Sean P. Marrelli

Endothelium-derived hyperpolarizing factor: a cousin to nitric oxide and prostacyclin.

There is now strong evidence that an endothelial mechanism, other than nitric oxide or prostacyclin, exists for dilating arteries and arterioles. This third pathway has been named endothelium-derived hyperpolarizing factor (EDHF) and should not be confused with endothelium-derived relaxing factor, which is nitric oxide. Currently, there are several ideas for the mechanism of EDHF, which may vary among vessels of different organs and species. During some pathologic states, EDHF can be up-regulated. This up-regulation often occurs as the dilator effects of endothelium-derived nitric oxide are suppressed. The up-regulated EDHF may serve in a protective capacity to help maintain blood flow to organs and tissues during these stressful states. Many anesthetics attenuate the dilator actions of EDHF; however, the full clinical implications of this anesthetic-related attenuation are not known. Like its cousins, nitric oxide and prostacyclin, EDHF is an important regulator of blood flow and should prove to be an important clinical consideration as we gain more knowledge of its mechanisms of action.

P2u receptor-mediated release of endothelium-derived relaxing factor/nitric oxide and endothelium-derived hyperpolarizing factor from cerebrovascular endothelium in rats.

BACKGROUND AND PURPOSE Stimulation of P2u purinoceptors by UTP on endothelium dilates the rat middle cerebral artery (MCA) through the release of endothelium-derived relaxing factor/nitric oxide (EDRF/NO) and an unknown relaxing factor. The purpose of this study was to determine whether this unknown relaxing factor is endothelium-derived hyperpolarizing factor (EDHF). METHODS Rat MCAs were isolated, cannulated, pressurized, and luminally perfused. UTP was added to the luminal perfusate to elicit dilations. RESULTS Resting outside diameter of the MCAs in one study was 209+/-7 micrometer (n=10). The MCAs showed concentration-dependent dilations with UTP administration. Inhibition of NO synthase with NG-nitro-L-arginine methyl ester (L-NAME) (1 micromol/L to 1 mmol/L) did not diminish the maximum response to UTP but did shift the concentration-response curve to the right. Scavenging NO with hemoglobin (1 or 10 micromol/L) or inhibition of guanylate cyclase with ODQ (1 or 10 micromol/L) had effects on the UTP-mediated dilations similar to those of L-NAME. In the presence of L-NAME, dilations induced by 10 micromol/L UTP were accompanied by 13+/-2 mV (P<0.009) hyperpolarization of the vascular smooth muscle membrane potential (-28+/-2 to -41+/-1 mV). Iberiotoxin (100 nmol/L), blocker of the large-conductance calcium-activated K channels, sometimes blocked the dilation, but its effects were variable. Charybdotoxin (100 nmol/L), also a blocker of the large-conductance calcium-activated K channels, abolished the L-NAME-insensitive component of the dilation to UTP. CONCLUSIONS Stimulation of P2u purinoceptors on the endothelium of the rat MCA released EDHF, in addition to EDRF/NO, and dilated the rat MCA by opening an atypical calcium-activated K channel.

Inward rectifier potassium channels in the rat middle cerebral artery.

Inward rectifier K+ channels (Kirs) were studied in the isolated perfused rat middle cerebral artery (MCA). The addition of 15 mM K+ (KCl) to the extraluminal bath dilated the MCAs. These dilations were blocked by selective inhibitors for the Kirs (40 μM BaCl2 or 40 mM CsCl) but not selective inhibitors for other K+channels (glibenclamide, tetraethylammonium, or 4-aminopyridine). Neither removal of the endothelium nor treatment with the nitric oxide synthase inhibitor ( N G-nitro-l-arginine methyl ester, 10 μM) affected the K+-induced dilation. The addition of BaCl2 to resting MCAs produced a dose-dependent constriction of 8-12%, indicating that, during resting conditions, Kirs aid in setting or determining the resting tone. The magnitude of the dilations produced by the addition of K+ or constrictions produced by BaCl2 were independent of pressure over a range of 40-100 mmHg. We conclude that Kirs, which produce a dilation when activated, exist on the vascular smooth muscle of the rat MCA. These Kirs aid in determining the resting tone of the vessel, and their function is independent of pressure over physiological pressure ranges.

