Tonic regulation of middle meningeal artery diameter by ATP-sensitive potassium channels.

Activation of ATP-sensitive potassium (KATP) channels in arterial smooth muscle (ASM) contributes to vasodilation evoked by a variety of endogenous and exogenous compounds. Although controversial, activation of KATP channels by neuropeptides such as calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase activating peptide (PACAP) in the trigeminovascular system, including the middle meningeal artery (MMA), has been linked to migraine headache. The objective of the current study was to determine if ongoing KATP channel activity also influences MMA diameter. In the absence of other exogenous compounds, the KATP channel inhibitors glibenclamide and PNU37883A induced constriction of isolated and pressurized MMAs. In contrast, KATP channel inhibition did not alter cerebral artery diameter. Consistent with tonic KATP activity in MMA, glibenclamide also induced ASM membrane potential depolarization and increased cytosolic Ca2+. Inhibitors of cAMP-dependent protein kinase (PKA) abolished basal KATP activation in MMA and caused a marked decrease in sensitivity to the synthetic KATP channel opener, cromakalim. In vivo MMA constriction in response to gibenclamide was observed using two-photon imaging of arterial diameter. Together these results indicate that PKA-mediated tonic KATP channel activity contributes to the regulation of MMA diameter.

Capillary K+-sensing initiates retrograde hyperpolarization to increase local cerebral blood flow.

Blood flow into the brain is dynamically regulated to satisfy the changing metabolic requirements of neurons, but how this is accomplished has remained unclear. Here we demonstrate a central role for capillary endothelial cells in sensing neural activity and communicating it to upstream arterioles in the form of an electrical vasodilatory signal. We further demonstrate that this signal is initiated by extracellular K+ -a byproduct of neural activity-which activates capillary endothelial cell inward-rectifier K+ (KIR2.1) channels to produce a rapidly propagating retrograde hyperpolarization that causes upstream arteriolar dilation, increasing blood flow into the capillary bed. Our results establish brain capillaries as an active sensory web that converts changes in external K+ into rapid, 'inside-out' electrical signaling to direct blood flow to active brain regions.

Rho kinase activity governs arteriolar myogenic depolarization.

Cerebral arterioles contribute critically to regulation of local and global blood flow within the brain. Dysfunction of these blood vessels is implicated in numerous cardiovascular diseases. However, treatments are limited due to incomplete understanding of fundamental control mechanisms at this level of circulation. Emerging evidence points to a key role of Rho-associated protein kinase in regulation of microvascular contractility. This study sought to decipher the mechanisms of Rho-associated protein kinase-mediated myogenic vasoconstriction in cerebral parenchymal arterioles. Here, we report that the Rho-associated protein kinase inhibitor H1152 strongly attenuated pressure-induced constriction, cytosolic [Ca2+] increases, and depolarization of isolated parenchymal arterioles. Further, the RhoA activator CN03 potentiated parenchymal arteriole myogenic constriction and depolarization, indicating important involvement of RhoA/Rho-associated protein kinase signaling in myogenic excitation-contraction mechanisms. Because of the well-established role of TRPM4 in pressure-induced depolarization, possible modulatory effects of Rho-associated protein kinase on TRPM4 currents were explored using patch clamp electrophysiology. TRPM4 currents were suppressed by H1152 and enhanced by CN03. Finally, H1152 elevated the apparent [Ca2+]-threshold for TRPM4 activation, suggesting that Rho-associated protein kinase activates TRPM4 by increasing its Ca2+-sensitivity. Our results support a novel mechanism whereby Rho-associated protein kinase-mediated myogenic vasoconstriction occurs primarily through activation of TRPM4 channels, smooth muscle depolarization, and cytosolic [Ca2+] increases in cerebral arterioles.

Synthetic Peptides as cGMP-Independent Activators of cGMP-Dependent Protein Kinase Iα.

