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Circulation Research. 2008;102:234-241
Published online before print November 8, 2007, doi: 10.1161/CIRCRESAHA.107.164145
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(Circulation Research. 2008;102:234.)
© 2008 American Heart Association, Inc.

Cellular Biology

Astrocyte-Derived CO Is a Diffusible Messenger That Mediates Glutamate-Induced Cerebral Arteriolar Dilation by Activating Smooth Muscle Cell KCa Channels

Anlong Li, Qi Xi, Edward S. Umstot, Lars Bellner, Michal L. Schwartzman, Jonathan H. Jaggar, Charles W. Leffler

From the Department of Physiology (A.L., Q.X., E.S.U., J.H.J., C.W.L.), University of Tennessee Health Science Center, Memphis; and the Department of Pharmacology (L.B., M.L.S.), New York Medical College, Valhalla.

Correspondence to Charles W. Leffler, PhD, Department of Physiology, 894 Union Ave, Memphis, TN 38163. E-mail cleffler{at}physio1.utmem.edu


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Astrocyte signals can modulate arteriolar tone, contributing to regulation of cerebral blood flow, but specific intercellular communication mechanisms are unclear. Here we used isolated cerebral arteriole myocytes, astrocytes, and brain slices to investigate whether carbon monoxide (CO) generated by the enzyme heme oxygenase (HO) acts as an astrocyte-to-myocyte gasotransmitter in the brain. Glutamate stimulated CO production by astrocytes with intact HO-2, but not those genetically deficient in HO-2. Glutamate activated transient KCa currents and single KCa channels in myocytes that were in contact with astrocytes, but did not affect KCa channel activity in myocytes that were alone. Pretreatment of astrocytes with chromium mesoporphyrin (CrMP), a HO inhibitor, or genetic ablation of HO-2 prevented glutamate-induced activation of myocyte transient KCa currents and KCa channels. Glutamate decreased arteriole myocyte intracellular Ca2+ concentration and dilated brain slice arterioles and this decrease and dilation were blocked by CrMP. Brain slice arteriole dilation to glutamate was also blocked by L-2-alpha aminoadipic acid, a selective astrocyte toxin, and paxilline, a KCa channel blocker. These data indicate that an astrocytic signal, notably HO-2–derived CO, is used by glutamate to stimulate arteriole myocyte KCa channels and dilate cerebral arterioles. Our study explains the astrocyte and HO dependence of glutamatergic functional hyperemia observed in the newborn cerebrovascular circulation in vivo.


Key Words: newborn • cerebrovascular circulation • functional hyperemia • heme oxygenase


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Local cerebral arteriole smooth muscle tone is temporally and spatially coordinated to match blood flow to neuronal activity. This vascular response is termed functional hyperemia or neurovascular coupling.1,2 In the cerebrovascular circulation, signals to vascular smooth muscle derive from endothelium, nerves, astrocytes, or pericytes, which interact to form a neurovascular unit.3 Astrocytes relay signals to neurons4 and other astrocytes.5,6 Parenchymal arterioles are ensheathed by astrocyte endfeet.7,8 Pial arterioles lay on a bed of astrocytes, the glia limitans, and are coated by astrocyte processes and endfeet.9–11 Recent articles have reviewed astrocytes functioning as intermediaries between neurons and cerebral blood vessels to adjust cerebral blood flow to neuronal activity.3,7,8 Potential astrocyte-derived vasoactive mediators of cerebrovascular responses include epoxyeicosatrienoic acids,12,13 K+,14,15 and ATP16, which appear to perform in concert.8

The principle excitatory cerebral neurotransmitter is glutamate that dilates cerebral arterioles to match blood flow to neural activity. Specific cells and mechanisms involved in coupling the glutamate release to arteriolar smooth muscle relaxation are uncertain. Astrocytes are uniquely positioned for sensing neuronal activity and regulating cerebral blood flow.7,8,17–19 They possess both metabotropic glutamate receptors (mGluRs) and ionotropic glutamate receptors (iGluRs). Glutamate stimulation of astrocytic mGluR and iGluR produces intracellular Ca2+ oscillations and localized and global elevations of astrocyte intracellular Ca2+ concentration ([Ca2+]i).20–24 In brain slices, glutamate elevates astrocyte [Ca2+]i but reduces adjacent vascular smooth muscle cell [Ca2+]i that produces vasodilation.22,25 However, the signaling mechanisms between astrocytes and vascular smooth muscle that result in this dilation to glutamate remain uncertain with eicosanoids,17,25 K+,2,14 and Ca2+24 among the mechanisms considered.

