 4. In experiments which distinguished the effects of EDHF, prostanoids and nitric oxide, hyperpolarizations and/or relaxations triggered by ACh were antagonized by muscarinic antagonists, the relative potencies (atropine approximately 4‐DAMP > pirenzepine) of which indicated that the release of all three endothelium‐derived factors was mediated by M3 receptors. 5. Our results suggest that ACh stimulates M3 receptors on endothelial cells, triggering the release of nitric oxide and prostanoids, which hyperpolarize underlying smooth muscle by activation of KATP channels, and the release of an EDHF, which hyperpolarizes smooth muscle through the activation of apamin‐sensitive K+ (KAS) channels.

TRPV4-dependent dilation of peripheral resistance arteries influences arterial pressure

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 approximately 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 approximately 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.

POTASSIUM CHANNELS IN VASCULAR SMOOTH MUSCLE

1. Regulation of smooth muscle membrane potential through changes in K+ channel activity and subsequent alterations in the activity of voltage‐dependent calcium channels is a major mechanism of vasodilation and vasoconstriction, both in normal and pathophysiological conditions. The contribution of a given K+ channel type to this mechanism of vascular regulation depends on the vascular bed and species examined.

Swelling‐activated cation channels mediate depolarization of rat cerebrovascular smooth muscle by hyposmolarity and intravascular pressure

1 Increases in intravascular pressure depolarize vascular smooth muscle cells. Based on the attenuating effects of Cl− channel antagonists, it has been suggested that swelling‐activated Cl− channels may be integral to this response. Consequently, this study tested for the presence of a swelling‐activated Cl− conductance in both intact rat cerebral arteries and isolated rat smooth muscle cells. 2 A 50 mosmol l−1 hyposmotic challenge (300 to 250 mosmol l−1) constricted rat cerebral arteries. This constriction contained all the salient features of a pressure‐induced response including smooth muscle cell depolarization and a rise in intracellular Ca2+ that was blocked by voltage‐operated Ca2+ channel antagonists. The hyposmotically induced depolarization was attenuated by DIDS (300 μm) and tamoxifen (1 μm), a response consistent with the presence of a swelling‐activated Cl− conductance. 3 A swelling‐activated current was identified in cerebral vascular smooth muscle cells. This current was sensitive to Cl− channel antagonists including DIDS (300 μm), tamoxifen (1 μm) and IAA‐94 (100 μm). However, contrary to expectations, the reversal potential of this swelling‐activated current shifted with the Na+ equilibrium potential and not the Cl− equilibrium potential, indicating that the swelling‐activated current was carried by cations and not anions. The swelling‐activated cation current was blocked by Gd3+, a cation channel antagonist. 4 Gd3+ also blocked both swelling‐ and pressure‐induced depolarization of smooth muscle cells in intact cerebral arteries. 5 These findings suggest that swelling‐ and pressure‐induced depolarization arise from the activation of a cation conductance. This current is inhibited by DIDS, tamoxifen, IAA‐94 and gadolinium.