PKG is a multifaceted signaling molecule and potential pharmaceutical target due to its role in smooth muscle function. A helix identified in the structure of the regulatory domain of PKG Iα suggests a novel architecture of the holoenzyme. In this study, a set of synthetic peptides (S-tides), derived from this helix, was found to bind to and activate PKG Iα in a cyclic guanosine monophosphate (cGMP)-independent manner. The most potent S-tide derivative (S1.5) increased the open probability of the potassium channel KCa1.1 to levels equivalent to saturating cGMP. Introduction of S1.5 to smooth muscle cells in isolated, endothelium-denuded cerebral arteries through a modified reversible permeabilization procedure inhibited myogenic constriction. In contrast, in endothelium-intact vessels S1.5 had no effect on myogenic tone. This suggests that PKG Iα activation by S1.5 in vascular smooth muscle would be sufficient to inhibit augmented arterial contractility that frequently occurs following endothelial damage associated with cardiovascular disease.

Transient receptor potential channels in the vasculature.

The mammalian genome encodes 28 distinct members of the transient receptor potential (TRP) superfamily of cation channels, which exhibit varying degrees of selectivity for different ionic species. Multiple TRP channels are present in all cells and are involved in diverse aspects of cellular function, including sensory perception and signal transduction. Notably, TRP channels are involved in regulating vascular function and pathophysiology, the focus of this review. TRP channels in vascular smooth muscle cells participate in regulating contractility and proliferation, whereas endothelial TRP channel activity is an important contributor to endothelium-dependent vasodilation, vascular wall permeability, and angiogenesis. TRP channels are also present in perivascular sensory neurons and astrocytic endfeet proximal to cerebral arterioles, where they participate in the regulation of vascular tone. Almost all of these functions are mediated by changes in global intracellular Ca(2+) levels or subcellular Ca(2+) signaling events. In addition to directly mediating Ca(2+) entry, TRP channels influence intracellular Ca(2+) dynamics through membrane depolarization associated with the influx of cations or through receptor- or store-operated mechanisms. Dysregulation of TRP channels is associated with vascular-related pathologies, including hypertension, neointimal injury, ischemia-reperfusion injury, pulmonary edema, and neurogenic inflammation. In this review, we briefly consider general aspects of TRP channel biology and provide an in-depth discussion of the functions of TRP channels in vascular smooth muscle cells, endothelial cells, and perivascular cells under normal and pathophysiological conditions.

Potassium channelopathy-like defect underlies early-stage cerebrovascular dysfunction in a genetic model of small vessel disease.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), caused by dominant mutations in the NOTCH3 receptor in vascular smooth muscle, is a genetic paradigm of small vessel disease (SVD) of the brain. Recent studies using transgenic (Tg)Notch3(R169C) mice, a genetic model of CADASIL, revealed functional defects in cerebral (pial) arteries on the surface of the brain at an early stage of disease progression. Here, using parenchymal arterioles (PAs) from within the brain, we determined the molecular mechanism underlying the early functional deficits associated with this Notch3 mutation. At physiological pressure (40 mmHg), smooth muscle membrane potential depolarization and constriction to pressure (myogenic tone) were blunted in PAs from TgNotch3(R169C) mice. This effect was associated with an ∼ 60% increase in the number of voltage-gated potassium (KV) channels, which oppose pressure-induced depolarization. Inhibition of KV1 channels with 4-aminopyridine (4-AP) or treatment with the epidermal growth factor receptor agonist heparin-binding EGF (HB-EGF), which promotes KV1 channel endocytosis, reduced KV current density and restored myogenic responses in PAs from TgNotch3(R169C) mice, whereas pharmacological inhibition of other major vasodilatory influences had no effect. KV1 currents and myogenic responses were similarly altered in pial arteries from TgNotch3(R169C) mice, but not in mesenteric arteries. Interestingly, HB-EGF had no effect on mesenteric arteries, suggesting a possible mechanistic basis for the exclusive cerebrovascular manifestation of CADASIL. Collectively, our results indicate that increasing the number of KV1 channels in cerebral smooth muscle produces a mutant vascular phenotype akin to a channelopathy in a genetic model of SVD.

