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.

Endothelial SK(Ca) and IK(Ca) channels regulate brain parenchymal arteriolar diameter and cortical cerebral blood flow.

Calcium-sensitive potassium (K(Ca)) channels have been shown to modulate the diameter of cerebral pial arteries; however, little is known regarding their roles in controlling cerebral parenchymal arterioles (PAs). We explored the function and cellular distribution of small-conductance (SK(Ca)) and intermediate-conductance (IK(Ca)) K(Ca) channels and large-conductance K(Ca) (BK(Ca)) channels in endothelial cells (ECs) and smooth muscle cells (SMCs) of PAs. Both SK(Ca) and IK(Ca) channels conducted the outward current in isolated PA ECs (current densities, ~20 pA/pF and ~28 pA/pF at +40 mV, respectively), but these currents were not detected in PA SMCs. In contrast, BK(Ca) currents were prominent in PA SMCs (~154 pA/pF), but were undetectable in PA ECs. Pressurized PAs constricted to inhibition of SK(Ca) (~16%) and IK(Ca) (~16%) channels, but were only modestly affected by inhibition of BK(Ca) channels (~5%). Blockade of SK(Ca) and IK(Ca) channels decreased resting cortical cerebral blood flow (CBF) by ~15%. NS309 (6,7-dichloro-1H-indole-2,3-dione3-oxime), a SK(Ca)/IK(Ca) channel opener, hyperpolarized PA SMCs by ~27 mV, maximally dilated pressurized PAs, and increased CBF by ~40%. In conclusion, these data show that SK(Ca) and IK(Ca) channels in ECs profoundly modulate PA tone and CBF, whereas BK(Ca) channels in SMCs only modestly influence PA diameter.

Astrocytic endfoot Ca2+ and BK channels determine both arteriolar dilation and constriction.

Neuronal activity is thought to communicate to arterioles in the brain through astrocytic calcium (Ca(2+)) signaling to cause local vasodilation. Paradoxically, this communication may cause vasoconstriction in some cases. Here, we show that, regardless of the mechanism by which astrocytic endfoot Ca(2+) was elevated, modest increases in Ca(2+) induced dilation, whereas larger increases switched dilation to constriction. Large-conductance, Ca(2+)-sensitive potassium channels in astrocytic endfeet mediated a majority of the dilation and the entire vasoconstriction, implicating local extracellular K(+) as a vasoactive signal for both dilation and constriction. These results provide evidence for a unifying mechanism that explains the nature and apparent duality of the vascular response, showing that the degree and polarity of neurovascular coupling depends on astrocytic endfoot Ca(2+) and perivascular K(+).

Regulation of intracerebral arteriolar tone by K(v) channels: effects of glucose and PKC.

Voltage-gated potassium (K(v)) channels in vascular smooth muscle cells (VSMC) are critical regulators of membrane potential and vascular tone. These channels exert a hyperpolarizing influence to counteract the depolarizing effects of intraluminal pressure and vasoconstrictors. However, the contribution of K(v) channel activity to the functional regulation of cerebral (parenchymal) arterioles within the brain is not known. Thus K(v) channel properties in parenchymal arteriolar SMCs were characterized. Isolated, pressurized parenchymal arterioles and arterioles in cortical brain slices exhibited robust constriction in the presence of the K(v) channel inhibitor 4-aminopyridine (4-AP). 4-AP also decreased the amplitude of K(v) currents recorded from SMCs. The steady-state activation and inactivation properties of K(v) currents suggested that these channels are composed of K(v)1.2 and 1.5 subunits, which was confirmed by RT-PCR. K(v) channels can be regulated by extracellular glucose, which may be involved in the functional hyperemic response in the brain. Thus the effects of glucose on K(v) channel activity and arteriolar function were investigated. Elevation of glucose from 4 to 14 mM significantly decreased the peak K(v) current amplitude and constricted arterioles. Arteriolar constriction was prevented by inhibition of protein kinase C (PKC), consistent with previous studies showing enhanced PKC activity in the presence of elevated glucose. In cortical brain slices, the dilation generated by neuronal activity induced by electrical field stimulation was decreased by 54% in 14 mM glucose when compared with the dilation in 4 mM glucose. In anesthetized mice the whisker stimulation-induced increase in local cerebral blood flow was also significantly decreased in 14 mM glucose, and this effect was similarly prevented by PKC inhibition. These findings point to a critical role for K(v) channels in the regulation of intracerebral arteriolar function and suggest that changes in perivascular glucose levels could directly alter vascular diameter resulting in a modulation of local cerebral blood flow.

Functional architecture of inositol 1,4,5-trisphosphate signaling in restricted spaces of myoendothelial projections.

Calcium (Ca(2+)) release through inositol 1,4,5-trisphosphate receptors (IP(3)Rs) regulates the function of virtually every mammalian cell. Unlike ryanodine receptors, which generate local Ca(2+) events ("sparks") that transmit signals to the juxtaposed cell membrane, a similar functional architecture has not been reported for IP(3)Rs. Here, we have identified spatially fixed, local Ca(2+) release events ("pulsars") in vascular endothelial membrane domains that project through the internal elastic lamina to adjacent smooth muscle membranes. Ca(2+) pulsars are mediated by IP(3)Rs in the endothelial endoplasmic reticulum of these membrane projections. Elevation of IP(3) by the endothelium-dependent vasodilator, acetylcholine, increased the frequency of Ca(2+) pulsars, whereas blunting IP(3) production, blocking IP(3)Rs, or depleting endoplasmic reticulum Ca(2+) inhibited these events. The elementary properties of Ca(2+) pulsars were distinct from ryanodine-receptor-mediated Ca(2+) sparks in smooth muscle and from IP(3)-mediated Ca(2+) puffs in Xenopus oocytes. The intermediate conductance, Ca(2+)-sensitive potassium (K(Ca)3.1) channel also colocalized to the endothelial projections, and blockage of this channel caused an 8-mV depolarization. Inhibition of Ca(2+) pulsars also depolarized to a similar extent, and blocking K(Ca)3.1 channels was without effect in the absence of pulsars. Our results support a mechanism of IP(3) signaling in which Ca(2+) release is spatially restricted to transmit intercellular signals.