KIR channels function as electrical amplifiers in rat vascular smooth muscle

Strong inward rectifying K+ (KIR) channels have been observed in vascular smooth muscle and can display negative slope conductance. In principle, this biophysical characteristic could enable KIR channels to ‘amplify’ responses initiated by other K+ conductances. To test this, we have characterized the diversity of smooth muscle KIR properties in resistance arteries, confirmed the presence of negative slope conductance and then determined whether KIR inhibition alters the responsiveness of middle cerebral, coronary septal and third‐order mesenteric arteries to K+ channel activators. Our initial characterization revealed that smooth muscle KIR channels were highly expressed in cerebral and coronary, but not mesenteric arteries. These channels comprised KIR2.1 and 2.2 subunits and electrophysiological recordings demonstrated that they display negative slope conductance. Computational modelling predicted that a KIR‐like current could amplify the hyperpolarization and dilatation initiated by a vascular K+ conductance. This prediction was consistent with experimental observations which showed that 30 μm Ba2+ attenuated the ability of K+ channel activators to dilate cerebral and coronary arteries. This attenuation was absent in mesenteric arteries where smooth muscle KIR channels were poorly expressed. In summary, smooth muscle KIR expression varies among resistance arteries and when channel are expressed, their negative slope conductance amplifies responses initiated by smooth muscle and endothelial K+ conductances. These findings highlight the fact that the subtle biophysical properties of KIR have a substantive, albeit indirect, role in enabling agonists to alter the electrical state of a multilayered artery.

Inhibition of TRPC1/TRPC3 by PKG contributes to NO-mediated vasorelaxation

Nitric oxide (NO) inhibits transient receptor potential channel 3 (TRPC3) channels via a PKG-dependent mechanism. We sought to determine 1) whether NO inhibition of TRPC3 occurs in freshly isolated smooth muscle cells (SMC); and 2) whether NO inhibition of TRPC3 channels contributes to NO-mediated vasorelaxation. We tested these hypotheses in freshly isolated rat carotid artery (CA) SMC using patch clamp and in intact CA by vessel myograph. We demonstrated TRPC3 expression in whole CA (mRNA and protein) that was localized to the smooth muscle layers. TRPC1 protein was also expressed and coimmunoprecipitated with TRPC3. Whole cell patch clamp demonstrated nonselective cation channel currents that were activated by UTP (60 microM) and completely inhibited by a TRPC channel inhibitor, La(3+) (100 microM). The UTP-stimulated current (I(UTP)) was also inhibited by intracellular application of anti-TRPC3 or anti-TRPC1 antibody, but not by anti-TRPC6 or anti-TRPC4 control antibodies. We next evaluated the NO signaling pathway on I(UTP). Exogenous NO [(Z)-1-{N-methyl-N-[6(N-methylammoniohexyl)amino]}diazen-1-ium-1,2-diolate (MAHMA NONOate)] or a cell-permeable cGMP analog (8-bromo-cGMP) significantly inhibited I(UTP). Preapplication of a PKG inhibitor (KT5823) reversed the inhibition of MAHMA NONOate or 8-bromo-cGMP, demonstrating the critical role of PKG in NO inhibition of TRPC1/TRPC3. Intact CA segments were contracted with UTP (100 microM) in the presence or absence of La(3+) (100 microM) and then evaluated for relaxation to an NO donor, sodium nitroprusside (1 nM to 1 microM). Relaxation to sodium nitroprusside was significantly reduced in the La(3+) treatment group. We conclude that freshly isolated SMC express TRPC1/TRPC3 channels and that these channels are inhibited by NO/cGMP/PKG. Furthermore, NO contributes to vasorelaxation by inhibition of La(3+)-sensitive channels consistent with TRPC1/TRPC3.

Altered Function of Inward Rectifier Potassium Channels in Cerebrovascular Smooth Muscle After Ischemia/Reperfusion

BACKGROUND AND PURPOSE Several recent studies have demonstrated that inward rectifier potassium channels (K(ir)s) are located on vascular smooth muscle of cerebral arteries in the rat. Activation of the K(ir)s dilates the arteries by relaxing the vascular smooth muscle. We tested the following hypothesis in the present study: function of inward rectifier potassium channels is altered after ischemia/reperfusion (I/R). METHODS Temporary (2-hour) focal ischemia was induced in male Long-Evans rats (3% isoflurane anesthesia) by the intraluminal filament model. After 24 hours of reperfusion, ipsilateral and contralateral middle cerebral arteries (MCAs) were harvested and mounted on micropipettes, pressurized to 85 mm Hg, and luminally perfused. RESULTS Resting diameters for contralateral (control) and ipsilateral (I/R) MCAs were not significantly different (215+/-4 microm and 211+/-5 microm [n = 6 and n = 7], respectively). Activation of the K(ir)s by abluminal administration of 15 mmol/L KCl to the control MCAs dilated the MCA by 34+/-4% (n = 8). Activation of the K(ir)s in I/R MCAs produced a dilation of only 11+/-3% (n = 8; P<0.001 compared with control). BaCl2 (75 micromol/L), a concentration-selective inhibitor of the K(ir)s, significantly attenuated the dilation produced by 15 mmol/L KCl in control MCAs but not in the I/R MCAs. Endothelial-mediated dilations elicited by the luminal administration of uridine triphosphate (10 micromol/L) produced similar dilations in both groups (32+/-5% for sham [n = 4] and 33+/-2% for I/R [n = 4]), indicating that dilator function in general was not altered in I/R vessels. CONCLUSIONS We conclude that Kir function is altered after I/R. The Kir altered function is likely to exacerbate the brain injury occurring after I/R.