TRPM4 channels couple purinergic receptor mechanoactivation and myogenic tone development in cerebral parenchymal arterioles.

Cerebral parenchymal arterioles (PAs) have a critical role in assuring appropriate blood flow and perfusion pressure within the brain. They are unique in contrast to upstream pial arteries, as defined by their critical roles in neurovascular coupling, distinct sensitivities to chemical stimulants, and enhanced myogenic tone development. The objective of the present study was to reveal some of the unique mechanisms of myogenic tone regulation in the cerebral microcirculation. Here, we report that in vivo suppression of TRPM4 (transient receptor potential) channel expression, or inhibition of TRPM4 channels with 9-phenanthrol substantially reduced myogenic tone of isolated PAs, supporting a key role of TRPM4 channels in PA myogenic tone development. Further, downregulation of TRPM4 channels inhibited vasoconstriction induced by the specific P2Y4 and P2Y6 receptor ligands (UTPγS and UDP) by 37% and 42%, respectively. In addition, 9-phenanthrol substantially attenuated purinergic ligand-induced membrane depolarization and constriction of PAs, and inhibited ligand-evoked TRPM4 channel activation in isolated PA myocytes. In concert with our previous work showing the essential contributions of P2Y4 and P2Y6 receptors to myogenic regulation of PAs, the current results point to TRPM4 channels as an important link between mechanosensitive P2Y receptor activation and myogenic constriction of cerebral PAs.

Postischemic reperfusion causes smooth muscle calcium sensitization and vasoconstriction of parenchymal arterioles.

BACKGROUND AND PURPOSE: Parenchymal arterioles (PAs) are high-resistance vessels in the brain that connect pial vessels to the microcirculation. We previously showed that PAs have increased vasoconstriction after ischemia and reperfusion that could increase perfusion deficit. Here, we investigated underlying mechanisms by which early postischemic reperfusion causes increased vasoconstriction of PAs. METHODS: Isolated and pressurized PAs from within the middle cerebral artery territory were studied in male Wistar rats that were either nonischemic control (n=34) or after exposure to transient middle cerebral artery occlusion (MCAO) by filament occlusion for 2 hours with 30 minutes of reperfusion (MCAO; n=38). The relationships among pressure-induced tone, smooth muscle calcium (using Fura 2), and membrane potential were determined. Sensitivity of the contractile apparatus to calcium was measured in permeabilized arterioles using Staphylococcus aureus α-toxin. Reactivity to inhibition of transient receptor potential melastanin receptor type 4 (9-phenanthrol), Rho kinase (Y27632), and protein kinase C (Gö6976) was also measured. RESULTS: After MCAO, PAs had increased myogenic tone compared with controls (47±2% versus 35±2% at 40 mm Hg; P<0.01), without an increase in smooth muscle calcium (177±21 versus 201±16 nmol/L; P>0.05) or membrane depolarization (-38±4 versus -36±1 mV; P>0.05). In α-toxin-permeabilized vessels, MCAO caused increased sensitivity of the contractile apparatus to calcium. MCAO did not affect dilation to transient receptor potential melastanin receptor type 4 or protein kinase C inhibition but diminished dilation to Rho kinase inhibition. CONCLUSIONS: The increased vasoconstriction of PAs during early postischemic reperfusion seems to be due to calcium sensitization of smooth muscle and could contribute to infarct expansion and limit neuroprotective agents from reaching their target tissue.

Increased pressure-induced tone in rat parenchymal arterioles vs. middle cerebral arteries: role of ion channels and calcium sensitivity.