CO could be a glutamate-induced, diffusible messenger used by astrocytes to signal for arteriole dilation. This hypothesis is rational because glutamatergic dilation of newborn pig cerebral arterioles, in vivo, is blocked by inhibition of HO that produces CO.26,27 Furthermore, astrocyte injury, in vivo, blocks piglet pial arteriolar dilation to glutamate9 making it reasonable to propose that astrocytes are the source of vasoregulatory CO involved in excitatory amino acid–induced cerebral vasodilation. CO dilates cerebral arterioles by activating smooth muscle cell large conductance Ca2+ activated potassium channels (KCa channels).28,29 Therefore, we investigated whether astrocyte-derived CO increases smooth muscle cell KCa channel activity, providing a mechanism by which glutamate can cause arteriole dilation. Specifically, the present study tested the hypothesis that glutamate stimulates astrocyte production of CO that increases arteriole smooth muscle cell KCa currents, leading to a decrease in intracellular Ca2+ ([Ca2+]i) and vasodilation.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Piglet Cerebral Arteriole Smooth Muscle Cell Isolation
Procedures were approved by the University of Tennessee Health Science Center Animal Care and Use Committee. Newborn pigs (1 to 3 days old, 1 to 2.5 kg; Nichols Hog Farm, Olive Branch, Minn) were anesthetized with ketamine hydrochloride (33 mg/kg im) and acepromazine (3.3 mg/kg im). The brain was removed and placed into ice-cold HEPES-buffered physiological saline solution (PSS). For electrophysiological studies, arterioles (50 to 200 µm in diameter) were dissected from the cerebral cortical surface. Individual vascular smooth muscle cells were enzymatically dissociated from cerebral arterioles using a procedure previously described.30

Astrocyte Culture
Piglets were anesthetized as above, and adult HO-2+/+ and HO-2–/– mice were killed by peritoneal injection of sodium pentobarbital (130 mg/kg). Mouse procedures were approved by New York Medical College Animal Care and Use Committee. Brains were removed and placed in ice-cold DMEM with antibiotic/antimycotic (100 U/mL penicillin, 100 mg/mL streptomycin, and 2.5 mg/mL amphotericin B). Astrocytes were isolated, grown, and identified as described previously.9 Primary culture astrocytes were dislodged by trypsin-EDTA and either transferred to DMSO (10%)/FBS (90%) and frozen in liquid nitrogen for future use or directly plated on glass cover slips (4 mmx4 mm) and grown to confluence. No differences were detectable in growth or phenotype between directly plated and frozen astrocytes. Astrocytes on cover slips were used for experiments with freshly isolated smooth muscle cells.

Astrocyte Culture on Cytodex Microcarrier Beads
Confluent mouse HO-2+/+ and HO-2–/– astrocytes were dislodged with trypsin-EDTA and mixed with Cytodex microcarrier beads (175 µm diameter, denatured collagen chemically coupled to a matrix of cross-linked dextran, GE Healthcare), transferred into spinner flasks and stirred intermittently (20 rpm 30 minutes on and 3 hours off) at 37°C and 5% CO2 in air. After 12 hours, the beads were stirred continuously (20 rpm) for 4 to 6 days before experiments. Cell density was approximately 106 cells/mL beads.

Patch-Clamp Electrophysiology
A glass coverslip with or without a confluent layer of astrocytes was placed in an experimental chamber. Freshly isolated cerebral arteriole smooth muscle cells were added to the chamber and allowed to settle for 10 minutes. Potassium currents were measured by using the whole cell perforated-patch-clamp configuration.28 Transient KCa currents were measured at a steady membrane potential of –40 mV and calculated from ≥5 minutes of continuous gap-free data. At –40 mV, a transient KCa current was defined as the simultaneous opening of 3 KCa channels. Single KCa channel currents were recorded at a steady membrane potential of 0 mV in the presence of thapsigargin (100 nmol/L).

