Marrelli, Sean P., Maxim S. Eckmann, and Michael S. Hunte. Role of endothelial intermediate conductance KCa channels in cerebral EDHF-mediated dilations.

Strong inward rectifying K + (K IR ) channels have been observed in vascular smooth muscle and can display negative slope conductance. In principle, this biophysical characteristic could enable K IR channels to 'amplify' responses initiated by other K + conductances. To test this, we have characterized the diversity of smooth muscle K IR properties in resistance arteries, confirmed the presence of negative slope conductance and then determined whether K IR 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 K IR channels were highly expressed in cerebral and coronary, but not mesenteric arteries. These channels comprised K IR 2.1 and 2.2 subunits and electrophysiological recordings demonstrated that they display negative slope conductance. Computational modelling predicted that a K IR -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 Ba 2+ attenuated the ability of K + channel activators to dilate cerebral and coronary arteries. This attenuation was absent in mesenteric arteries where smooth muscle K IR channels were poorly expressed. In summary, smooth muscle K IR 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 K IR have a substantive, albeit indirect, role in enabling agonists to alter the electrical state of a multilayered artery.

Marrelli SP, O'Neil RG, Brown RC, Bryan RM Jr. PLA2 and TRPV4 channels regulate endothelial calcium in cerebral arteries. Am J Physiol Heart Circ Physiol 292: H1390 -H1397, 2007. First published October 27, 2006; doi:10.1152/ajpheart.01006.2006.-We previously demonstrated that endothelium-derived hyperpolarizing factor (EDHF)-mediated dilations in cerebral arteries are significantly reduced by inhibitors of PLA 2. In this study we examined possible mechanisms by which PLA 2 regulates endothelium-dependent dilation, specifically whether PLA 2 is involved in endothelial Ca 2ϩ regulation through stimulation of TRPV4 channels. Studies were carried out with middle cerebral arteries (MCA) or freshly isolated MCA endothelial cells (EC) of male Long-Evans rats. Nitro-L-arginine methyl ester (L-NAME) and indomethacin were present throughout. (15,17). Activation of the IK Ca channels promotes endothelial cell hyperpolarization, which then appears to be directly transferred to the surrounding smooth muscle (25).In addition, our group recently demonstrated a critical role for cytoplasmic phospholipase A 2 (PLA 2 ) in EDHF-mediated dilation in rat MCA (33). In that study, we found that inhibitors of cytoplasmic PLA 2 (cPLA 2 ) significantly reduced EDHFmediated dilations to the purinergic receptor agonist uridine triphosphate (UTP). The specific role of PLA 2 in EDHFmediated dilation, however, was not determined.The purpose of the present study was to examine the mechanism by which PLA 2 contributes to EDHF-mediated dilation. We evaluated two hypotheses regarding endothelial Ca 2ϩ regulation and EDHF-mediated dilation. Our first hypothesis states that PLA 2 is critical for normal endothelial Ca 2ϩ regulation following UTP stimulation. In our proposed model, the critical role of PLA 2 activation would be to promote the sustained or plateau component of endothelial [Ca 2ϩ ] i to sufficient levels for IK Ca activation (17). To examine the mechanism of Ca 2ϩ regulation further, we examined the role of transient receptor potential (TRP) channels in mediating Ca 2ϩ influx in MCA endothelium. Our second hypothesis states that TRPV4 channels contribute to endothelial Ca 2ϩ influx and are regulated by products generated subsequent to activation of PLA 2 . For these studies we measured endothelial [Ca 2ϩ ] i and Ca 2ϩ influx in pressurized MCA, [Ca 2ϩ ] i in isolated endothelial cells, and message for candidate TRP channels by cellspecific PCR. METHODSIsolated/pressurized artery preparation. Rat MCA were harvested and mounted in a pressurized vessel chamber as described previously (14,17). MCA were pressurized to a mean transmural pressure of 85 mmHg, and flow was established through the lumen of the artery (100 l/min) with PSS of the following composition (in mM): 119 NaCl, 4.7 KCl, 21 NaHCO 3, 1.18 KH2PO4, 1.17 MgSO4, 0.026 EDTA, 5.5 glucose, and 1.6 CaCl2. PSS was warmed (37°C) and gassed with 5% CO2-20% O2-75% N2 to maintain a pH of 7.4. Nitro-L-arginine methyl ester (L-NAME) and indomethacin (10 M each) were included in...