Brain parenchymal arterioles (PAs) are high-resistance vessels that branch off pial arteries and perfuse the brain parenchyma. PAs are the target of cerebral small vessel disease and have been shown to have greater pressure-induced tone at lower pressures than pial arteries. We investigated mechanisms by which brain PAs have increased myogenic tone compared with middle cerebral arteries (MCAs), focusing on differences in vascular smooth muscle (VSM) calcium and ion channel function. The amount of myogenic tone and VSM calcium was measured using Fura 2 in isolated and pressurized PAs and MCAs. Increases in intraluminal pressure caused larger increases in tone and cytosolic calcium in PAs compared with MCAs. At 50 mmHg, myogenic tone was 37 ± 5% for PAs vs. 6.5 ± 4% for MCAs (P < 0.01), and VSM calcium was 200 ± 20 nmol/l in PAs vs. 104 ± 15 nmol/l in MCAs (P < 0.01). In vessels permeabilized with Staphylococcus aureus α-toxin, PAs were not more sensitive to calcium, suggesting calcium sensitization was not at the level of the contractile apparatus. PAs were 30-fold more sensitive to the voltage-dependent calcium channel (VDCC) inhibitor nifedipine than MCAs (EC50 for PAs was 3.5 ± 0.4 vs. 82.1 ± 2.1 nmol/l for MCAs;P < 0.01); however, electrophysiological properties of the VDCC were not different in VSM. PAs had little to no response to the calcium-activated potassium channel inhibitor iberiotoxin, whereas MCAs constricted ∼15%. Thus increased myogenic tone in PAs appears related to differences in ion channel activity that promotes VSM membrane depolarization but not to a direct sensitization of the contractile apparatus to calcium.

Local control of TRPV4 channels by AKAP150-targeted PKC in arterial smooth muscle.

Transient receptor potential vanilloid 4 (TRPV4) channels are Ca(2+)-permeable, nonselective cation channels expressed in multiple tissues, including smooth muscle. Although TRPV4 channels play a key role in regulating vascular tone, the mechanisms controlling Ca(2+) influx through these channels in arterial myocytes are poorly understood. Here, we tested the hypothesis that in arterial myocytes the anchoring protein AKAP150 and protein kinase C (PKC) play a critical role in the regulation of TRPV4 channels during angiotensin II (AngII) signaling. Super-resolution imaging revealed that TRPV4 channels are gathered into puncta of variable sizes along the sarcolemma of arterial myocytes. Recordings of Ca(2+) entry via single TRPV4 channels ("TRPV4 sparklets") suggested that basal TRPV4 sparklet activity was low. However, Ca(2+) entry during elementary TRPV4 sparklets was ∼ 100-fold greater than that during L-type CaV1.2 channel sparklets. Application of the TRPV4 channel agonist GSK1016790A or the vasoconstrictor AngII increased the activity of TRPV4 sparklets in specific regions of the cells. PKC and AKAP150 were required for AngII-induced increases in TRPV4 sparklet activity. AKAP150 and TRPV4 channel interactions were dynamic; activation of AngII signaling increased the proximity of AKAP150 and TRPV4 puncta in arterial myocytes. Furthermore, local stimulation of diacylglycerol and PKC signaling by laser activation of a light-sensitive Gq-coupled receptor (opto-α1AR) resulted in TRPV4-mediated Ca(2+) influx. We propose that AKAP150, PKC, and TRPV4 channels form dynamic subcellular signaling domains that control Ca(2+) influx into arterial myocytes.

Prostaglandin E2, a postulated astrocyte-derived neurovascular coupling agent, constricts rather than dilates parenchymal arterioles.

It has been proposed that prostaglandin E(2) (PGE(2)) is released from astrocytic endfeet to dilate parenchymal arterioles through activation of prostanoid (EP(4)) receptors during neurovascular coupling. However, the direct effects of PGE(2) on isolated parenchymal arterioles have not been tested. Here, we examined the effects of PGE(2) on the diameter of isolated pressurized parenchymal arterioles from rat and mouse brain. Contrary to the prevailing assumption, we found that PGE(2) (0.1, 1, and 5 µmol/L) constricted rather than dilated parenchymal arterioles. Vasoconstriction to PGE(2) was prevented by inhibitors of EP(1) receptors. These results strongly argue against a direct role of PGE(2) on arterioles during neurovascular coupling.

Ryanodine receptors, calcium signaling, and regulation of vascular tone in the cerebral parenchymal microcirculation.