CO Measurement
After overnight incubation in L-glutamine and serum-free DMEM, astrocytes grown on Cytodex microcarrier beads (1 mL beads diluted to 1.7 mL with Krebs solution) were placed into amber vials with 13C18O (31CO, ISOTEC) as the internal standard. After 1 hour incubation at 37°C, 100 µL of head space gas was collected for CO detection by gas chromatography/mass spectrometry as described before.31,32

Detection of HO-2 Expression
Proteins from Laemmli buffer solubilized samples were separated by 12% SDS PAGE, transferred to PVDF membranes and blocked with 10% BSA in PBS-Tween 20 (0.1%). Immunoblotting was performed using the following antibodies: primary 1:2500 rabbit anti-mouse HO-2 antibodies (Stressgen), secondary 1:5000 goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology); primary 1:5000 mouse anti-mouse {alpha}-actin (Sigma Aldrich), secondary 1:10 000 goat anti-mouse IgG-HRP (Santa Cruz Biotechnology). The immunocomplexes were visualized with ECL Plus Western blot detection reagent (GE Healthcare) and quantified by digital densitometry using Gel-Pro Analyzer software (Media Cybernetics).

Brain Slice Preparation, [Ca2+]i, and Arteriolar Diameter Measurements
Newborn pig brains were removed and placed in cold (4 to 8°C) M199 with 10 mmol/L HEPES, and 10 mmol/L glucose, pH 7.37. Tangential slices were cut (200 µm thick) from the cerebral cortical surface and placed in M199. Diameters of cortical surface arterioles were measured in bicarbonate-buffered PSS (21% O2, 5% CO2) superfused (1.5 mL/min, 35°C) brain slices using an inverted microscope, CCD camera, and edge detection software (IonWizard, IonOptix). [Ca2+]i was measured in cerebral arterioles of piglet brain slices using fura-2 with a method published previously.33 The cerebral arteriolar wall in the brain slice was selected for data acquisition.

To selectively injure astrocytes, slices were incubated with L-2-alpha aminoadipic acid (L-AAA, 2 mmol/L). We,9 and others,10,11 have shown previously that L-AAA treatment in vivo produces histological evidence of injury to the superficial glia limitans and loss of astrocyte-dependent cerebrovascular responses without altering responses in general. Control slices were incubated with D-AAA, the inactive isomer.

Chemicals
Cr (III) mesoporphyrin IX chloride (CrMP) was purchased from Frontier Scientific. Papain was purchased from Worthington Biochemical. All other chemicals were obtained from Sigma Chemical unless otherwise stated.

Statistical Analysis
Values are means±SEM. For comparisons among more than 2 groups, results were subjected to a 1-way ANOVA for repeated measures with Tukey post hoc test to isolate differences between groups. Student t tests were used for comparison between each 2 groups of paired or unpaired data, as appropriate. P<0.05 was considered significant.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Cerebral arteriole smooth muscle cells were allowed to settle either on astrocytes or empty coverslips before measurement of whole cell currents. Using patch-clamp electrophysiology, transient KCa current frequency and KCa channel activity were recorded in smooth muscle cells. At –40mV, a physiological membrane potential, glutamate (20µmol/L) reversibly increased ({approx}75%) transient KCa current frequency in cerebral arteriole smooth muscle cells that were in contact with astrocytes (Figure 1). Conversely, glutamate had no effect on the transient KCa current frequency of smooth muscle cells that were not in contact with astrocytes (Figure 1).


Figure 1
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  		Figure 1. Glutamate (20µmol/L) elevates transient KCa current frequency in cerebrovascular smooth muscle cells in contact with astrocytes (A) but not in cerebrovascular smooth muscle cells alone (B). Summarized data are shown in C. n=7 and n=12 for smooth muscle cells alone and with astrocytes, respectively. *P≤0.05 compared with zero.

Transient KCa current amplitudes are not normally distributed (failed Kolmogorov and Smirnov for a Gaussian distribution at P<0.0001). Therefore, transient KCa currents were divided into groups that are logical based on numbers of simultaneous channel openings (Table): small transient KCa currents (3 to 7 channel openings [1 channel=2.8pA34]), midsized transient KCa currents (8 to 14 channel openings), and large transient KCa currents (15 to 30 channel openings). As can be seen in the Table, the distribution is skewed toward smaller transient KCa currents, then medium sized transient KCa currents, and fewer large transient KCa currents. Glutamate increased the number of small (1.6-fold), medium (1.7-fold), and large (3.1-fold) transient outward currents in smooth muscle cells in contact with astrocytes, but did not change the number of outward currents of any amplitude in smooth muscle cells that were alone.