The effects of stimulating P2Y1 or P2Y2 purinoceptors on the endothelium of isolated middle cerebral arteries (MCAs), third-order branches of the MCA (bMCAs), and penetrating arterioles (PAs) of the rat were studied. After pressurization and development of spontaneous tone (25% contraction), resting diameters for MCAs, bMCAs, and PAs were 203 ± 5 ( n = 50), 99 ± 2 ( n = 42), and 87 ± 2 μm ( n = 53), respectively. Luminal application of the P2Y1-selective agonist 2-methylthioadenosine 5′-triphosphate elicited dose-dependent dilations (or loss of intrinsic tone) in MCAs but not in bMCAs or PAs. The dilation in MCAs was completely blocked by removal of the endothelium or by nitro-l-arginine methyl ester (10−5 M), an inhibitor of NO synthase. Luminal application of the P2Y2-selective agonist ATP elicited dilations in MCAs, bMCAs, and PAs. Removal of the endothelium abolished the dilations in all vessel groups. Dilations in MCAs have been shown to involve both NO and endothelium-derived hyperpolarizing factor (EDHF). The dilations in bMCAs and PAs had a minor NO component and prominent EDHF component; that is, 1) the dilations to ATP were not diminished by the combined inhibition of NO synthase and cyclooxygenase, 2) the dilations were accompanied by significant hyperpolarizations of the vascular smooth muscle (∼15 mV), and 3) the dilations were completely abolished by the calcium-activated potassium channel blocker charybdotoxin. We concluded that the role of NO in purinoceptor-induced dilations diminishes along the cerebrovascular tree in the rat, whereas the role of EDHF becomes more prominent.

Endothelial-mediated dilations to selective P2Y1 and P2Y2 purinoceptor agonists [2-methylthioadenosine triphosphate (2MeS-ATP) and uridine 5′-triphosphate (UTP), respectively] were evaluated in middle cerebral arteries (MCAs) of rats after 2 h of ischemia followed by 24 h of reperfusion (I/R). MCAs were harvested, pressurized to 85 mmHg, and luminally perfused. 2MeS-ATP, which dilates by the synthesis and release of nitric oxide (NO), had significantly reduced maximum dilations following I/R. Reduced smooth muscle sensitivity to NO may explain the reduced dilation to 2MeS-ATP. In contrast, the dilations elicited by UTP were potentiated in that the concentration of agonist necessary to produce one-half of the maximum dilation was reduced by 75%. The potentiated dilation to UTP was the result of an endothelial factor having all the characteristics of the endothelium-derived hyperpolarizing factor (EDHF). That is, it was neither NO nor a cyclooxygenase metabolite, and its actions involved calcium-activated potassium channels and smooth muscle hyperpolarization. We conclude that the effect of I/R on endothelial-mediated dilations depends on the receptor system and the mechanism of dilation. Dilations elicited by 2MeS-ATP were attenuated, while dilations UTP were potentiated due to the upregulation of the EDHF mechanism.

The effect of luminal shear stress was studied in cerebral arteries and arterioles. Middle cerebral arteries (MCA) and penetrating arterioles (PA) were isolated from male Long-Evans rats, mounted in a tissue bath, and pressurized. After the development of spontaneous tone, inside diameters were 186 +/- 5 microm (n = 28) for MCA and 65 +/- 3 microm (n = 37) for PA. MCA and PA constricted approximately 20% with increasing flow. Flow-induced constriction persisted in MCA and PA after removal of the endothelium. After removal of the endothelium, the luminal application of a polypeptide containing the Arg-Gly-Asp amino acid sequence (inhibitor of integrin attachment) abolished the flow-induced constriction. Similarly, an antibody specific for the beta(3)-chain of the integrin complex significantly inhibited the flow-induced constriction. The shear stress-induced constriction was accompanied by an increase in vascular smooth muscle Ca(2+). For example, a shear stress of 20 dyn/cm(2) constricted MCA 8% (n = 5) and increased Ca(2+) from 209 +/- 17 to 262 +/- 29 nM (n = 5). We conclude that isolated cerebral arteries and arterioles from the rat constrict to increased shear stress. Because the endothelium is not necessary for the response, the shear forces must be transmitted across the endothelium, presumably by the cytoskeletal matrix, to elicit constriction. Integrins containing the beta(3)-chain are involved with the shear stress-induced constrictions.

The present study was designed to evaluate the role of endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) in the difference between P2Y(1)- and P2Y(2)-mediated vasodilatations in cerebral arteries. Rat middle cerebral arteries were cannulated, pressurized, and luminally perfused. The endothelium was selectively loaded with fura 2, a fluorescent Ca(2+) indicator, for simultaneous measurement of endothelial [Ca(2+)](i) and diameter. Luminal administration of 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP), an endothelial P2Y(1) agonist, resulted in purely nitric oxide (NO)-dependent dilation and [Ca(2+)](i) increases up to approximately 300 nM (resting [Ca(2+)](i) = 145 nM). UTP, an endothelial P2Y(2) agonist, resulted in dilations that were both endothelium-derived hyperpolarizing factor (EDHF)- and NO-dependent with [Ca(2+)](i) increases to >400 nM. In the presence of N(G)-nitro-L-arginine-indomethacin to inhibit NO synthase and cyclooxygenase, UTP resulted in an EDHF-dependent dilation alone. The [Ca(2+)](i) threshold for NO-dependent dilation was 220 vs. 340 nM for EDHF. In summary, the differences in the mechanism of vasodilatation resulting from stimulation of endothelial P2Y(1) and P2Y(2) purinoceptors result in part from differential [Ca(2+)](i) responses. Consistent with this finding, these studies also demonstrate a higher [Ca(2+)](i) threshold for EDHF-dependent responses compared with NO.

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.