The cerebral blood supply is delivered by a surface network of pial arteries and arterioles from which arise (parenchymal) arterioles that penetrate into the cortex and terminate in a rich capillary bed. The critical regulation of CBF, locally and globally, requires precise vasomotor regulation of the intracerebral microvasculature. This vascular region is anatomically unique as illustrated by the presence of astrocytic processes that envelope almost the entire basolateral surface of PAs. There are, moreover, notable functional differences between pial arteries and PAs. For example, in pial VSMCs, local calcium release events ("calcium sparks") through ryanodine receptor (RyR) channels in SR membrane activate large conductance, calcium-sensitive potassium channels to modulate vascular diameter. In contrast, VSMCs in PAs express functional RyR and BK channels, but under physiological conditions, these channels do not oppose pressure-induced vasoconstriction. Here, we summarize the roles of ryanodine receptors in the parenchymal microvasculature under physiologic and pathologic conditions, and discuss their importance in the control of CBF.

Purinergic receptors regulate myogenic tone in cerebral parenchymal arterioles.

Myogenic tone is a fundamental aspect of vascular behavior in resistance arteries. This contractile response to changes in intravascular pressure is critically involved in blood flow autoregulation in tissues such as the brain, kidneys, and heart. Myogenic tone also helps regulate precapillary pressure and provides a level of background tone upon which vasodilator stimuli act to increase tissue perfusion when appropriate. Despite the importance of these processes in the brain, little is known about the mechanisms involved in control of myogenic tone in the cerebral microcirculation. Here, we report that pharmacological inhibition of P2Y4 and P2Y6 pyrimidine receptors nearly abolished myogenic tone in cerebral parenchymal arterioles (PAs). Molecular suppression of either P2Y4 or P2Y6 receptors using antisense oligodeoxynucleotides reduced myogenic tone by 44%±8% and 45%±7%, respectively. These results indicate that both receptor isoforms are activated by increased intravascular pressure, which enhances the activity of voltage-dependent calcium channels and increases myogenic tone in PAs. Enhancement or inhibition of ectonucleotidase activity had no effect on parenchymal arteriolar myogenic tone, indicating that this response is not mediated by local release of nucleotides, but rather may involve direct mechanical activation of P2Y receptors in the smooth muscle cells.

Acidosis dilates brain parenchymal arterioles by conversion of calcium waves to sparks to activate BK channels.

RATIONALE: Acidosis is a powerful vasodilator signal in the brain circulation. However, the mechanisms by which this response occurs are not well understood, particularly in the cerebral microcirculation. One important mechanism to dilate cerebral (pial) arteries is by activation of large-conductance, calcium-sensitive potassium (BK(Ca)) channels by local Ca(2+) signals (Ca(2+) sparks) through ryanodine receptors (RyRs). However, the role of this pathway in the brain microcirculation is not known. OBJECTIVE: The objectives of this study were to determine the mechanism by which acidosis dilates brain parenchymal arterioles (PAs) and to elucidate the roles of RyRs and BK(Ca) channels in this response. METHODS AND RESULTS: Internal diameter and vascular smooth muscle cell Ca(2+) signals were measured in isolated pressurized murine PAs, using imaging techniques. In physiological pH (7.4), vascular smooth muscle cells exhibited primarily RyR-dependent Ca(2+) waves. Reducing external pH from 7.4 to 7.0 in both normocapnic and hypercapnic conditions decreased Ca(2+) wave activity, and dramatically increased Ca(2+) spark activity. Acidic pH caused a dilation of PAs which was inhibited by about 60% by BK(Ca) channel or RyR blockers, in a nonadditive manner. Similarly, dilator responses to acidosis were reduced by nearly 60% in arterioles from BK(Ca) channel knockout mice. Dilations induced by acidic pH were unaltered by inhibitors of K(ATP) channels or nitric oxide synthase. CONCLUSIONS: These results support the novel concept that acidification, by converting Ca(2+) waves to sparks, leads to the activation of BK(Ca) channels to induce dilation of cerebral PAs.