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  		Table 1. Table. Effect of Glutamate on Piglet Cerebral Arteriole Smooth Muscle Cell (SMC) Transient KCa Currents

To investigate KCa channel regulation by glutamate, transient KCa currents were inhibited by depleting SR Ca2+ with thapsigargin (100nmol/L), a SR Ca2+ ATPase inhibitor that blocks Ca2+ sparks.30 Glutamate increased ({approx}65%) single KCa channel activity when smooth muscle cells were in contact with astrocytes (Figure 2). In contrast, glutamate did not change KCa channel activity in smooth muscle cells without astrocytes.


Figure 2
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  		Figure 2. Glutamate (20µmol/L) activates KCa channels in cerebrovascular smooth muscle cells in contact with astrocytes (A) but not in cerebrovascular smooth muscle cells alone (B). Summarized data are shown in C. n=6 and n=13 for smooth muscle cells alone and with astrocytes, respectively. *P≤0.05 compared with zero.

To investigate the contribution of astrocyte HO to the responses, astrocytes were pretreated with the HO inhibitor CrMP (20µmol/L, 1 hour). Coverslips with CrMP-treated astrocytes were rinsed and then added to the recording chamber to avoid exposure of smooth muscle cells to the HO inhibitor. Cerebral arteriole smooth muscle cells were allowed to settle on CrMP-treated astrocytes. In contrast to the effect of glutamate on vascular smooth muscle cells in contact with untreated astrocytes, where transient KCa current frequency increased {approx}75% and single KCa channel open probability increased {approx}65%, glutamate changed neither transient KCa current frequency nor KCa channel activity of smooth muscle cells in contact with CrMP-pretreated astrocytes (Figure 3).


Figure 3
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  		Figure 3. Chromium mesoporphyrin (20µmol/L; CrMP) pretreatment of astrocytes blocks glutamate (20 µmol/L) activation of transient KCa current frequency (A & B, n=8) and KCa channel activity (C & D, n=6) in smooth muscle cells in contact with astrocytes.

As an alternative approach for depression of astrocyte CO production, as well as for investigation of the HO subtype involved in glutamate-induced CO production, we used HO-2 knockout mice. Wild-type (HO-2+/+) homogenates showed strong expression of HO-2, whereas there was no detectable HO-2 protein expression in HO-2 knockout (HO-2–/–) mice (Figure 4A). Glutamate more than doubled CO production by HO-2+/+ astrocytes (Figure 4B). In contrast, glutamate had no effect on CO production by HO-2–/– astrocytes (Figure 4B).


Figure 4
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  		Figure 4. A, HO-2 protein expression in HO-2+/+ and HO-2–/– mice. B, Glutamate (20µmol/L) stimulates CO production by HO-2+/+ mouse astrocytes but not by HO-2–/– mouse astrocytes (n=3 separate mouse brain preparations each for HO-2+/+ and HO-2–/– with 5 or 6 tubes for each preparation). *P<0.05 compared with basal production.

To examine the effect of HO-2 expression on the ability of astrocytes to activate KCa channels in cerebral arterial smooth muscle cells, piglet cerebral arteriole smooth muscle cells were allowed to settle on either HO-2+/+ or HO-2–/– mouse astrocytes. Transient KCa current frequency and KCa channel activity were recorded. As was the case when using piglet astrocytes, glutamate elevated transient KCa current frequency (2.3-fold) as well as KCa channel activity (2-fold) in piglet smooth muscle cells in contact with HO-2+/+ mouse astrocytes (Figure 5). In contrast, glutamate did not alter transient KCa current frequency or KCa channel activity in piglet smooth muscle cells in contact with HO-2–/– astrocytes (Figure 5).


Figure 5
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  		Figure 5. Glutamate activates transient KCa current frequencies and single channel activity in piglet cerebral microvascular smooth muscle cells in contact with mouse HO-2+/+ (A & B, n=8) but not HO-2–/– (C & D, n=6) astrocytes. *P<0.05 compared with without glutamate.