Transient receptor potential canonical type 3 channels facilitate endothelium-derived hyperpolarization-mediated resistance artery vasodilator activity

AIMS Microdomain signalling mechanisms underlie key aspects of artery function and the modulation of intracellular calcium, with transient receptor potential (TRP) channels playing an integral role. This study determines the distribution and role of TRP canonical type 3 (C3) channels in the control of endothelium-derived hyperpolarization (EDH)-mediated vasodilator tone in rat mesenteric artery. METHODS AND RESULTS TRPC3 antibody specificity was verified using rat tissue, human embryonic kidney (HEK)-293 cells stably transfected with mouse TRPC3 cDNA, and TRPC3 knock-out (KO) mouse tissue using western blotting and confocal and ultrastructural immunohistochemistry. TRPC3-Pyr3 (ethyl-1-(4-(2,3,3-trichloroacrylamide)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate) specificity was verified using patch clamp of mouse mesenteric artery endothelial and TRPC3-transfected HEK cells, and TRPC3 KO and wild-type mouse aortic endothelial cell calcium imaging and mesenteric artery pressure myography. TRPC3 distribution, expression, and role in EDH-mediated function were examined in rat mesenteric artery using immunohistochemistry and western blotting, and pressure myography and endothelial cell membrane potential recordings. In rat mesenteric artery, TRPC3 was diffusely distributed in the endothelium, with approximately five-fold higher expression at potential myoendothelial microdomain contact sites, and immunoelectron microscopy confirmed TRPC3 at these sites. Western blotting and endothelial damage confirmed primary endothelial TRPC3 expression. In rat mesenteric artery endothelial cells, Pyr3 inhibited hyperpolarization generation, and with individual SK(Ca) (apamin) or IK(Ca) (TRAM-34) block, Pyr3 abolished the residual respective IK(Ca)- and SK(Ca)-dependent EDH-mediated vasodilation. CONCLUSION The spatial localization of TRPC3 and associated channels, receptors, and calcium stores are integral for myoendothelial microdomain function. TRPC3 facilitates endothelial SK(Ca) and IK(Ca) activation, as key components of EDH-mediated vasodilator activity and for regulating mesenteric artery tone.

Altered Endothelial Ca2+ Regulation After Ischemia/Reperfusion Produces Potentiated Endothelium-Derived Hyperpolarizing Factor–Mediated Dilations

Background and Purpose— Endothelium-derived hyperpolarizing factor (EDHF)–mediated dilations are potentiated after several pathologies, including ischemia/reperfusion (I/R). However, no study to date has addressed the mechanism by which this potentiation occurs. This study tested the hypothesis that potentiated EDHF-mediated dilations are due to altered endothelial Ca2+ handling after I/R. Methods— Rat middle cerebral arteries (MCAs) were isolated after 2 hours of MCA occlusion and 24 hours of reperfusion (or sham surgery). This model has been previously demonstrated to produce potentiated EDHF-mediated dilations. MCAs were studied in a pressurized/perfused vessel chamber equipped for the simultaneous measurement of endothelial Ca2+ (with fura 2) and artery diameter. Measures were made after luminal administration of UTP (P2Y2 purinoceptor agonist), 2 MeS-ATP (P2Y1 purinoceptor agonist), and Br-A23187 (receptor-independent Ca2+ ionophore) for sham and I/R MCAs. Results— I/R resulted in significantly potentiated UTP-mediated dilations (through a P2Y2 purinoceptor) and endothelial Ca2+ responses in the presence of NG-nitro-l-arginine methyl ester (L-NAME) and indomethacin. Endothelial Ca2+ and diameter responses were also significantly potentiated with 2 MeS-ATP (through a P2Y1 purinoceptor) when L-NAME and indomethacin were absent. Br-A23187, a receptor-independent Ca2+ ionophore, produced significantly potentiated endothelial Ca2+ responses after I/R in the presence of L-NAME/indomethacin. Evaluation of artery diameter as a function of endothelial Ca2+ demonstrated no differences between sham and I/R groups. Conclusions— These findings demonstrate that I/R results in augmented endothelial Ca2+ responses that appear to be downstream of the receptor level. Moreover, these data suggest that this augmented Ca2+ response contributes to the potentiated EDHF-mediated dilations after I/R.