Fundamental increase in pressure-dependent constriction of brain parenchymal arterioles from subarachnoid hemorrhage model rats due to membrane depolarization.

Intracerebral (parenchymal) arterioles are morphologically and physiologically unique compared with pial arteries and arterioles. The ability of subarachnoid hemorrhage (SAH) to induce vasospasm in large-diameter pial arteries has been extensively studied, although the contribution of this phenomenon to patient outcome is controversial. Currently, little is known regarding the impact of SAH on parenchymal arterioles, which are critical for regulation of local and global cerebral blood flow. Here diameter, smooth muscle intracellular Ca(2+) concentration ([Ca(2+)](i)), and membrane potential measurements were used to assess the function of intact brain parenchymal arterioles isolated from unoperated (control), sham-operated, and SAH model rats. At low intravascular pressure (5 mmHg), membrane potential and [Ca(2+)](i) were not different in arterioles from control, sham-operated, and SAH animals. However, raising intravascular pressure caused significantly greater membrane potential depolarization, elevation in [Ca(2+)](i), and constriction in SAH arterioles. This SAH-induced increase in [Ca(2+)](i) and tone occurred in the absence of the vascular endothelium and was abolished by the L-type voltage-dependent calcium channel (VDCC) inhibitor nimodipine. Arteriolar [Ca(2+)](i) and tone were not different between groups when smooth muscle membrane potential was adjusted to the same value. Protein and mRNA levels of the L-type VDCC Ca(V)1.2 were similar in parenchymal arterioles isolated from control and SAH animals, suggesting that SAH did not cause VDCC upregulation. We conclude that enhanced parenchymal arteriolar tone after SAH is driven by smooth muscle membrane potential depolarization, leading to increased L-type VDCC-mediated Ca(2+) influx.

Impact of subarachnoid hemorrhage on local and global calcium signaling in cerebral artery myocytes.

BACKGROUND: Ca2+ signaling mechanisms are crucial for proper regulation of vascular smooth muscle contractility and vessel diameter. In cerebral artery myocytes, a rise in global cytosolic Ca2+ concentration ([Ca2+]i) causes contraction while an increase in local Ca2+ release events from the sarcoplasmic reticulum (Ca2+ sparks) leads to increased activity of large-conductance Ca2+-activated (BK) K+ channels, hyperpolarization and relaxation. Here, we examined the impact of SAH on Ca2+ spark activity and [Ca2+]i in cerebral artery myocytes following SAH. METHODS: A rabbit double injection SAH model was used in this study. Five days after the initial intracisternal injection of whole blood, small diameter cerebral arteries were dissected from the brain for study. For simultaneous measurement of arterial wall [Ca2+]i and diameter, vessels were cannulated and loaded with the ratiometric Ca2+ indicator fura-2. For measurement of Ca2+ sparks, individual myocytes were enzymatically isolated from cerebral arteries and loaded with the Ca2+ indicator fluo-4. Sparks were visualized using laser scanning confocal microscopy. RESULTS: Arterial wall [Ca2+]i was significantly elevated and greater levels of myogenic tone developed in arteries isolated from SAH animals compared with arteries isolated from healthy animals. The L-type voltage-dependent Ca2+ channel (VDCC) blocker nifedipine attenuated increases in [Ca2+]i and tone in both groups suggesting increased VDCC activity following SAH. Membrane potential measurement using intracellular microelectrodes revealed significant depolarization of vascular smooth muscle following SAH. Further, myocytes from SAH animals exhibited significantly reduced Ca2+ spark frequency (~50%). CONCLUSIONS: Our findings suggest decreased Ca2+ spark frequency leads to reduced BK channel activity in cerebral artery myocytes following SAH. This results in membrane potential depolarization, increased VDCC activity, elevated [Ca2+]i and decreased vessel diameter. We propose this mechanism of enhanced cerebral artery myocyte contractility may contribute to decreased cerebral blood flow and development of neurological deficits in SAH patients.

Transient receptor potential channels and vascular function.