We used newborn pig cerebral cortex slices to examine contributions of HO and astrocytes to glutamate regulation of arteriolar [Ca2+]i and diameter in structurally intact brain. Glutamate (30µmol/L) decreased the fura-2 ratio (340/380 nm) in arterioles, indicating a decrease of [Ca2+]i (Figure 6). Pretreatment of slices with CrMP markedly inhibited the effect of glutamate on [Ca2+]i. In addition, glutamate dilated piglet neocortical slice arterioles (from 111±10 to 123±11 µm; Figure 7). In contrast, glutamate did not alter the diameters of arterioles in brain slices pretreated with CrMP, suggesting CO generated by HO is required for glutamate-induced vasodilation. CrMP-treated slice arterioles constricted normally to the thromboxane receptor agonist U46619 (data not shown). Paxilline, a selective KCa channel blocker,35 reversed glutamate-induced dilation, suggesting the dilation was mediated by KCa channel activation. Also, after treatment of brain slices with L-AAA (2 mmol/L, 2 hours), a selective astrocyte toxin, arterioles did not dilate to glutamate (Figure 7D). In contrast, arterioles within slices incubated with the inactive isomer, D-AAA, dilated similarly to glutamate as did arterioles in untreated slices. L-AAA did not depress overall arteriolar reactivity as indicated by similar dilations to sodium nitroprusside of arterioles in L-AAA (12%) and D-AAA (15%) treated slices. These data show that astrocytes, HO-2, and KCa channels are required for glutamate-induced cerebral arteriolar dilation in newborn pigs.


Figure 6
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  		Figure 6. Glutamate (30 µmol/L) decreases intracellular Ca2+ in brain slice arteriole smooth muscle and this effect is blocked by chromium mesoporphyrin (CrMP, 20µmol/L). A, Representative traces of fura-2 fluorescence ratio in an arteriole of an untreated brain slice and a slice treated with CrMP. B, Collated data of brain slice arteriole responses to glutamate without and with CrMP (n=13 and 11, respectively). *P<0.05 compared with diameter prior to glutamate. #P<0.05 compared with or without CrMP.


Figure 7
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  		Figure 7. Chromium mesoporphyrin (CrMP, 20µmol/L), paxilline (20µmol/L), and L-2-alpha aminoadipic acid (L-AAA, 2 mmol/L) block glutamate (30µmol/L)-induced dilation of piglet cerebral cortical brain slice arterioles. A, Representative trace illustrating the effect of glutamate on the diameter of an arteriole in an untreated slice (a) and a slice treated with CrMP (b). B, Representative trace of glutamate-induced arteriolar dilation and the effect of paxilline on that dilation. C, Collated data of brain slice arteriole responses to glutamate without and with CrMP or paxilline (n=26, 11, and 11, respectively). D, Brain slice arteriolar diameter responses to glutamate and sodium nitroprusside (SNP, 140µmol/L) after treatment with D-AAA (n=10) or L-AAA (n=9). *P<0.05 compared with control diameter. #P<0.05 compared with or without CrMP, paxilline, or L-AAA, as appropriate.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
Major findings of the present study are that: (1) Glutamate activates transient KCa currents and KCa channel activity in cerebral arteriolar smooth muscle cells that are in contact with astrocytes but not in smooth muscle cells alone; (2) Glutamate increases CO production by intact astrocytes but not by genetically HO-2 deficient astrocytes; (3) Pharmacological inhibition of HO or genetic ablation of astrocyte HO-2 eliminates glutamate-induced KCa channel activation in cerebral arteriolar smooth muscle cells in contact with these astrocytes; (4) Glutamate decreases brain slice arteriolar smooth muscle [Ca2+]i through a mechanism that is attenuated by pharmacological inhibition of HO; and (5) Glutamate-induced dilation of piglet neocortical brain slice arterioles is blocked by either HO inhibition, KCa channel inhibition, or astrocyte injury.

Between the present and a recent study,9 we use 3 approaches, isolated cells, brain slices, and cranial windows in vivo. Each has different strengths and weaknesses and the combination is mutually complimentary. Brain slices have no descending pressure or blood flow. But slices allow arteriole wall [Ca2+]i measurements and remove both neuronal influences from beyond the study area and blood borne mediators. The results from slices mirror the in vivo experiments9 that show glutamate causes arteriolar dilation and brain CO production9 which are inhibited by astrocyte injury. In vivo, arterioles have normal pressure and flow and CO production can be measured, but specific cell types involved in communication or CO production, channels involved, and [Ca2+]i measurements are not possible. Isolated cells allow specific communication between myocytes and astrocytes to be investigated, inhibitor treatment of only astrocytes, using HO-2 knockout astrocytes with wild-type arteriolar smooth muscle, and measurement of CO production specifically by astrocytes. Thus, the combined 3 approaches lead to a more complete picture of the functional control mechanisms.