Role of Endothelium in Shear Stress–Induced Constrictions in Rat Middle Cerebral Artery

Background and Purpose— Luminal shear stress has been reported to constrict cerebral arteries and arterioles of several species. Although the endothelium is not required for this response, it is not known whether the endothelium enhances or attenuates shear stress-induced constrictions. Methods— Middle cerebral arteries (MCAs) were isolated from male Long-Evans rats, mounted in a tissue bath, and pressurized to 80 mm Hg in the absence of luminal flow. In some MCAs, the endothelium was selectively loaded with fura 2 for the measurement of endothelial Ca2+ concentration. Luminal shear stress was increased by adjusting luminal flow while maintaining a constant intraluminal pressure. Results— After the development of spontaneous tone in MCAs without luminal flow, inside diameters were ≈190 &mgr;m. MCAs constricted ≈15% when luminal flow was increased to produce a shear stress of 50 dyne/cm2. The shear stress-induced constrictions were more pronounced in vessels without intact endothelium. Scavenging reactive oxygen species with 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron) or superoxide dismutase/catalase significantly inhibited the shear stress-induced constrictions in vessels with intact endothelium and in vessels in which the endothelium had been removed. In intact vessels, endothelial Ca2+ increased 33 nmol/L (from 133±11 to 166±12 nmol/L) when shear stress was increased to 50 dyne/cm2. The presence of NG-nitro-l-arginine methyl ester (L-NAME), L-NAME+indomethacin, or L-NAME+indomethacin+charybdotoxin had no significant effect on the shear stress-induced constrictions in MCAs with intact endothelium. Conclusions— We conclude that the endothelium plays a role in attenuating the shear stress-induced constrictions in rat MCAs. The attenuation does not appear to be by release of NO, prostacyclin, or endothelium-derived hyperpolarizing factor. The endothelium apparently attenuates the constriction by an unknown dilating factor, by a dilating process, or simply by attenuating the mechanical force of the shear stress as it is transmitted to the abluminal side of the vessel.

Pharmacologically induced hypothermia via TRPV1 channel agonism provides neuroprotection following ischemic stroke when initiated 90 min after reperfusion

Traditional methods of therapeutic hypothermia show promise for neuroprotection against cerebral ischemia-reperfusion (I/R), however, with limitations. We examined effectiveness and specificity of pharmacological hypothermia (PH) by transient receptor potential vanilloid 1 (TRPV1) channel agonism in the treatment of focal cerebral I/R. Core temperature (T(core)) was measured after subcutaneous infusion of TRPV1 agonist dihydrocapsaicin (DHC) in conscious C57BL/6 WT and TRPV1 knockout (KO) mice. Acute measurements of heart rate (HR), mean arterial pressure (MAP), and cerebral perfusion were measured before and after DHC treatment. Focal cerebral I/R (1 h ischemia + 24 h reperfusion) was induced by distal middle cerebral artery occlusion. Hypothermia (>8 h) was initiated 90 min after start of reperfusion by DHC infusion (osmotic pump). Neurofunction (behavioral testing) and infarct volume (TTC staining) were measured at 24 h. DHC (1.25 mg/kg) produced a stable drop in T(core) (33°C) in naive and I/R mouse models but not in TRPV1 KO mice. DHC (1.25 mg/kg) had no measurable effect on HR and cerebral perfusion but produced a slight transient drop in MAP (<6 mmHg). In stroke mice, DHC infusion produced hypothermia, decreased infarct volume by 87%, and improved neurofunctional score. The hypothermic and neuroprotective effects of DHC were absent in TRPV1 KO mice or mice maintained normothermic with heat support. PH via TRPV1 agonist appears to be a well-tolerated and effective method for promoting mild hypothermia in the conscious mouse. Furthermore, TRPV1 agonism produces effective hypothermia in I/R mice and significantly improves outcome when initiated 90 min after start of reperfusion.