TRP (transient receptor potential) channels play important roles in the regulation of normal and pathological cellular function. In the vasculature, TRP channels are present both in ECs (endothelial cells) and vascular SMCs (smooth muscle cells) and contribute to vasomotor control mechanisms in most vascular beds. Vascular TRP channels are activated by various stimuli, such as mechanical perturbation, receptor activation and dietary molecules. Some of the specific roles of these channels in normal and impaired vascular function have emerged in recent years and include participation in vascular signalling processes, such as neurotransmission, hormonal signalling, NO production, myogenic tone and autoregulation of blood flow, thermoregulation, responses to oxidative stress and cellular proliferative activity. Current research is aimed at understanding the interactions of TRP channels with other vascular proteins and signalling mechanisms. These studies should reveal new targets for pharmacological therapy of vascular diseases, such as hypertension, ischaemia and vasospasm, and vascular proliferative states.

(D)-Amino acid analogues of DT-2 as highly selective and superior inhibitors of cGMP-dependent protein kinase Ialpha.

The cGMP-dependent protein kinase type I (PKG I) is an essential regulator of cellular function in blood vessels throughout the body. DT-2, a peptidic inhibitor of PKG, has played a central role in determining the molecular mechanisms of vascular control involving PKG and its signaling partners. Here, we report the development of (d)-amino acid DT-2 derivatives, namely the retro-inverso ri-(d)-DT-2 and the all (d)-amino acid analog, (d)-DT-2. Both peptide analogs were potent PKG Ialpha inhibitors with K(i) values of 5.5 nM (ri-(d)-DT-2) and 0.8 nM ((d)-DT-2) as determined using a hyperbolic mixed-type inhibition model. Also, both analogs were proteolytically stable in vivo, showed elevated selectivity, and displayed enhanced membrane translocation properties. Studies on isolated arteries from the resistance vasculature demonstrated that intraluminally perfused (d)-DT-2 significantly inhibited vasodilation induced by 8-Br-cGMP. Furthermore, in vivo application of (d)-DT-2 established a uniform translocation pattern in the resistance vasculature, with exception of the brain. Thus, (d)-DT-2 caused significant increases in mean arterial blood pressure in unrestrained, awake mice. Further, mesenteric arteries isolated from (d)-DT-2 treated animals showed a markedly reduced dilator response to 8-Br-cGMP in vitro. Our results clearly demonstrate that (d)-DT-2 is a superior inhibitor of PKG Ialpha and its application in vivo leads to sustained inhibition of PKG in vascular smooth muscle cells. The discovery of (d)-DT-2 may help our understanding of how blood vessels constrict and dilate and may also aid the development of new strategies and therapeutic agents targeted to the prevention and treatment of vascular disorders such as hypertension, stroke and coronary artery disease.

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.

Transient receptor potential (TRP) channels, vascular tone and autoregulation of cerebral blood flow.

Members of the transient receptor potential (TRP) channel superfamily are present in vascular smooth muscle cells and play important roles in the regulation of vascular contractility. The TRPC3 and TRPC6 channels are activated by stimulation of several excitatory receptors in vascular smooth muscle cells. Activation of these channels leads to myocyte depolarization, which stimulates Ca2+ entry via voltage-dependent Ca2+ channels (VDCC), leading to vasoconstriction. The TRPV4 channels in arterial myocytes are activated by epoxyeicosatrienoic acids, and activation of the channels enhances Ca2+ spark and transient Ca2+-sensitive K+ channel activity, thereby hyperpolarizing and relaxing vascular smooth muscle cells. The TRPC6 and TRPM4 channels are activated by mechanical stimulation of cerebral artery myocytes. Subsequent depolarization and activation of VDCC Ca2+ entry is directly linked to the development of myogenic tone in vitro and to autoregulation of cerebral blood flow in vivo. These findings imply a fundamental importance of TRP channels in the regulation of vascular smooth muscle tone and suggest that TRP channels could be important targets for drug therapy under conditions in which vascular contractility is disturbed (e.g. hypertension, stroke, vasospasm).