CO is produced physiologically by catabolism of heme to CO, iron, and biliverdin.36 HO catalyzes this reaction with oxidation of NADPH. HO is expressed as 3 known isoforms: HO-1, HO-2, and a third isoform (HO-3) with much lower heme degrading activity and expression.37 The present study shows that glutamate increases CO production by HO-2+/+ mouse astrocytes but not HO-2–/– astrocytes that do not express HO-2. CO reduces vascular smooth muscle tone both in vitro38,39 and in vivo.27,40 Indications of the functional significance of CO are supported by alterations of responses to important physiological cerebrovascular regulatory stimuli on HO inhibition. In the newborn pig in vivo, pial arteriolar dilations to glutamate, hypotension, and hypoxia are inhibited after topical treatment with CrMP that blocks CO production.26,41

Although CO has been demonstrated to increase transient KCa currents when applied directly to isolated vascular smooth muscle cells or arterioles, the present study is the first to show that a HO-2 product produced by another cell, an astrocyte, can activate transient KCa currents in an associated arteriolar smooth muscle cell. Because HO products other than CO do not activate KCa channels,28 the HO-2 product activating the KCa channel must be CO. Of additional significance, the signal, glutamate, is a primary mediator of brain function that does not directly affect KCa currents in cerebrovascular myocytes (Figure 1). The signal from astrocytes is necessary to provide the functional hyperemic response.

Our studies using cranial windows in vivo, found that either astrocyte injury or inhibition of CO production blocks dilation of newborn pig pial arterioles to topical glutamate application.9,26 In the present study, we show that in the absence of astrocytes or with astrocytes pretreated with CrMP, glutamate does not increase either smooth muscle cell transient KCa currents or KCa channel activity. To confirm that HO-2 is involved in this signaling pathway, piglet smooth muscle cells were allowed to contact mouse HO-2+/+ and HO-2–/– astrocytes. Glutamate increased transient KCa currents as well as KCa channel activity of smooth muscle cells attached to HO-2+/+ astrocytes, but not those attached to HO-2–/– astrocytes. We show that glutamate-induced dilation of brain slice arterioles is abrogated by either blocking the enzyme that produces CO or by blocking the KCa channel that CO activates. Further, HO inhibition prevented glutamate-induced reduction of [Ca2+]i in brain slice arterioles. All these data are consistent with the hypothesis that glutamate stimulates astrocyte HO-2, producing the diffusible gasotransmitter, CO, which activates KCa channels in adjacent cerebral microvascular smooth muscle cells.

In contrast to rat hypothalamus42 and newborn pig cerebral cortex,26 the sole reported influence of the HO/CO system on vascular tone in adult rat cerebral cortex has been constrictor.43 Endogenous CO appears to have a tonic inhibitory effect on NO production in rat cerebrum because the HO inhibitor, zinc protoporphrin (ZnPP) dilated pial arterioles over a 1-hour period, and enhanced cerebral NO production as estimated using diaminofluorescein-2.43 The dilation was blocked by L-NAME and by addition of CO to the superfusion solution. Conceivably, contributions of CO to cerebrovascular regulation could be biphasic with basal production providing tonic inhibition of NOS with resultant increased tone, whereas acute CO elevations in response to stimulation lead to dilation. Also, available data on responses of isolated adult cerebral arteries to CO suggest that species differences exist. Dilation to exogenous CO added to the bathing solution has not been detected in rabbit, rat, or mouse cerebral arteries in vitro.44,45 On the other hand, CO dilates dog basilar artery segments.46 In addition, in pigs, responses of pial arterioles to CO are age-dependent with juvenile pigs, and adult rats, having reduced dilations compared with baby pigs.47

CO causes dilation by activating smooth muscle cell KCa channels.26,29,48 Absence of dilation of rat cerebral arteries and arterioles to CO in vitro (above) is surprising because CO dilates peripheral rat arterioles via KCa channel activation.49,50 In vascular smooth muscle cells, several KCa channels are activated by spatially localized intracellular Ca2+ transients, termed Ca2+ sparks, that elevate the local [Ca2+] into the micromolar range.51 Transient KCa currents induce membrane hyperpolarization that reduces voltage-dependent Ca2+ channel activity, and thus decreases global [Ca2+]i, producing dilation. CO increases transient KCa currents by enhancing the coupling of Ca2+ sparks to KCa channels.28 The KCa channel {alpha}-subunit contains a heme-binding pocket and binding of heme inhibits KCa channel activity.52 CO, by binding to channel-bound heme, changes the association of heme with the KCa channel and causes activation.48 Therefore, the KCa channel is functionally a heme-protein, with heme acting as the binding site for CO.48

Species or age differences exist in Ca2+ spark-to-KCa channel coupling efficiency that could influence arteriole smooth muscle sensitivity to CO and possibly even the role of CO in regulation of cerebrovascular circulation. In adult rat cerebrovascular smooth muscle cells, virtually all Ca2+ sparks produce transient KCa currents.53,54 Conversely, not all Ca2+ sparks produce transient KCa currents in either newborn pig28,55 or adult human56 cerebral arterioles. Therefore, the ability of CO to hyperpolarize and dilate rat cerebral arterioles may be less44 than in newborn pig cerebral arterioles because CO increases the coupling of Ca2+ sparks to KCa channels and rat Ca2+ spark-to-KCa channel coupling is essentially unitary at physiological membrane potentials.

In the present study, rather than report transient KCa current amplitude as the mean of all transient KCa currents, we divided transient KCa currents into smaller (7 to 20 pA), larger (21 to 40 pA), and largest (>40 pA) currents. As can be seen in the Table, transient KCa current frequency in all size categories increased on treatment with glutamate. In piglets, smaller amplitude Ca2+ sparks do not cause transient KCa currents.28,34 CO increases the number of smaller amplitude transient KCa currents because smaller, uncoupled Ca2+ sparks become coupled to KCa channels.28 The addition of these smaller currents depresses the overall mean, which explains the minimal change in mean transient KCa current amplitude, even though large amplitude transient KCa currents are markedly increased. An accurate depiction of the effect of glutamate on transient KCa current amplitude requires accounting for the increased number of smaller currents. Glutamate increases frequencies of transient KCa currents across the amplitude spectrum because glutamate uses CO to increase Ca2+ spark-to-KCa channel coupling.

The present study shows glutamate increases CO production by astrocytes, but coupling mechanisms are not known. Stimulation of mGluR and iGluR increase Ca2+ oscillations in astrocytes,20–24 electrical field stimulation increases brain slice astrocyte endfoot [Ca2+]i,14,22 and inhibition of calmodulin blocks glutamate stimulation of CO production in cerebral microvessels and cortical neurons.57,58 Therefore, it is reasonable to speculate that glutamate stimulates CO production in astrocytes via elevation of astrocytic [Ca2+]i. Increasing [Ca2+]i could increase HO-2 activity directly to elevate CO production or by a variety of indirect actions including delivery of electrons from NADPH via cytochrome P450 reductase, delivery of heme to HO-2, or activation of kinases that elevate HO-2 activity. Glutamate also could increase HO-2 activity via Ca2+-independent kinase pathways. In endothelium, vascular smooth muscle, and neurons HO-2 activity can be increased by phosphorylation.31,58 mGluR elevates diacylglycerol that stimulates protein kinase C, which could phosphorylate and activate HO-2.

In summary, the present study indicates that the neurotransmitter glutamate activates transient KCa currents and KCa channel activity in newborn pig cerebral arteriole smooth muscle cells by increasing CO production by associated astrocytes. Either inhibition of CO production or treatment with astrocyte toxin prevents glutamate-induced arteriolar dilation in piglet brain slices. These data support the conclusion that astrocytes use CO as a diffusible messenger to cause cerebral arteriole dilation and enhance local blood flow to match increased glutamatergic neuronal activity.


*    Acknowledgments
 
Sources of Funding

This research was supported by NHLBI/NIH and NEI/NIH.

Disclosures

None.


*    Footnotes
 
Original received April 23, 2007; resubmission received September 13, 2007; revised resubmission received October 10, 2007; accepted October 31, 2007.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

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X. Qin, H. Kwansa, E. Bucci, S. Dore, D. Boehning, D. Shugar, and R. C. Koehler
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